stem cell transplant

How was BMT discovered as a treatment for blood diseases?

2011-09-19T04:29:00.000-07:00

BMTs done in the late 1950s to the late 1960s were often disappointing. Most patients who had transplants were terminally ill and often died soon after the transplant. Many patients receiving BMT were given bone marrow that was not typed correctly. Some patients died of graft versus host disease (GVHD), in which the transplanted tissue attacks the person it was supposed to cure, or succumbed to infections that took advantage of the patient's weakened immune system.

In the early 1970s there were dramatic improvements in the survival rates of patients undergoing the procedure. This was due to HLA typing, which was established around that time. BMTs were also more successful because leukemia patients received transplants while in remission and because supportive care had improved. The results of BMTs continued to improve through the 1970s and 1980s as new antibacterial, antifungal, and antiviral agents were developed. At the same time, medications called growth factors were developed, which increased the patients neutrophil counts so they had fewer infections. Today, the cure rate for acute leukemia patients receiving BMT is about 50-70%; for chronic myelogenous leukemia, it's about 70-80%; and for aplastic anemia, it's 60-80%. In the relatively short time of about four decades, the bone marrow transplant procedure has become a successful form of treatment for a number of illnesses. Many patients who would die otherwise can be saved through the BMT. The use of blood as a stem cell source redefined this procedure to stem cell transplant (SCT), but the basic idea is the same: stem cells from a healthy donor are used to replace diseased tissue.

What is a cord blood transplant?

2011-09-19T04:13:00.000-07:00

Cord blood is blood collected from a baby's placentaand umbilical cord at the time of birth. Although it is not yet common for people to preserve this blood, more parents arc choosing to do so because scientists have discovered that the blood in the umbilical cordhas many stem cells that can be transplanted later. Several hundred cord blood transplants have been performed to date, and the data suggest that these transplants may have a lower risk of GVHD. Cord blood can be collected from both the umbilical cord and the placenta after birth. If the umbilical cord is clamped within 30 seconds of vaginal delivery while the placenta is still inside the mother, an experienced physician can collect up to 100 milliliters of cord blood without harming either the infant or the mother. Cord blood is generally not manipulated before it is frozen and stored, so the stem cells can be preserved.

Considering the advantages of cord blood transplants lower risk of GVHD for the recipient and completely painless collection for the donor infant it seems strange that so few have been performed, until you understand the reasons why doctors are not collecting cord blood. Congenital malformations in the child are one rationale and a sensible one, considering that no one knows how the genetic problems of the donor child might affect the already sick recipient. Cord blood banks test all collected cord blood for infections and for possible congenital malformations. But the other primary reason is simply that the mother has not given her informed consent for the procedure. However, as more people become aware of the possibilities of cord blood transplants, preserving cord blood is becoming more common. However, the number of stem cells obtained from one newborn baby is relatively small and usually not enough to successfully transplant an adult patient. Most cord blood transplants have been performed in children.

With cord blood collected for an unrelated transplant, doctors study the medical histories of both the biological mother and father. Also, before the cells may be used for transplant, the donor infant must be evaluated at 6 and 12 months of age for any evidence of congenital or infectious diseases that might not have been obvious at birth. Cord blood contamination (inadvertent contamination with bacteria or other infectious agents during the collection procedure) rates are reported to be 3-15%. This depends on what kind of collection system was used. The possibility of contamination means that the cord blood must undergo stringent microbiological testing before it is used.

Spectral Karyotyping and Fluorescent in situ Hybridization - Stem Cell

2010-03-24T02:24:00.000-07:00

Spectral karyotyping (SKY) is a hybridization-based diagnostic technique originally developed to diagnose chromosomal aberrations associated with cancer and genetic disease. SKY can be used to detect specific inter- and intra-chromosomal genomic rearrangements, and unambiguously determine both the total number and individual identity of all chromosomes in a metaphase nucleus.

Fluorescence in situ hybridization (FISH) is a similar technology, but it can be used on non-dividing cells at interphase, while SKY requires metaphase chromosomes. FISH is used for preimplantation genetic diagnosis of single blastomeres, and is very useful when studying ESC differentiation, as the terminally differentiated cell populations are typically post-mitotic and thus cannot be karyotyped using SKY. However, FISH also has certain drawbacks compared to SKY: it does not allow enumeration of every chromosome in a single experiment because each chromosome must be assessed separately with a different FISH probe and it is difficult to detect genomic rearrangements.

The advantages of SKY and FISH for hESCs is their ability to generate information at the single cell level. Often in the case of complex processes such as cellular differentiation and disease progression, valuable data from single cells may be obscured by the heterogeneity of the cell population, a limitation which both SKY and FISH have the potential to overcome.

Overview

The underlying procedure behind metaphase cell analysis using SKY is straightforward. SkyPaint (Applied Spectral Imaging) is hybridized to metaphase chromosome spreads from the cells of interest. SkyPaint is a mixture of probes specific to single chromosomes, each of which contains a spectrally unique combination of fluorescent nucleotides thus allowing the user to “paint” each chromosome a different color. After acquiring a metaphase spread image using a microscope equipped with an interferometer that reads emissions across the entire visible spectrum, individual chromosomes are assigned using SkyView software (Applied Spectral Imaging). SkyView analyzes the spectral image in two dimensions and displays each chromosome with a distinct classification color from which it creates a karyotype table.
FISH is a technique used to identify the presence of a single nucleic acid sequence (often specific to a particular chromosome) through hybridization of fluorescently labeled DNA probes to denatured chromosomal DNA in cytological material. Interphase nuclei are hybridized with the FISH probe, though metaphase spreads can be used as well. FISH probes can be purchased commercially or made by the user. The hybridized nuclei can then be viewed using a fluorescent microscope.

Because probes for both SKY and FISH are generated using a direct labeling technique (the fluorophore is covalently attached to the nucleotide) they can be stripped from the template DNA by heat denaturation, and new probes hybridized to the same nuclei afterwards.
The following protocol describes basic techniques that can be used for both FISH and SKY analyses. Places where the protocols differ are noted in the text.

Procedures

To obtain a sufficient number of metaphase spreads for SKY from hESCs it is often necessary to harvest the cells to be karyotyped 1-2 days after splitting them. When cells are harvested later, there may not be enough dividing cells to obtain an adequate number of metaphase spreads. In terms of cell density, it is possible to do karyotyp-ing with a few thousand cells but it is always better to have more. We recommend taking two confluent wells of a six-well plate of hESCs and splitting them 1:2 to make four wells. One or two days later harvest all four wells for karyotyping. The following procedure can be used for generating material for FISH as well, though it is not crucial to have dividing cells.

Day 1: Cell preparation and harvest

1. Add colcemid to the cells at a final concentration of 0.1 |ig/mL and return cells to the incubator for 5-6 h (necessary for SKY, optional for FISH).
Note: Colcemid is added to the cells to arrest them in mitosis. Because they have an extended cell cycle, longer colcemid incubations (5-6 h) are needed for hESCs compared with other cell types.
2. Trypsinize cells using 0.05% trypsin/EDTA to obtain a single cell suspension; some remaining clumps are fine.
3. Wash cells with 10 mL of PBS and aspirate the supernatant. Flick the pellet so it is easy to resuspend.
4. Add 10 mL of 0.075 M KCl to the tube, making sure that the cells are well suspended. Incubate cells in a water bath at 37°C for 15 min.
5. After the 15 min incubation, add three drops of fixative dropwise with a transfer pipette to the cells while flicking the tube between drops.
6. Spin cells at 1000 rpm for 5 min at room temperature.
7. Aspirate most of the supernatant off and then flick the tube to resuspend the pellet.
8. Add 5 mL of fixative dropwise while slowly vortexing the tube. It is VERY important not to make too many cell clumps at this point.
9. Incubate at 4°C overnight.

Day 2: Making chromosome spreads

Making chromosome spreads is not necessary for FISH but this technique can be used to adhere the nuclei to the slide.
1. Let the cells warm up to room temperature and wash twice with fixative. Resuspend cells in 1 mL of fixative.
Note: It may be necessary to spin the cells down later and resuspend them in a smaller or larger volume of fixative, depending on how many cells you have. Try 1 mL to start and if the spreads on the slide look too sparse or too close together (see step 6), adjust the volume appropriately and repeat the slide-making process.
2. Open the lid on the 80°C water bath and let some of the steam dissipate. Make sure the heating plate is positioned close to the water level (1 cm) in the water bath. See Figure 6.3 for a schematic illustration of the water bath and heating plate set-up.
3. Flick cells to resuspend then take 20 \iL of cell suspension and pipette it onto the slide. Hold the slide level for about 15-20 s - you should see the center of the slide become granular as the fixative evaporates.
4. Quickly flip the slide over (cell side down) and briefly hold it to the steam coming from the water bath (about 5 cm above the water level in the bath).
5. Immediately place the slide, cell side up, on the metal heating plate in the water bath until the liquid on the slide beads up and is mostly evaporated. Immediately remove the slide and look at it under a microscope.
6. Check the spreads for two things: (1) spread density and (2) chromosome color/contrast.
7. Make 5-10 good slides/sample. Put them in a slide box and store them at room temperature for 1-7 days. This “aging” time will improve the results.
8. Fill the tube containing the remaining cell suspension to 5 mL with fixative and store at 4°C. Cells can be stored like this for at least one year.

Day 3: Slide pretreatment

Slides should be aged at room temperature in the dark for 1-7 days.
1. Wash slides in 2 X SSC at room temperature for 5 min.
2. Add 25 |jL of 100 mg/mL pepsin to 50 mL of 0.01 M HCl solution that has been pre-warmed to 37°C. This gives a final pepsin concentration of 50 |ig/mL. Make sure the pepsin is thoroughly mixed into the solution.
3. Incubate slides in the pepsin solution for 5 min at 37°C.
Note: Chromosome spreads and interphase nuclei from hESCs often need longer pepsin pretreatments to remove cytoplasmic debris compared with other cells. Pepsin concentration and incubation time should be determined empirically depending on the cell type. Be careful not to expose the slides to the pepsin solution for too long as this will denature the chromosomes and make them difficult to hybridize.
4. Wash slides twice with PBS for 5 min at room temperature.
5. Incubate slides for 5 min at room temperature in PBS with 50 mM MgCl2.
Incubate slides in 50 mM MgCl2 in PBS containing 1% formaldehyde for 10 min at room temperature.
7. Wash slides in PBS for 5 min at room temperature.
Dehydrate slide in 70%, 80%, 100% EtOH sequence, 1 min each.
9. Air dry slides.

Slides can be hybridized immediately or stored for at least a year in a dessicator at -20°C.
Paint and probe preparation

1. Place 10 |jL of SkyPaint or the manufacturer’s suggested amount of FISH probe in a small microfuge tube at 37°C. Vortex tube every 3-5 min for 30 min. Protect paint and probe from light!
2. Denature SkyPaint or probe for 10 min in a water bath (or thermocycler) at 80°C, then leave for 60 min at 37°C.
3. Denature slide in denaturation solution at 73°C for 1.5 min.
4. Immediately dehydrate slide in 70%, 80%, 100% EtOH sequence, 1 min each.
5. Air dry slide.
Note: Denaturation separates the homologous chromosomes of the target genome and rapid dehydration holds them in the single-stranded state prior to hybridization.
6. Place the slide on a 37°C slide warmer for 5 min prior to addition of the SkyPaint.
Hybridization
1. Apply SkyPaint or probe (from Paint and probe preparation section) to a coverslip (24 X 24 mm). Apply coverslip to slide immediately after adding paint or probe, and seal the edges with rubber cement.
2. Place slide in a pre-warmed humidified box and allow hybridization to proceed overnight in a 37°C incubator. Two day hybridizations are fine as well.
Note: Sealing the slide with rubber cement and the use of a humidified hybridization chamber are both done to prevent the probe mix from evaporating. Hybridization is done at 37°C because this temperature is low enough to promote the binding of complementary sequences but high enough to deter the binding of mismatched sequences.
3. Make up the wash solutions for the next day and preheat to 45°C.

Day 4: Washes

1. Carefully remove the rubber cement seal using forceps.
2. Place slide in 2 X SSC until the coverslip falls off.
3. Wash slide three times in formamide solution at 45°C for 5 min.
4. Wash slide three times in 1 X SSC at 45°C for 5 min.
5. Wash slide once in 4 X SSC + 0.1% Tween-20 for 5 min.
6. Make up staining reagent: combine 10 \iL of reagent 3; 5 \iL of reagent 4 (both from the Concentrated Antibody Detection (CAD) kit from Applied Spectral Imaging); and 1 mL of 4 X SSC. Vortex solution for 10 s and spin in a microfuge for 2 min to pellet fluorescent aggregates.
Note: This step is not necessary for FISH. Proceed to step 9 of this section.
7. Remove as much water as possible from the slide without letting it dry out then add 100 \iL of staining solution to the slide. Cover the slide with a coverslip.
Place slide in a humidified chamber at 37°C for 30 min in the dark.
9. Remove coverslip and wash slide three times in 4 X SSC + 0.1% Tween-20 at 45°C for 5 min.
10. Incubate slide in 4 X SSC + 0.5 |ig/mL DAPI for 5 min at room temperature.
11. Immediately dehydrate slide in 70%, 80% and 100% EtOH sequence, 1 min each.
12. Air dry slide in the dark.
Note: Washes are done to remove unbound and weakly hybridized paint or probe from the slide. High stringency washes may reduce background but could also remove specifically bound paint or probe. Reduced stringency washes may increase background fluorescence. Stringency can be increased or decreased by changing the temperature, formamide concentration, and salt concentration.
Interpretation
1. Apply Vectashield (antifade) and add coverslip (24 X 50 mm).
2. View slide using a microscope equipped with an interferometer and SKY software for SKY analysis. Only a fluorescent microscope is needed for FISH analysis.
Storage
Store slide at -20°C in the dark.
Recipes
Denaturing solution (70% formamide/2 X SSC solution)
For 70 mL: add 49 mL of formamide and 14 mL of water to 7mL of 20 X SSC. Important: Adjust pH to 7.0. Between periods of use, store at 4°C. Use each batch of denaturant for 7 days and then discard.
Formamide solution (50% formamide/2 X SSC)
For 150 mL: add 75 mL of formamide to 75 mL of 4 X SSC and adjust pH to 7.0. Between periods of use, store at 4°C. Use each batch for 7 days and then discard.
Ethanol sequence solutions
For final concentrations of 70%, 80%, and 100%, prepare v/v dilutions of 100% ethanol with H2O. Use dilution for up to 7 days and then discard. If solution evaporates or becomes diluted, replace with fresh solution. Between periods of use, store at room temperature.
4 X SSC + DAPI
For 50 mL: add 5.0 \iL of DAPI (5 mg/mL stock) to 50 mL 4 X SSC.
4 X SSC + 0.1% Tween-20
For 500 mL: add 100 mL of 20 X SSC, 400 mL of H2O and 0.5 mL of Tween-20. Mix well.
20 X SSC
For 1L: dissolve 175.3 g of sodium chloride and 88.2 g of sodium citrate in 1L of water. pH to 7.0.
Fixative
For 20 mL add 15 mL of methanol to 5 mL of glacial acetic acid. Make fixative solution up fresh EACH DAY.

Supplies and Reagents
SkyPaint available from Applied Spectral Imaging
FISH probe available commercially from Vysis, Cambio or other companies
Vectashield Antifade Mounting Medium (Vector Labs catalog no. H1000)
Coverslips (24 X 24 mm and 24 X 50 mm)
Slides (Fisher catalog no. 12-544-7)
Rubber cement
Pair of forceps
Corning Falcon Tubes (50 mL)
Concentrated Antibody Detection Kit (CAD Kit) from Applied Spectral Imaging
Formamide (Sigma catalog no. F7503)
DAPI (Sigma catalog no. 32670)
Colcemid (Invitrogen catalog no. 15212-012)
Pepsin (Sigma catalog no. P7000)
Methanol (Fisher catalog no. A454-1)
Glacial acetic acid (Fisher catalog no. A38-212)
Formaldehyde (Sigma catalog no. F1268)
0.05% Trypsin/EDTA solution (Invitrogen catalog no. 25300-054)
Tween-20 (Sigma catalog no. T8787).
Equipment
- Slide warmer Micropipettors (10, 20, 200 (jlL)
- Microcentrifuge
Water baths with temperature control (two or more)
- Vortex mixer
- Heated block or PCR machine
Fluorescent microscope with interferometer and SKY software available from Applied Spectral Imaging
- -20°C freezer and 4°C refrigerator
- 80°C water bath with metal plate.

Pitfalls and Advice
Feeder cells in hESC cultures
For SKY it is not necessary to separate the hESCs from feeder layer cells because the feeder cells are not dividing and thus will not contribute to the metaphase population. When analyzing cells via FISH the feeder layer cells will be included in the analysis, but they are usually karyotypically grossly abnormal because of the methods used for mitotic inactivation. If feeder cells appear to be confusing the results, feeder cells stably expressing green fluorescent protein (GFP) can be used and then sorted out before cells are harvested. Alternatively, individual ESC colonies can be picked off the feeder layer prior to trypsinization to generate a cell population with very few feeder cells. It is also possible to culture the cells to be karyotyped under feeder-free conditions.

Chromosome spreads
Making chromosome spreads is an art. Getting a good spread depends on many variables (e.g. humidity), which cannot be easily controlled. Here are some things to try if you are not getting good spreads:
- Wash cell suspension with fixative again.
Slow down the evaporation process by not placing the slide on the heated metal plate - just allow it to dry slowly.
To decrease the water content in the chromosomes, do not expose the slide to steam.
Try using a different fixative. We have had some luck with 1:1 glacial acetic acid to methanol.
Repeat the procedure on a different day when (presumably) atmospheric conditions are different.
Spectral karyotyping
Background staining can be divided into two general categories, large chunks of paint that are scattered across the slide and fluorescent haze that coats the slide evenly.
Chunks of paint
This type of background typically occurs when the SkyPaint is not fully solubilized before hybridization. Placing the tube of SkyPaint at 37°C and vortexing periodically for 30-60 min will clear this up.
Fluorescent haze
This type of background is typically due to improper slide preparation or pretreatment, which can leave residual cytoplasmic debris on the slide that can non-specifically bind labeled SkyPaint. A longer pepsin pretreatment can decrease this type of background.
Fluorescent in situ hybridization
Background fluorescence
This can be divided into two general categories, depending on where the background staining is originating:
Originating from the chromosomes: This type of background typically comes from non-specific hybridization of probe DNA to the target genome. Addition of Cot-1 carrier DNA, hybridization at a higher temperature, additional washes at a higher stringency, and the use of less probe during the hybridization can decrease this background.
Originating among and around the chromosomes: This type of background is typically due to improper slide preparation or pretreatment, which can leave residual cytoplasmic debris on the slide which can non-specifically bind labeled probe. A longer pepsin pretreatment or preparation of a new cell suspension and can decrease this type of background.
Weak staining
Wash was too stringent - decrease wash temperature and increase salt concentration.
DNA was not adequately denatured prior to hybridization - repeat the slide and probe denaturation.
Increase the signal using tyramide amplification (available from Molecular Probes and Perkin Elmer).
Intense staining
Wash more, or at a higher stringency (increased temperature, increased formamide concentration, decreased salt concentration).
Use less probe - sometimes if there are not very many metaphase spreads and/or nuclei on the slide use half the recommended amount of probe.

Classical Cytogenetics: Karyotyping

2010-02-18T01:21:00.000-08:00

Embryonic stem cells (ESCs) are arguably the most stable normal diploid cells that can be maintained in long-term culture. However, aneuploidies and other chromosomal abnormalities have been reported to occur in both human and mouse ESCs. Aneuploid mouse ESCs do not contribute to the germline in chimeric animals. But since this ultimate test of normalcy cannot be applied to hESCs, the cultures must be routinely evaluated for chromosomal abnormalities.

When hESCs are cultured using high-quality validated reagents, and passaged using gentle techniques, the cells can maintain a normal karyotype for years of continuous culture. However, under certain conditions that are believed to stress the cells, such as enzymatic passaging or culture in the absence of feeder cells, cultures tend to acquire karyotypic abnormalities. hESCs that acquire an extra chromosome 12 and the long arm of chromosome 17 (17q) appear to have a growth advantage and can eventually dominate the cultures. While we do not know when accumulated genotypic changes tip the scales to make a hESC culture no longer useful for experimental or therapeutic applications, we do know that the higher the percentage of abnormal cells in our cultures, the more cells are drifting towards an abnormal phenotype, and the less reproducible and dependable are the results.

There are several methods for assessing chromosomal stability, including the classical cytogenetics approaches described here, and the SKY, FISH, and SNP methods described in other chapters. These methods differ in resolution and the types of abnormalities they can detect. The best resolution obtainable by classic cytogenetic methods is estimated to be about 10 Mb, while spectral karyotyping (SKY) resolves at 1–2 Mb, and single nucleotide polymorphism (SNP) and copy number polymorphism (CNP) mapping can give 30 kb resolution. However, more resolution is not necessarily better; for example, SNP genotyping cannot be used to detect translocations or inversions, and SKY cannot detect inversions or duplications.

Cytogenetics is currently the most accessible method for detecting chromosomal abnormalities. But there are shortcomings to this technique. First, cytogenetic analysis can only be applied to metaphase-stage cells, so a rapidly dividing population is required. And second, while most hospitals have laboratories that perform karyotyping as a service, such laboratories routinely examine metaphases from 20 cells, 6 of which are analyzed and 14 are counted. This gives only a hint of the composition of the cell population, and many hESC researchers prefer analysis of 100 metaphases. For this reason, and to lower costs, some research laboratories are learning to perform their own karyotyping, at least at the gross level of counting chromosomes to detect aneuploidies.

It is relatively easy to count the chromosomes to determine the modal chromosome number and, with training, one may be able to identify chromosomes by their individual size and banding pattern. It is unlikely that an untrained eye will be able to identify the translocations or deletions that do not change the chromosome count, but may drastically modify the genome. So while we suggest that a research laboratory should routinely count chromosomes, we recommend that the detailed karyotype of the culture be obtained from a trained cytogeneticist every 10–15 passages.

OVERVIEW

The basic conventional cytogenetic method involves chromosome harvest, slide preparation, banding of the chromosomes, analysis of banding patterns, and interpretation of the results. In this chapter we will describe:

How to prepare a culture to maximize the number of metaphase chromosomes
How to prepare slides containing chromosome spreads
How to stain chromosomes
How to interpret the cytogenetic report.

Chromosome harvest consists of arresting the cell cycle at metaphase, hypotonic treatment of the cells and their fixation. After fixation, the chromosomes are spread onto glass slides, air-dried, and aged before banding. Chromosomes are stained and visualized as a continuous series of light and dark bands. A band is defined as that part of a chromosome that is clearly distinguishable from its adjacent segments by appearing darker or lighter. Slide preparation profoundly affects the quality of banding and it is one of the most challenging steps in chromosome preparation and analysis.

Different banding patterns, such as G-, Q-, R-, C-, T-, or NOR-banding, can be generated for analysis. The G-banding method (using the Giemsa dye mixture) is the most commonly used staining method, and it can generate up to 1000 bands per haploid human genome. Each band has a specific number assigned to indicate its location on the human chromosome. The nomenclature of band assignment and chromosome aberrations is sanctioned by the International System for human Cytogenetic Nomenclature (ISCN 2005).

PROCEDURES

Metaphase harvest of hESCs

A culture with actively dividing cells is the best way to obtain high-quality metaphases. Since hESCs are usually actively dividing, it is relatively easy to obtain many quality metaphase chromosomes from a culture. We suggest harvesting the cells for karyotyping roughly one day before they would normally be passaged. This strategy should yield a high number of dividing cells and therefore a sufficient number of metaphase chromosomes in order to make an accurate analysis of the culture.
This procedure describes harvesting cells from a 35 mm dish or one well of a six-well plate.

1. Add 1/100 volume of colcemid stock solution to the culture. 2. Return the culture to the incubator for 2–3 h.
3. Aspirate the medium and wash with 1 mL of PBS.
4. Trypsinize the cells with 0.3 mL trypsin, recover with 0.6 mL complete medium, and transfer into a microfuge tube.
NOTE: Good metaphase spreads require a single cell suspension.
5. Spin in a microfuge at 3000 rpm for 5 min at room temperature.
6. Aspirate the medium carefully, leaving about 50–100 ?L. Resuspend the cell pellet by tapping the tube.
7. Add 1.5 mL of hypotonic solution and let stand at room temperature for 15 min.
NOTE: The timing is important; if the cells are incubated too long they may burst and the chromosomes will spill out of the cell membrane. If too short, the chromosomes may be too tightly packed to analyze.
8. Gently invert the tube several times in order to resuspend the cells and then add a few drops of fixative (3:1 methanol:glacial acetic acid). Mix by inverting the tube several times.
9. Spin in a microfuge at 3000 rpm for 5 min at room temperature.
10. Aspirate the hypotonic solution carefully, leaving about 50–100 ?L. Resuspend the cell pellet well by tapping the tube.
11. Add 1 mL of fixative (3:1 methanol:glacial acetic acid); mix well.
12. Spin in a microfuge at 3000 rpm for 5 min at room temperature.
13. Aspirate the fixative carefully, leaving about 50–100 ?L. Resuspend the cell pellet well.
14. Repeat steps 10 and 11: Add 1 mL fixative (3:1 methanol:glacial acetic acid), mix well, then spin in a microfuge at 3000 rpm for 5 min at room temperature.
15. Add an appropriate amount of fixative to the pellet for making slides immediately.

NOTE: Fixed cells may be stored at 4°C for up to a week before slides are made.
Slide preparation: making chromosome spreads There are many ways to make slides. Below we describe a protocol that has been used successfully to make high-quality chromosome spreads and will provide a good starting point from which one can develop an individualized method.

Set-up for chromosome spreads

1. Prepare a Coplin jar with slides soaking in 100% methanol. It is a good idea to use single frosted slides, so the slides can be easily marked.
2. Make fresh fixative (3:1 methanol:glacial acetic acid).
3. Prepare a slide-making area with 2–3 sheets of paper towels, a folded Kimwipe for slide cleaning, and a pencil for marking the slides. Place the Coplin jar with slides soaking in 100% methanol and a beaker of deionized or distilled water on one side and fixative with a Pasteur pipette on the other side (if you are righthanded, the slides and water should be on the left and the fixative should be on the right). The chromosome harvest can be placed either in the middle or on the same side as the fixative, with a Pasteur pipette alongside.

4. Remove a slide from the Coplin jar of methanol and use a folded Kimwipe to polish the surface of the side to be used for the cells. It is important to keep track of the polished side of the slide so that the chromosome harvest is dropped on the polished side; frosted end slides are useful for this purpose.

Chromosome spreads

If you are right-handed, hold the slide with your left hand so that you can hold a pipette in your right hand. The descriptions below are for a right-handed person. NOTE: The following steps will be performed at nearly the same time, so you will need to coordinate left and right hands. We recommend that you practice these steps several times before using cell samples that may be limited.

1. Left hand: Dip the slide back into the methanol jar briefly, remove and swirl the slide in a beaker of deionized or distilled water. The rinse should be just long enough for a uniform film of water to coat the polished side of the slide. You will be able to observe this easily as you lift the slide out of the water.
2. Right hand: Gently but thoroughly mix the prepared cells with a glass Pasteur pipette.
3. Right hand: Draw the cell mixture into the Pasteur pipette and allow the suspension to sit in the pipette, so that it is ready for dropping onto the slides. At the same time, proceed with the next step with the other hand.
4. Left hand: Hold the frosted end of the slide between fingers and thumb (index and middle fingers on the polished side and thumb on the back side) and keep the polished side up as you lift the slide out of the beaker of water. Keep the long edge of the slide in contact with the paper towel and tilt the top edge of the slide forward quickly to drain off the excess water. Then tilt the top edge backwards until your thumb is rested on the paper towels (approximately 30 degrees between the back side of the slide and the paper towels).
5. Right hand: Hold the Pasteur pipette horizontally about 2–7 cm above the slide. Drop three drops of chromosome harvest, evenly spaced, along the slide, starting from the free end of the slide and moving toward the end you are holding. The drops should land on the slide slightly above the midline of the length of the slide – about one-third of the slide width from the top of the slide.
NOTE: The number of drops per slide can be adjusted according to the density of the chromosome harvest after the test slide is evaluated. If it requires more than four drops of chromosome suspension, spin down the suspension in the microfuge and reduce the amount of fixative accordingly.
6. Right hand: Fill a Pasteur pipette with fresh fixative and flow it across the top of the slide immediately after dropping the chromosome harvest (Figure 5.1B).
7. Left hand: Tilt the slide forward and tap gently on the paper towels to drain off the fixative.
8. Right hand: Wipe off the back and the long edges quickly, and mark the slide with a pencil on the frosted end.

Preparation for staining and banding

Drying: The drying process can affect how the chromosomes spread and the banding quality. In general, it is sufficient to dry the slide without much manipulation when the humidity is about 50%. If necessary, the humidity can be manipulated by using a damp paper towel or hot plate as drying surface as needed. The morphology of the chromosomes should be evaluated with a phase contrast microscope.

Prior to staining, bake the slides at 90°C for 30 min.

G-Banding of chromosomes

Chromosomes can be stained with dyes that result in a specific banding pattern on each chromosome. This banding pattern is used to determine the identity and integrity of individual chromosomes and the karyotype of a cell. Giemsa (“G”) is the dye mixture that is most commonly used to stain chromosomes. G-banding allows the specific identification of individual chromosomes as well as segments of each individual chromosome.

The “bands” are differently stained regions (and subregions) that are recognizable in chromosomes, and are given numerical designations, from proximal to distal on the chromosome arms. The short and long arms of chromosomes are designated as p and q, respectively.

A cytogeneticist can identify deletions, translocations, inversions, and duplications of chromosomes by analyzing G-banded chromosome spreads. G-bands are provided in reference materials that describe genes (for example, NIH’s NCBI Entrez Gene: www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB gene, and the European Bioinformatics, and the Online Mendelian Inheritance in Man database and provide disease information linked to banding data. The nomenclature of band assignment and chromosome aberrations is provided by the International System for human Cytogenetic Nomenclature (ISCN 2005).

Interpreting results

Professional cytogeneticists examine at least 20 metaphases. Generally, six metaphases are analyzed and the other 14 are counted. However, if an abnormal chromosome is observed, the cytogeneticist will search the slide for this and other abnormalities and may end up evaluating more than 20 spreads to determine whether this particular abnormality represents the clonal expansion of a cell within the culture or a random change that does not represent a significant shift to a neuploidy. The International System for human Cytogenetic Nomenclature (ISCN, 2005) establishes the rules for identifying and naming individual chromosomes and chromosomal abnormalities that are used by cytogeneticists to determine the karyotype of a cell. A book containing this information is updated and published periodically.

Karyotype

The karyotype, by convention, provides the following information: modal number, sex chromosome, abnormal abbreviation (1st chrom; 2nd chrom) (arm band number; arm band number):

Modal number: total count of number of chromosomes in each cell of a given cell line

Sex chromosomes: complement of X and Y chromosomes

Band number: numerical description of the location of a band on a chromosome arm, in order, from the centromere to the end of the chromosome. These numbers are a standard determined by the ISCN, revised in 2005.

Karyogram

A karyogram is made by taking a photograph of a G-banded metaphase. Then the individual chromosomes are cut out of the photograph (originally, the “cuts” were made by scissors, but now software is usually used) and arranged in a standardized template by size, specific banding pattern, and centromere location. By convention, the short (p) arm is at the top of the chromosome image (Figure 5.2).

Resolution

One of the variables in classical karyotyping by G-banding is the “resolution.” The resolution of the karyotype is related to the number of bands that are visible and therefore the smallest segment of the genome that can be detected using this method. The most common method to determine the resolution of banded chromosomes is to count the number of bands visible on chromosome 10 and then estimate the resolution from the chart in Table 5.1.

ALTERNATIVE PROCEDURES

DAPI staining

This is a simple method used to stain DNA and count chromosomes, but it will not allow identification of individual chromosomes.
1. Add a drop of mounting medium containing DAPI to the slide.
2. Seal the coverslip.
3. Count chromosomes under UV light using 100x oil immersion lens.

PITFALLS AND ADVICE

Cultures

The cultures should be subconfluent and actively dividing for best results. One effective method of obtaining enough metaphase chromosomes is to harvest hESCs for karyotyping the day before they would normally be passaged. During the metaphase of mitosis, the chromosomes reach their highest level of condensation and become identifiable under the microscope. The chromosomes are less condensed at early metaphase and become more condensed as the cell progresses towards the end of metaphase. Since the goal of harvesting the cells is to obtain as many quality metaphase chromosomes as possible in order to make an accurate analysis of the culture, colcemid is added to the cultures as it blocks the cells in metaphase. Longer treatment with colcemid will increase the mitotic index, but prolonged treatment will lead to higher fraction of cells with condensed, short chromosomes, and the resolution of G-bands will be low. In order to obtain both a good mitotic index and good chromosome length, the optimum length of time the cells are incubated with colcemid can be determined empirically.

Chromosome spreads

Unlike mouse chromosomes, human chromosomes generally have distinct arms visible on both sides of the centromere. However, chromosomes often overlap in the metaphase spread, so it can be difficult for the untrained eye to identify individual chromosomes; this is especially difficult for the smaller chromosomes, 21, 22, and Y. Hypotonic solution is used to swell the cells and allow the chromosomes to separate. If the cells are left too long in the hypotonic solution the cells will burst and it becomes impossible to determine which chromosomes belong together. Drying the slides immediately after the cells are dropped to make the spreads seems to be the most critical variable in making good slides. Best results are obtained when drying in an atmosphere of 50% humidity at 22°C. This can be achieved by monitoring the humidity and temperature in the working area using a portable hygrometer/thermometer and adjusting humidity by adding or removing wet paper towels in the immediate slide-making area.

Even after one becomes proficient in making slides, variables such as temperature and humidity may be difficult to control. In general, a “test” slide is made to determine whether the density of the cell suspension is adequate and whether the condition of slide drying is appropriate for the specific harvest and for the given day.

Cryopreservation of Human Embryonic Stem Cells

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Cryopreservation is used to stabilize cultures at specific points in time with specific genetic characteristics. Without the ability to cryopreserve our cell lines, we are forced to continuously subculture them, during which time the cells may accumulate genetic changes and become heterogeneous. Cryopreservation allows us to produce a bank of stock vials at specific passages with specific genetic characteristics. Using validated stock vials to initiate new experiments maximizes the long-term usefulness of a cell line and minimizes experimental variation.

During cryopreservation, most of the water is removed from the interior of cells and is converted to ice. This stops metabolism and allows cells to be stored at low temperatures for long periods of time. However, recovery of hESCs from cryopreservation is sometimes very poor, and because of the slow growth rate of hESCs, the time from thawing of the vial to having cultures suitable for experimentation can be weeks to months.

OVERVIEW

The methods outlined here work well in some laboratories but not others, for reasons that are not clear. This is an area of active research, to maximize the viability and maintain pluripotency of hESCs after cryopreservation. The most important issue is to make sure that the cells used for cryopreservation are in excellent condition, actively proliferating, and that the cultures have very little differentiation. Freezing the cells at high density appears to improve their viability after thawing, but since hESCs do not survive well after being dissociated into single cells, the exact densities cannot be easily quantified.

We provide two different methods in this chapter. The main method is a modification of a standard slow freezing protocol that works well for at least some hESC lines. For this protocol, we recommend that the entire population of a culture vessel be placed into 1–3 vials, and when thawed, the cells should be placed in the same size vessel. The second method, vitrification, is offered as an alternative procedure. Vitrification is a rapid freezing technique that minimizes formation of damaging ice crystals. While vitrification requires considerable skill, for some hESC lines it gives consistent results, and for researchers who master this method it is a recommended technique for cryopreserving small numbers of cells from newly derived hESC lines.

PROCEDURES

Slow freezing of cells

1. Prepare actively proliferating, high-density cells as you would for passaging. Change the culture medium just before harvesting the cells.
2. Label 1.8 mL cryovials with cell line name, date, and passage number.
3. Mix 2x stock cryopreservation medium (see Recipes) and keep on ice.
4. Dislodge the colonies from the plate mechanically using a sterile pipette tip. Alternatively, treat with 200 U/mL of collagenase IV for 5–10 min at 37°C. Remove collagenase and replace with ESC medium (3 mL for each well of a six-well dish).
5. For each well of a six-well dish, collect the cells in 3 mL of ESC medium and transfer to a 15 mL conical tube.
6. Centrifuge 5 min at 0.2 rcf (usually about 1000 rpm). Aspirate supernatant, leaving a small amount of medium.
7. Gently resuspend the pellet in protein-containing ESC medium (usually 1.5mL for each well of a six-well dish or one half of the final freezing volume). Use a 5 mL pipette to gently triturate the cells.
8. Drop wise, add an equivalent volume of ice-cold 2 stock cryopreservation medium, mixing constantly by tapping the tube.
9. Place 1.0 mL of cell mixture in each cryovial.
10. Rapidly transfer the vials to a precooled (4°C) Nalgene freezing container (containing isopropanol), and place immediately in a freezer at –70 to –80°C.
NOTE: Do not leave the cells in DMSO at room temperature for long periods of time. 11. Transfer cells to liquid nitrogen for long-term storage.

Thawing cells

There is a growth lag upon thawing the cells and it may take several days in order to be able to visualize the colonies. It is advisable to observe the cultures under 4 magnification 24 h after thawing, but not exchange the medium for at least 48 h. There will be a lot of floating debris and dead cells upon thawing the cells – this is normal.
1. Gently thaw the vial of cells by shaking it gently in a 37°C water bath and remove while a sliver of ice still remains.
2. Spray the tube with 70% EtOH and dry with a Kimwipe.
3. In the biosafety hood, aseptically remove the vial contents and place into a 15 mL conical tube.
4. Slowly, with gentle tapping, add 10 mL of room temperature culture medium.
5. Spin very gently at 0.2 rcf (1000 rpm) for 5 min.
6. Remove the supernatant.
7. Tap the tube to dislodge the pellet.
8. Add 3 mL of ESC medium to the tube and transfer to one well of a six-well dish that has been prepared with an inactivated feeder layer or extracellular matrix.
9. Place plate into the incubator and allow the cells to attach to the plate.
10. Allow 3–7 days for the cells to attach. During this time replace half of the medium every other day.
11. The medium should be replaced daily starting 4–7 days after thawing the cells, or when the cells appear to be attached.

PITFALLS AND ADVICE

Centrifugation

Centrifugation can damage sensitive cells. The major factors that can be important are: relative centrifugal force (200xg, 400xg, 800xg), time (5, 10, and 15 min), length of the column of liquid (3 mL, 6 mL, and 12 mL in 15 mL tubes) and the type of centrifuge (fixed angle or swinging bucket).

Cryoprotectants

DMSO could contribute to hESC death and differentiation. First, addition and removal of DMSO causes osmotic stress that may affect the survival of delicate cells. Second, DMSO itself has been shown to be a potent inducer of apoptosis and differentiation. Alternate cryoprotectants that are being investigated are permeable agents such as ethylene glycol, propylene glycol, glycerol and erythritol and non-permeable sugars and sugar-alcohols such as D-glucose and fructose, sucrose, trehalose, mannose, raffinose, adonitol, glucitol, and sorbitol.

ALTERNATIVE PROCEDURES

Freezing cells by open pulled straw vitrification

Open pulled straw (OPS) vitrification is not a trivial technique. It requires preparation of three different media and careful work under a dissecting scope. In this method, hESC colonies are dissected, and 10–12 individual undifferentiated pieces of colonies are carefully collected and placed into sequential vitrification media with increasing concentrations and combinations of cryoprotectants. The cells are then placed into straws and frozen by plunging into liquid nitrogen. In spite of the extra time spent preparing and the great care to freeze only undifferentiated clumps of cells, this method results in a very high (x90%) percentage recovery of the frozen cellular aggregates, with very low percentage of differentiation in the cultures following recovery from cryopreservation. It is possible that since there is low percentage of cell death and rapid recovery following OPS vitrification, the selective pressures that may be at play during more traditional cryopreservation methods may not be as much of an issue with this method. NOTE: This method was adapted from methods used at the Monash Institute of Medical Research Laboratory of Embryonic Stem Cell Biology.

Freezing cells by vitrification

For this method, hESC colonies are dissociated using a combination of dispase (10 mg/mL in serum-containing medium) and mechanical dissection into clumps of x100–200 cells each.

The sequential incubations are performed on a 37°C heated stage of a dissecting microscope. This procedure should not take more than 3min from the time the cells are placed into vitrification solution 1 (VS1) until they are placed into liquid nitrogen. Work quickly.

Set-up for vitrification

Label 4.5 mL cryovials with cell line, passage number, and date. Puncture vials with an 18G needle through the top and on the side so that liquid nitrogen can fill the vial. Use a four-well plate with three wells containing 1 mL each of holding medium (HM), VS1, VS2 vitrification solutions on a heating stage of a dissecting microscope (Figure 4.2). Transfer of the cells will be done in droplets of VS2 on the inside of the lid of the four-well dish.

Mouse Embryonic Fibroblast Feeder Cells

2010-02-17T13:53:00.000-08:00

Mouse embryonic fibroblasts (MEFs) have been used reliably as feeder cells for mouse embryonic stem cells (mESCs) since the early 1980s when the first mESC lines were being derived and cultivated. The first published derivation of hESC used MEF feeder layers, and many laboratories continue to use them routinely for long-term hESC culture.

MEFs are primary cells derived from day 12.5–14.5 fetuses, and are primary cells that do not continue to proliferate indefinitely. Once the cells begin to senesce they seem to lose their capacity to support undifferentiated growth and proliferation of hESC, so they are used optimally between passage 3 and passage 6. Usually large batches are made, tested, and cryopreserved so that this process needs to be repeated only occasionally.

Batches of MEFs need to be prepared on a routine basis and each newly prepared batch should be tested for robust recovery from cryopreservation, and support of undifferentiated proliferation of hESC cultures. They must also be tested for mycoplasma and should be subjected to mouse antibody pathogen (MAP) testing, usually by an outside service.

It is important that MEFs are mitotically inactivated before being co-cultured with hESCs, or the MEFs will become a growing contaminant cell type that is difficult to remove. There are two common ways of inactivating MEFs: irradiation and mitomycin C treatment. The cells can be cryopreserved either before or after mitotic inactivation.

NOTE: Several suppliers provide prepared stock vials of MEFs from various mouse strains with or without selectable markers. Depending on the quantity of MEFs that will be required, it may be cost-effective to purchase MEFs that have passed quality control by a supplier.

PROCEDURES

Isolation of MEF feeder cells

You will need to plan well in advance in order to prepare your own batch of MEFs. It is important to follow your institutional, local, state, and federal regulations regarding the use of laboratory mice. There are usually institutional guidelines covering the use of vertebrate animals, governed by the Institutional Animal Care and Use Committee (IACUC), which reviews proposed use of vertebrate animals in research. Obtain IACUC approval, then obtain the desired mouse strain and set up matings as approved by IACUC.

Preparation

Animal facility

- Mice: female(s) 13.5 dpc (days post coitum)
- Surgical instruments: sterilized
- Ethanol: 70%
- One 50 mL conical tube containing 25 mL D-PBS.

Tissue culture laboratory

- Prepare the tissue culture hood
- Three 10 cm sterile Petri dishes containing 15 mL of sterile D-PBS per pregnant female mouse.

Isolation of MEF

The protocol below is a method that has worked well to produce high-quality MEFs from various mouse strains including, 129, C57B/6, FVB/N, and CF-1.

Day 1: Preparing and plating MEFs

1. In the animal facility: using approved euthanasia method, sacrifice a 13.5 dpc female mouse. At 13.5 dpc, the female will be very visibly pregnant.

2. Place the female on her back on a clean bench or inside a hood. Spray the abdomen with 70% ethanol. Using sterile forceps and scissors make a small lateral incision under the diaphragm. With two hands pull the skin back, revealing the peritoneum. Make an incision in the peritoneum and remove the uterus.

3. Using aseptic techniques, remove the uterus, containing 13.5 dpc embryos and place in the sterile conical tube containing 25 mL of sterile D-PBS.

4. In the tissue culture lab: Using aseptic techniques in the tissue culture hood, remove the uterus from the 50 mL conical tube and place in a dish containing 15 mL D-PBS.

5. Dissect the embryos from the uterus and carefully remove each from their yolk sac and placenta.

6. Place embryos into a fresh Petri dish containing 15 mL of D-PBS.

7. Remove the head and internal organs (dark red tissue in the abdomen) using a pair of small sharp scissors and watchmaker forceps.

8. Rinse each carcass well, by placing in a fresh Petri dish containing 15 mL of D-PBS and gently swirling the dish to remove any remaining blood.

9. Place dissected embryos in 10 cm dish containing 10 mL of 0.05% trypsin and using very sharp, fine scissors mince the tissues into fine pieces.

10. Add another 5 mL of trypsin and triturate the solution until it easily moves into and out of a 5 mL pipette.

11. Place the Petri dish into the incubator for x5 min, just long enough to dissociate the cells, but not so long as to produce a “stringy sludge” of DNA. As soon as you notice stringy material in the plate stop reaction by adding 15 mL of feeder cell medium and place the entire solution into a 50 mL conical tube.

12. Allow the large pieces of tissue to settle to the bottom of the tube for 5 min.

13. Carefully remove the supernatant to a clean 50 mL conical and add D-MEM to final volume of 50 mL. Mix by gently inverting the tube several times.

14. Spin the tube at 1000 rpm for 3–5 min.

15. Aspirate the supernatant and discard.

16. Resuspend the cell pellet in 10 mL of feeder cell medium, put 5 mL into each of two 150 cm2 tissue culture flasks containing 20 mL of feeder cell medium.

17. Place in the incubator overnight.

Day 2: Observe the cultures

The flask should be 60–70% covered with healthy fibroblasts.
1. Aspirate the medium.

2. Rinse once or twice with 10 mL of D-PBS to remove any debris and red cells that were carried along in the isolation process (the fibroblasts will remain firmly attached to the tissue culture flask).

3. Place 25 mL of feeder cell medium in each 150 cm flask and return to the incubator overnight.

Day 3: The cells should be confluent and ready to passage

Passage the cells using 0.05% trypsin/EDTA 1:3 and return to the incubator. 1. Rinse the flasks with 15 mL of D-PBS.

2. Add 5 mL of 0.05% trypsin/EDTA.

3. Incubate for 3–5 min at 37°C.

4. Add 10 mL of feeder cell medium and gently triturate to mix the cells.

5. Add 5 mL of cells to each of three 150 cm2 flasks containing 20 mL of feeder cell medium.

6. Return to incubator.

Day 4–5: Observe the cultures for growth following passaging and make sure there is no contamination

By this point the cultures should have very characteristic fibroblast morphology. Harvest cells for batch freezing when confluent; this may be on day 4 or day 5. Harvest cells with trypsin/EDTA.

1. Rinse each flask with 15 mL of D-PBS.

2. Add 5 mL of 0.05% trypsin/EDTA.

3. Incubate for 3–5 min at 37°C.

4. Add an equal volume of feeder cell culture medium to inactivate trypsin.

5. Triturate the cell suspension and distribute to conical tubes for centrifugation. Centrifuge at 1000xg (0.2 rcf) for 5 min. Aspirate supernatant and resuspend cells in a small volume (about 1 mL for each flask).

6. Count the cells using a hemocytometer and test for viability with trypan blue. The cells should be nearly 100% viable.

7. Prepare cryovials – usually this procedure generates 30–50 vials at 3–5 x 106 cells/vial from each pregnant female. This is “passage 2.”

8. Dilute the cell suspension to about 6 x 106cells/mL. Add an equal volume of 2x cryopreservation solution and distribute 1 mL to each vial.

9. Slow freeze to – 80°C, using a freezing container (usually with isopropanol) or Styrofoam box. Transfer the vials to liquid nitrogen if possible.

Test MEF stocks for pathogens and growth recovery after freezing

A week or so after preparing the MEF, thaw a vial to test for viability and pathogens. Mycoplasma tests can be performed inhouse or by a service lab (see chapter on hESC culture methods). In addition, the cells should be tested to be sure that they do not harbor mouse pathogens. Mouse antibody pathogen (MAP) testing is usually performed by a service lab such as RADIL (www.radil.org).

Thawing and culture of feeder cells

The protocol below describes the culture of actively dividing cultures. If you are using mitotically inactivated feeder cells, follow the supplier’s instructions for thawing, plating, and use.

1. Prepare feeder cell medium.

2. Place 15 mL of medium into a 75 cm2 tissue culture flask and put the flask into the incubator 15–30 min prior to thawing the cells in order to allow the medium to equilibrate.

3. Thaw the vial of cells by gently shaking it in a 37°C water bath without submerging the vial below the O-ring on the cap.

4. As soon as the contents have thawed, less than 2 min, remove the vial from the water bath and spray it with 70% ethanol. Dry the vial with a clean Kimwipe, and move to a tissue culture hood where the rest of the procedures will be performed aseptically.

5. Transfer the contents of the vial to the prepared flask and incubate the culture over night at 5% CO2 in humidified air at 37°C.

6. The next morning, observe the culture. The flask should be 40–60% confluent with a healthy culture of feeders.

7. Replace the medium with 15 mL of fresh feeder cell medium and return to the incubator.

8. Monitor the cultures daily and passage 1:3 when the cultures are 80–90% confluent.

Passaging feeder cells

1. Aspirate the feeder cell medium.

2. Wash the flask with 5–10 mL of D-PBS.

3. Add 3–4 mL of trypsin-EDTA to the flask and incubate at 37°C for 3–5 min.

NOTE: Use less time for human fibroblasts, slightly more for MEFs. Gently shake the flask to make sure that the entire surface area is coated with trypsin.

4. Gently shake the flask to remove the cells, add 10 mL of culture medium, rinse the surface of the flask and transfer 5 mL to each of two new flasks.

5. Add 10 mL of medium to all three flasks and return to the incubator for further culture.

6. Monitor the culture daily and passage 1:3 when the cultures are 80–90% confluent.

Inactivating feeder cells

The feeder layer plays a complex role in helping to maintain hESCs in an undifferentiated state. The feeder layer must be healthy and rapidly dividing prior to inactivation in order to provide the best substrate for the growth of the hESCs.

Following inactivation, feeder layers remain adequate for the culture of hESCs for 5–7 days. In order to keep the feeder layer healthy, it is advisable to change the medium on the inactivated cells every 3 days. Always observe the feeder layer under the microscope prior to using them for the culture of hESCs in order to confirm that the cell layer is still intact and the cells have not begun to deteriorate. Since hESCs are usually passaged every 6–7 days, the feeder layer can start to deteriorate before the hESCs are ready to passage if “old” feeder layer dishes are used. For best results, inactivate the feeders the day before passaging the ESCs.

Inactivation of the feeder cells is accomplished by either irradiation or treatment with mitomycin C. Inactivated feeder cells are usually plated on gelatin (collagen I)- coated dishes to aid in their attachment.

NOTE: Although the cells are unable to undergo mitosis, they still replicate their chromosomes and can become multiploid. When karyotyping hESCs, occasionally a feeder cell is included in the cell count. Mouse cells are distinguishable by their acrocentric chromosomes.

Inactivation by gamma irradiation

1. Trypsinize the feeder layer as you would for passage.

2. Remove the cells from the flask and wash with feeder cell medium up to 10 mL.

3. Place cells in a sterile 15 mL conical tube and irradiate for a total of 30–40 Gy (3000–4000 rads).

NOTE: 3000–4000 rads is standard for irradiation of mouse embryo fibroblasts. Higher or lower levels of irradiation are sometimes suggested, but the important issue is that the cells are alive but unable to proliferate. See Pitfalls and Advice section for more detailed information.

4. Following irradiation, dilute the feeders to 3 x 105 in feeder cell medium, and re-plate the cells on the appropriate configuration of gelatinized culture dishes to meet experimental goals, and incubate overnight.

5. The next morning aspirate the feeder cell medium, rinse with D-PBS, and replenish the culture dish with hESC medium.

Inactivation by mitomycin C treatment

NOTE: Mitomycin C is a cytotoxic antitumor agent and must be handled carefully; it works by cross-linking the DNA, which blocks cell division. Follow your institution’s rules for safe handling and disposal. Handlers should wear latex or nitrile protective gloves and work in a biological safety or fume hood. One effective method is to inactivate the mitomycin C with an equal volume of household bleach. Inactivation is rapid.

1. Remove the feeder cell medium.

2. Add 10 mL/75 cm2 of mitomycin C medium.

NOTE: Make sure the entire flask is covered with mitomycin C medium so that the inactivation is complete and all cells are exposed for the entire incubation time. 3. Incubate for 3 h at 37°C in 5% CO2.

4. Remove mitomycin C solution and inactivate it with bleach or other recommended procedure.

5. Wash inactivated feeder layer three times with 10 mL each of PBS.

6. Trypsinize the cells to remove from flask, resuspend in feeder cell medium, and re-plate the cells on the appropriate configuration of gelatin-coated (see below) culture dishes to meet experimental goals, and incubate overnight.

NOTE: at this point, inactivated feeder cells can be cryopreserved for later use. Be sure to indicate on the freezing vial that the cells are already inactivated.

7. The next morning, wash the dishes of inactivated fibroblasts with PBS and refeed with either feeder cell medium or hESC medium in preparation for hESC culture.

Substratum support for feeder cell layers

In order to provide better support for the long culture periods required for hESC culture, inactivated feeder cells are plated on gelatinized dishes. 1. Coat culture dishes with 0.1% gelatin solution.

2. Incubate 1 h to overnight at 37°C.

3. Just prior to plating inactivated feeder cells, remove gelatin and rinse the dish with D-PBS.

4. Plate the inactivated feeders on the gelatin coated dishes and allow them to attach in the incubator for at least 4 h before culturing with ESCs.

Photomicrographs MEF feeder layers

Figure 3.1 shows the morphology of MEF feeder layers. MEFs do not form the whorls of cells that are typical of other fibroblasts used as feeder layers, such as human foreskin fibroblasts. The bottom photo shows high-density hESC (WA09 line) colonies cultured on MEF feeder layers.

ALTERNAT I V E PROCEDURES

MEF cells from commercial sources

There are several commercial sources for MEFs from various mouse strains and containing various selectable drug-resistant markers. Depending on the level of use, this can be a convenient and economical alternative to the de novo preparation of MEF feeder layers.

PITFALLS AND ADVICE

General advice

Rules of thumb:
- If the provider of the hESCs recommends a certain plating density, follow their instructions – at least initially.

- If no instructions are given, a slightly less than confluent layer seems to be optimal. The feeder layer should have healthy cell bodies spread out on the tissue culture plate. Before inactivation the MEF culture should be doubling every 24–30 h, requiring passaging at 1:3 every 3 days.

- MEF feeders should be used between passages 3 and 7. When the MEFs start to slow down in their proliferation or the cultures contain many multinucleate cells or floating debris, dispose of them and thaw a fresh vial.

- Some laboratories have a strong preference for MEFs derived from particular mouse strains. Others indicate that the strain is unimportant. As a rule, we suggest that the most important characteristic of any cells used for feeder layers is that they be rapidly growing, free of pathogens, and low passage when they are inactivated.

MEF cell plating density

The density of the feeder layer plays a role in the appearance of the hESC colonies, the rate at which the media components are depleted and the concentration of the feeder-derived culture components. However, there is as yet no predetermined optimal recommended density for the plating the MEF feeder layer. Each commercial hESC line has a slightly different recommended plating density for the MEF feeders:

- WiCell: 0.75 x 105/cm
- Bresagen: 2.4 x 105/cm
- mESCs: 2.5–3.0 x 105/cm dilute to 3 105/mL for plating. Table 3.1 gives an example of a plating guide that can be adapted as the researcher determines the optimal conditions for the cell lines being cultured.

Determining timing and dose for inactivation of feeder cells

The exact time of irradiation will depend on the irradiator and the cell type used. We have found that MEFs perform better when irradiated at 30 Gy and human fibroblasts 40 Gy. If the chamber of the irradiator is large enough to accommodate the culture dishes, the cells can be irradiated after they have attached to the dishes that will be used for passage of the hESC cultures.

To determine the effective dose to mitotically inactivate the cells, test at least three exposure periods on identical tubes of cells. After irradiation, dilute the cells so that you can plate 100 cells per 10 cm plate. Plate the cells on gelatin and observe the dishes for about a week. If any cells have failed to inactivate, you will see clones, and will have to use a longer exposure.

How to rid ESC cultures of contaminating feeder cells

Having hESC cultures contaminated with mitotically active feeders should be avoidable if care is taken to inactivate them thoroughly. But should valuable cultures appear to contain living feeder cells there is a straightforward solution; if the feeder cells are primary cells, they have a limited lifespan in culture and will eventually senesce. If the fibroblasts are an immortal line, they can be diluted by panning the more adhesive fibroblasts in dissociated cultures on a series of tissue culture dishes, or by serially passaging a small portion of the center of a hESC colony. If the feeders are a different species, “immunosurgery” (complement-mediated lysis) has been used successfully to remove them.

Human Feeder Cells,Feeder-free, and Defined Culture Systems

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Feeder cells support the growth of cells in culture by contributing an as yet undefined and complex mixture of extracellular matrix (ECM) components and growth factors. Feeder cells used for the co-culture of embryonic stem cells (ESCs) are usually fibroblasts and are usually (but not always) mitotically inactivated so that they remain viable but cannot replicate and overgrow the ESC culture.

Mouse embryonic fibroblasts (MEFs) are the most commonly used feeder cell type and have reliably served as feeder cells for co-culture with mouse embryonic stem cells (mESC) since they were first derived in 1981. Following the methods published in the early 1980s, most researchers used MEFs (or other mouse fibroblastic cells) to derive new lines of human and other primate ESCs, and they continue to be the most frequently used feeder cell for culturing hESCs. Even though they are primary cells, MEFs are inexpensive to prepare or purchase, and when properly cultured provide excellent support for growth of undifferentiated hESCs. See Chapter 3 for a description of methods for producing MEFs.

The search for improved methods and efforts to develop non-xenogenic culture systems has led to the use of various human-derived feeder cells, extracellular matrix components, and growth factors. The development of fully defined culture systems is an important milestone for the hESC field, which will greatly improve the usefulness of hESCs in both basic science programs and, over the longer term, their use in human therapeutic applications. The use of defined culture systems will eliminate much of the inherent variability in culture media, whose components are sourced from animals, and are likely to improve our ability to predictably and reliably direct differentiation. The acceptance of such systems is not solely dependent on identification of an ideal mix of factors and chemicals; if the cost of the perfect medium is too high, it will price most researchers out of the field. The movement now is therefore toward reasonable compromises that combine the best possible components at an affordable cost.

OVERVIEW

In this chapter we provide examples of alternatives to culture of hESC on MEF feeder layers. These methods are still the focus of a great deal of research and testing, and no ideal culture system has been developed to fulfill the needs of most researchers. The direction is toward animal product-free systems with all components defined, but a word of caution is warranted. The methods that have been most well tested are either not completely animal-free nor defined, are too expensive for most laboratories, and/or simply do not work for multiple hESC lines. We provide two practical approaches: feeder-free systems and human feeder cell-based systems.

The most well-documented feeder-free culture system uses MEFs to condition the hESC culture medium and a BD Matrigel substratum, which is derived from a mouse sarcoma cell line. While this feeder-free system eliminates the direct co-culture of hESCs with feeder cells, it includes animal products and relies on a commercial product that is not completely predictable.

An alternative to building a culture system from a combination of purified human factors and animal-derived products is to use human fibroblast feeder layers instead of MEFs. This solves the problem of the animal origin of unknown conditioning factors provided by MEF feeder layers. But simply using human feeder layers does not resolve the issue of completely animal-free culture, since the culture medium used generally contains animal products. Nor does it solve the problem of defining the composition of the medium since the human fibroblasts are still an undefined component.

PROCEDURES

Mouse embryo fibroblast (MEF)-conditioned medium/BD Matrigel culture system

This feeder-free hESC culture system consists of a MEF-conditioned hESC medium and a BD Matrigel substratum. This has been used for years of continuous culture of hESCs without loss of pluripotency markers or the development of karyotypically abnormal clones in the hESC cultures.

MEF-conditioned medium (MEF-CM)

MEF feeders are cultured and inactivated as described in Chapter 3. MEFs inactivated by gamma irradiation or mitomycin C work equally well.

1. Plate inactivated MEFs at 5 105 cells/cm2 in MEF medium and allow them to attach to the flask overnight in the incubator.

NOTE: MEF medium contains fetal bovine serum (FBS), and MEFs will not attach well to the tissue culture flask in hESC medium without FBS.

2. The next day replace MEF medium with hESC medium (0.5 mL/cm2) containing 4 ng/mL hFGF2. Allow MEFs to condition the hESC medium for 24 h.

3. Medium/flask guide:
- 38 mL of medium/75 cm2 flask
- 75 mL of medium/150 cm2 flask
- 112 mL of medium/225 cm2 flask.

4. Collect the conditioned hESC medium (MEF-CM) from feeder cell flasks daily, for up to 7 days.

NOTE: Harvest MEF-CM about the same time each day in an effort to minimize the variability of the medium.

5. Replace the medium in the flasks with hESC medium.

6. Filter MEF-CM through a 2 ?m low protein binding filter and aliquot in 10 mL, 25 mL, and 50 mL aliquots in labeled and dated sterile tubes. Store at 20°C. MEF-CM is stable for least six months at x20°C.

NOTE: Prior to using MEF-CM to feed hESC cultures, supplement with an additional 8 ng/mL of hFGF2.

BD Matrigel

BD Matrigel is a derived from the Engelbreth-Holm-Swarm mouse tumor cell line. It is very rich in extracellular matrix components and comprises approximately 60% laminin, 30% collagen IV, 8% entactin, heparan sulfate proteoglycan, and low levels of growth factors.
It is a liquid at 4°C, but polymerizes quickly room temperature.

Stock solution (1:2)

1. Prior to preparation of gel, thaw Matrigel slowly on ice at 4°C overnight. NOTE: Keep on ice until use. When preparing gel, use pre-cooled pipettes, plates, and tubes. BD Matrigel Matrix will gel rapidly at 22–35.

2. Keep on ice and add 10 mL of cold knockout D-MEM to the bottle containing 10 mL of Matrigel.

3. Keeping the mixture on ice, mix well by pipetting up and down with a 10mL pipette.

4. Aliquot 1–2 mL into pre-chilled tubes (on ice) and store aliquots at x20°C.

Preparation of culture plates

1. Slowly thaw the frozen aliquots at 4°C on ice.

2. Dilute BD Matrigel aliquots 1:15 in cold knockout D-MEM (1:30 final).

3. Add 1 mL of diluted BD Matrigel/well of a six-well plate.

4. Incubate the plates for at least 1 h at room temperature or overnight at 4°C.

NOTE: If stored at 4°C, coated plates can be used for up to one week after coating.

5. Allow the plate containing the Matrigel to sit at room temperature for at least 10 min before removing excess Matrigel solution, in order for the gel to polymerize.

Culture of hESCs – collagenase passaging

1. Observe cultures daily.

2. Change medium daily. Feed hESCs with 4 mL of MEF-CM supplemented with additional 8 ng/mL of hFGF2.

3. Passage when cells are confluent, removing differentiated cells.

NOTE: In the MEF-CM/Matrigel system, hESCs are maintained at high density and passaged at 1:3 to 1:6 every week.

4. Aspirate medium and add 1 mL of collagenase IV (1 mg/mL) per well of sixwell plate.

5. Incubate 5–10 min at 37°C. Monitor the culture during the incubation and stop collagenase treatment when the edges of the colonies begin to curl up and slightly loosen from the plate.

6. Aspirate the collagenase and rinse with 2 mL of PBS. Take care not to wash off loosened colonies.

7. Add 2 mL of MEF-CM to each well.

8. Gently scrape the well using a cell scraper or 10 mL pipette.

9. Collect most of the cells from the well and transfer to a 15 mL conical tube.

10. Gently pipette the cells to break up the clumps into groups of 50–100 cells, but do not make single-cell suspension.

11. Remove excess Matrigel from the new plates.

12. Seed the cells onto new plates at 1:3 to 1:6 dilutions. Place the plates in the incubator.

13. Observe the cultures and change medium daily.

NOTE: See Chapter 1 for other passaging methods.

Animal product-free culture conditions

Most hESC lines have been derived and maintained using medium containing bovine sera (FBS) or bovine-serum-derived products (such as Invitrogen’s KnockOut serum replacement, KSR) and co-culture with MEFs or mouse-derived Matrigel extracellular matrix. But concerns about problems with xenografts have motivated development of alternative culture systems that do not require the use of animal-derived products.

Several groups have begun to use human cells as feeder layers (Table 2.1), but to be completely “xeno-free” the culture medium must be composed of defined components, not animal derived; substrata must consist of either a chemically treated growth surface or synthetic or recombinant extracellular matrix components (Table 2.2). Human growth factors and ECM have been combined in culture “systems” (Table 2.3) designed to support hESCs in the absence of animal-derived products.

Transfer of existing hESC lines to new culture conditions should be done slowly, with gradual changes of conditions over multiple passages. For example, to transfer a hESC line from MEF feeder layers to human feeder layers, try using a combination of MEF-conditioned medium and human feeder layers at first; then gradually, over multiple passages, reduce the concentration of MEF-conditioned medium until the cells grow well without it.

PITFALLS AND ADVICE

Strains of mouse used to make MEF-CM

MEFs from several mouse strains have been found to support hESC culture. However, the strains currently favored are isolated from the CF-1 strain or 129 strains. Other strains that have been used to support hESC growth in co-culture are FVB/N, B6/129 hybrids, and C57BL/6. The most critical variable seems to be the quality of the MEFs, which should be used between passage 3 and passage 6 and before the culture consists of many large multi-nucleated fibroblasts. CF-1 MEFs require a higher dose of radiation to inactivate them (60–80 Gy) than 129 or B6 MEFs (30–40 Gy).

Adapting hESC cultures to growth on BD Matrigel in MEF-CM

Transferring cells from fibroblast co-culture to Matrigel/MEF-CM system may require a couple of passages to allow the cells to adapt to culture without feeders. Passage to low-density feeders on BD Matrigel and conditioned medium for the first couple of passages may ease the transition to feeder-free culture.

Growth factors

It is important to keep in mind that protein growth factors are easily degraded if not treated properly. Care should be taken when diluting and aliquoting all proteins. Keep them in a solution containing low concentrations of albumin – usually 0.1% BSA (diluted from fraction V, Sigma catalog no. A7979) in PBS. When pipetting or filtering low concentration solutions one may want to coat pipettes and filters with 0.1% BSA to lower the likelihood of the growth factor sticking to the pipettes and filters.

BD Matrigel

The source of this ECM mixture is the Engelbreth-Holm-Swarm mouse tumor. Its major components are laminin, collagen IV, heparan sulfate proteoglycans, and entactin. At room temperature, BD Matrigel matrix polymerizes to produce biologically active matrix material resembling the mammalian cellular basement membrane.

While quality control measures are used to minimize the variability between production lots, lot-to-lot variations are inherent in any cell-derived product. Culture results may vary depending on the production lot, the concentration that is plated, the length of time the plates are incubated with Matrigel, and how the plates are stored prior to use.

Matrigel comes as a solution measured in weight/volume. More consistent results may be achieved by calculating milligram per milliliter concentration rather than using a strict final dilution of 1:30 regardless of the concentration of the stock lot. An alternative to preparing Matrigel-coated plates in the laboratory is to purchase pre-coated plates, which have been coated with optimized concentration and prepared and tested for culture with hESCs (e.g. BD BioCoat BD Matrigel six-well plates for ES culture, BD Biosciences Catalog number 354671).

EQUIPMENT

- Tissue culture incubator: 37°C, 5% CO2 in humidified air
- Class II biosafety cabinet
- Microscope: Phase contrast with 4x, 10x, 20x objectives
- Centrifuge: low speed 300–1000 rpm
- Access to 4°C, x20°C, x80°C, and cryogenic freezers.

Human Embryonic Stem Cell Culture

2010-02-17T12:44:00.000-08:00

Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures will require daily attention. Once hESC cultures are established, they can, with skill and the methods described below, be kept in continuous culture for years.

A word of caution for those with experience culturing mouse embryonic stem cells: they are not the same! Both mouse and human ESCs are diploid, they are pluripotent, and they are relatively stable in culture. However, the stability of mouse ESC lines is regularly measured because the objective of almost all genetic manipulation is to make new lines of mice. If an ESC line can generate a mouse, as we term it, “go germline,” we know that it is clearly pluripotent. This has given us an operational definition for pluripotence and stability in culture for mouse ESCs.

hESC lines were originally derived using very similar culture medium and conditions as those developed for the derivation and culture of mouse ESC lines. However, these methods were suboptimal for hESCs, and have evolved considerably in the years since hESC lines were derived. Compared with mouse ESCs, hESCs are very difficult to culture – they grow slowly, and most importantly, since we have no equivalent assays for germline competence, we cannot assume that the cells that we have in our culture dishes are either stable or pluripotent. This makes it far more critical to assay the cells frequently, using characterization methods such as the karyotyping, immunocytochemistry, gene expression analysis, and fluorescence activated cell sorting (FACS) methods provided in this manual.

In this chapter we outline protocols for the culture of hESCs, starting as one would usually do, by being handed a culture by an experienced colleague. Other chapters focus on cryopreservation and establishing hESC cultures from frozen stocks, and on the variety of culture conditions, including the preparation of various types of feeder layers, conditioned medium, and extracellular matrix substrata.
The methods we recommend are those that are the most straightforward and have worked well in our hands; these are offered as the recommended methods and reagents. We also offer alternative methods and reagents that work but are not routinely used in most laboratories. The key variables that we outline in this chapter are:

- Culture medium
– Basal medium
– Serum or serum substitute

- Passaging cells
– Manual passage
– Non-enzymatic dissociation
– Enzymatic dissociation.

While optimizing and standardizing conditions in your lab, it is important to keep in mind that changing one thing in a system may have unexpected impact on the entire system.

PROCEDURES

Tips for successfully culturing hESCs

- Feed cells every day, except for 1 or 2 days following passage.

- Examine the cultures every day under 4 and 10 phase contrast. This will allow you to become familiar with the morphologies of undifferentiated and differentiated cells and colonies.

- When they are cultured on feeder layers some hESC lines tend to undergo spontaneous differentiation in the centers of the colonies. When passaging, take care to avoid passaging these differentiated “centers” to the new culture.

- Most hESC lines double every 31–35 h.

- Store medium at 4°C and discard any unused medium after 10 days. Best results are achieved when medium is prepared in small batches once a week.

The single most important skill in successful culturing of hESCs may be the ability to recognize the morphology of undifferentiated cells under a variety of conditions.

Phase contrast micrographs from the same culture, 4 days after it was passaged onto a feeder layer (human foreskin fibroblasts, ATCC HS27). (A) Typical colonies with smooth, phase-bright edges, with the fibroblast feeder layer forming whorls around the colonies (10x magnification). In contrast, in the same culture there are colonies with obvious differentiation at the edges (B – 4x magnification) and in the center. (C – 10 magnification). In selecting colonies for passage and expansion, only the ones shown in (A) would be acceptable. The others should not be passaged to the next culture dish.

For routine expansion of hESCs, we recommend that the cells be cultured at a relatively low density so that individual colonies can be easily monitored and selected against differentiation. hESCs can be cultured to high density (Figure 1.2), but a higher proportion of differentiated cells must be expected.

Passaging hESCs

hESCs, unlike mouse ESCs, do not survive well when dissociated to single cells. Therefore, the most reliable method for passaging undifferentiated hESC cultures is manual dissection of the colonies. This method may seem tedious, but it is virtually foolproof and we recommend that novices use this method until they have familiarity with the cells and can easily recognize differentiation in the culture. We also recommend manual passaging for producing cell banks of low-passage hESCs. Enzymatic dissociation methods are provided in Alternative Procedures.

NOTE: Using the number of passages as a measure of the age of an hESC line is an unfortunate historical accident. Because of the inconsistencies in hESC culture procedures in different labs, cells are passaged at different time intervals, ranging from 4 to 7 days. Therefore the number of passages for one line might be twice that of another, even though the cells have been in culture for exactly the same amount of time. For example, in a year of continuous culture, a cell line could be passaged as few as 52 and as many as 90 times. A better measure would be the number of doublings, but to count the number of cells in a culture is difficult since the cells form tight clusters and are not passaged as single cells.

General guidelines

- The cells should be passaged at about 1:3 every 5–7 days.

- Prepare the feeder layer or extracellular matrix (ECM) substrata the day before passaging.

- Depending on the cell line, passaging on Friday may be a good routine. The cells can usually be left undisturbed for 2 days following passaging, which allows them to settle down on the substrata, attach and begin dividing before the medium is changed.

- There will be considerable variation in the size of colonies in a single dish. Compared with their mouse counterparts, hESCs do not substantially pile up on each other, and their colonies can grow to a large diameter while remaining undifferentiated. Culture conditions affect the flatness of the colonies, but as an approximation, they are ready to split when the diameter fills the 10x field when observed under the microscope. As shown in Figure 1.3, a colony about half the diameter of the 10x field contains about 4400 cells. A colony filling the field would contain about 15 000 cells.

- For routine passaging by any method, do not make a single-cell suspension; dissociate the colonies into smaller colonies of a few hundred cells.

- Examine the culture daily for colony morphology under the phase contrast or dissecting microscope.

- With experience, one can get a good overview of colony morphology by holding the dish up to a light and looking at the bottom of the dish. The differentiated colonies will have ragged edges and hollow centers.

- On the bottom of the dish, mark colonies that are badly differentiated or parts of the colony that you do not wish to transfer to a new culture dish.

- To be certain that the colonies selected are undifferentiated, it is advisable to dissect the colonies while viewing the dish under a dissecting microscope with illumination from the base. But this is not absolutely necessary, and some prefer to passage the cells without magnification.

Mechanical dissociation

1. Evaluate the culture under 4x or 10x phase contrast optics.
2. The cells can be split among 3–6 dishes of the same size as the original culture, depending on the density of the original culture. If you wish to put the cells in different-sized dishes, calculate the amount of volume to add based on surface area of each type of dish.
3. Mark (or remove) overtly differentiated colonies so as not to disturb these during the dissociation process.
4. Remove the medium from the dish and replace with fresh hESC medium.
5. Dissect the colonies by hand, either under a low-power dissecting microscope (in a horizontal flow hood) or without a microscope, in the tissue culture hood.

NOTE: Several implements can be used to slice up or break up the colonies. Because they are inexpensive and sterile, we recommend either a 20 ?L pipettor which has a sterile filter tip attached, or a sterile 23G needle.
6. Figure 1.4 shows the method used for slicing the colonies into about 100 pieces. The colony is cut into strips, and then into squares. Each piece of the colony has a few hundred cells.
7. Break up each colony by moving the tip around and across each colony in a crosshatch or a spiral motion.

NOTE: Since the colonies are large at the time of passage, it is relatively easy to see individual colonies on the plate and, with practise, one can quickly dissociate an entire plate in less than 20 min.
8. After all of the colonies are dissected, use a 5 mL pipette to transfer the culture medium containing the dissected colonies to a 15 mL conical tube. Rinse the plate with hESC medium and add this to the same 15 mL tube.
9. Bring up the final volume in the tube to 8–10 mL with hESC medium.
10. Gently triturate the cell clumps using a sterile 10 mL pipette and divide the suspension into the prepared culture dishes on feeder layer or ECM-coated plates. Do not make a single-cell suspension; triturate gently, trying to achieve a relatively uniform suspension of cell clumps containing a few hundred cells each.

ALTERNATIVE PROCEDURES

Enzymatic dissociation

Enzymatic dissociation methods vary widely, and the exact conditions need to be developed for each laboratory. Most importantly, cultures that have been maintained by manual passaging cannot be passaged by enzymatic dissociation unless exceptional care is taken to adapt and monitor the cells.

The type of enzyme used for dissociation is critical. For example, passaging with trypsin appears to put more selective pressure on the cultures than other methods, resulting in a higher incidence of drift of hESC lines toward aneuploidy. But some hESC lines have been derived using trypsin from the outset; these lines can be rountinely passaged using whatever enzymatic technique is provided by the supplier.

Microbial collagenase is preferred by many laboratories, perhaps because of the way in which it is used. Collagenase is used to loosen the hESC colonies from the dishes, not to dissociate them to single cells, and the cell clumps have to be further dissociated by trituration.

NOTE: Keep in mind that enzymes are not highly purified recombinant products, and they may contain animal products. Trypsin is prepared from porcine (pig) tissue, and collagenase is a crude microbial product.

Collagenase dissociation

1. Remove the culture medium.
2. Rinse culture with Dulbecco’s PBS (D-PBS).
3. Treat the culture with 200 U/mL of collagenase IV for 5–10 min at 37°C until the edges of the colonies start to curl up – observe the culture under the microscope.
4. Remove the collagenase and replace with 2 mL of hESC medium (if using a sixwell or 35 mm dish).
5. Using a 5 mL pipette, gently dislodge the “good” colonies from the plate and place them in a 15 mL conical tube. Alternatively, one could remove the differentiated colonies prior to treating the culture dish with collagenase.
6. Gently triturate the cell clumps using a sterile 10 mL pipette and plate on feeder layer- or ECM-prepared dishes. Try to achieve a relatively uniform suspension of cell clumps containing several hundred cells each.
7. The cells can be split among 3–6 dishes of the same size as the original culture, depending on density of the original culture. If you wish to put the cells in different sized dishes, calculate the dilution based on surface area of each type of dish.

Non-enzymatic cell dissociation

Ca2x- and Mg2-free saline solutions containing EDTA or EGTA have not been as widely used for hESC dissociation as the methods described above, but they should offer advantages for assays that require intact cell surface proteins such as flow cytometry and immunocytochemistry. Commercial formulations are available, such as Cell Dissociation Buffer (Invitrogen catalog no. 13150016), which contains glycerol as well as a proprietary mixture of salts and chelators.

If you decide to try this method, remove all of the protein-containing medium and rinse the cells briefly with the dissociation buffer. Add enough buffer to cover the cells and monitor them under the microscope until the edges of the colonies begin to lift, then triturate the cells gently to dissociate. If the cells are to be recultured, don’t dissociate them into single cells, and be certain to check the karyotype of the cells after 10 passages; until you prove otherwise, you should assume that any untested passaging method is selecting for chromosomal abnormalities.

Accutase and Accumax (Millipore/Chemicon catalog no. SCR005 and SCR006)

These products are proprietary mixtures of proteolytic and collagenolytic enzymes in EDTA that the manufacturer states is free of mammalian- or bacterial-derived products. Accumax also contains DNAse. If you test this method, start with a 5-minute room temperature incubation and monitor the cells under the microscope. While the manufacturer indicates that inactivation of the enzymes with protein is not necessary, we recommend that protein-containing medium be used to dilute out the enzyme after the cells are dissociated, to prevent clumping and sticking of the cells to the pipettes.

Trypsin-like Enzyme (TrypLE Select, Invitrogen catalog no. 12563-029)

This is a single enzyme, a recombinant fungal serine protease with trypsin-like activity. Anecdotal reports suggest that hESC line that have been mechanically passaged can be successfully transitioned to single-cell enzymatic passaging using TrypLE Select. If you decide to try this method, we recommend a saline rinse, then a 5-minute incubation in the 1x enzyme solution as provided by the manufacturer. Monitor the cells under the microscope and add protein-containing medium to the culture before triturating.

HyQTase (HyClone catalog no. SV30030.01)

This is a cell detachment solution in D-PBS with EDTA. The composition is proprietary. According to the manufacturer, HyQTase is composed of a naturally derived complex of proteolytic and collagenolytic enzymes in D-PBS containing EDTA. According to the manufacturer it can be used for either serum-containing or serumfree cultures. The manufacturer states that it does not contain mammalian or bacterial derived products and is non-recombinant.

PITFALLS AND ADVICE

Monitoring drift in hESC cultures

Since hESC cultures are often kept in continuous culture for months, even years, it is very important to monitor for drift in the cultures. The best way to avoid drift is to generate a large bank of frozen cells as soon as possible after the cultures are first expanded. The importance of this cannot be overemphasized; the value of discoveries based on hESC cells depends on the reproducibility of results. See Chapter 26 for methods for setting up an hESC lab.

Genetic drift

We know that hESCs acquire chromosomal abnormalities over long periods of culture, so karyotyping or other genetic analysis methods must be performed on a regular basis. For detailed information about how to monitor genetic drifts, see Chapters 5–7 and 26. Keep in mind that changes during the time the cells are cultured in your lab can only be detected if you analyze the cells very soon after you obtain them.

Developmental drift

hESCs can also drift toward a more differentiated state over periods of extended culture. Since there is no assay for pluripotence equivalent to germline transmission of mouse ESCs, surrogate markers, such as antibody markers, should be routinely checked, especially if the morphology of the cells seems to be different from the earlier cultures. The gold standard for measuring the pluripotency of an hESC line is to transplant it to an immune-deficient mouse to form a teratoma tumor (Chapters 12 and 13). Keep in mind that it will require histological expertise to identify cell types and tissues in the tumors. In vitro, differentiation of hESCs using embryoid body culture will allow at least a cursory analysis of hESC differentiation potential. However, embryoid bodies never achieve the maturity of cells that develop in teratomas, and since the methods used to assess differentiation in vitro usually involve a small number of markers assayed by PCR (Chapter 10) or immunocytochemistry (Chapter 9), it is more difficult to judge the full range of pluripotence.

The best approach to monitoring developmental drift is to pick a particular method and differentiated cell type to check periodically (see Chapters 14 on embryoid body and neuroepithelial differentiation, as well as the specific chapters on neuronal, cardiac, and hematopoietic cells, Chapters 15–18).

Contamination of cultures

hESCs are usually cultured without antibiotics; with good culture technique, bacterial contamination should not be a problem. However, we recommend that antibiotics be used while new investigators are being trained in the techniques. Antibiotics such as penicillin and streptomycin do not have any effect on mycoplasma. Mycoplasma is a serious problem in laboratories that culture multiple cell lines or have inadequately trained personnel. Cultures must be monitored for mycoplasma on a regular basis, and contaminated cultures destroyed. Methods for mycoplasma detection are provided in the quality control section of this chapter, and in Chapter 26.

EQU I PMENT

- Tissue culture hood: Class II A/B3
- Tissue culture incubator, 37°C, 5% CO2, in humidified air
- Inverted phase contrast microscope with 4x, 10x, and 20x objectives
- Centrifuge, low speed 300–1000 rpm
- Water bath, 37°C
- Pipettors, such as Eppendorf p-2, p-20, p-200, p-1000
- Pipette aid, automatic pipettor for use in measuring and dispensing media
- Aspirator in the hood, with flask
- Refrigerator, 4°C
- Freezers: –20°C, –80°C, and –140°C.

Supplies

- 5 mL, 10 mL, 25 mL sterile disposable pipettes
- Six-well culture dishes
- 15 mL sterile conical tubes
- 50 mL sterile conical tubes
- Sterile 9x Pasteur pipettes
- Pipette tips for Eppendorf or similar pipettor.

KnockOut™ serum replacement (Invitrogen catalog no. 108280-028)

This product has a short shelf-life and should be aliquoted into 50 mL tubes and stored at –20°C. Thaw at 37°C just prior to use.

Additional information

KnockOut serum replacement (KSR) is a brand name for an Invitrogen product that is composed of BSA, transferrin, insulin, and other protein and non-protein components. The exact formulation is proprietary, but its composition was published (July 16, 1998) in an International Application Published under the Patent Cooperation Treaty (PCT), designated WO98/20679. See Epoline (ofi.epolin.org) to view the entire patent application.

L-Glutamine (200mM)

L-Glutamine (Invitrogen catalog no. 25030-081) is unstable and must be stored frozen at –20°C. Thaw the bottle completely just prior to use and aliquot in 10 mL tubes. Do not refreeze tubes, store at –4°C and discard unused glutamine after two weeks.

2-Mercaptoethanol

2-Mercaptoethanol (2-ME) has been used in ESC culture media since the first derivation of mouse ESCs in 1981. Originally included as a reducing agent because of concern about oxidation of culture components, it continues to be used in hESC media. Since the final concentration is 0.1 mM, and the pure solutions of 2-ME are 14.3 M, it is necessary to start with a stock solution.

Several companies sell diluted solutions of 2-ME; the 55 mM solution in PBS (Invitrogen catalog no. 21985-023) is a convenient concentration for a stock. If you wish to make your own stock, we suggest that you make a 1000x stock from the generally available concentrated solution (14.3 M).

For 1000x stock: dilute 35 ?L of 14.3 M 2-ME (Sigma catalog no. M7522) into 5 mL of PBS to make a 0.1 M stock solution. Filter before use.

QUALITY CONTROL METHODS

Lot-to-lot variability of reagents

It is important to keep in mind the actual source of the materials and reagents used in the culture and maintenance of hESCs. Since many are derived from animal sources, there is inherent lot-to-lot variability of the product. While vendors make every effort to control the variability by setting production specifications, these are usually ranges and as long as the product falls within the approved range, the product passes inspection and is distributed.

Ideally, you should have your own quality control methods to test new lots of products. At the very least, record the lot numbers of reagents used; if an experimental result cannot be replicated, or a cell line fails to thrive, you will save considerable time if the problem is traceable to a bad reagent.

Monitoring for mycoplasma contamination

Mycoplasma are the smallest forms of bacteria (0.2–0.3 ?m in diameter) and they can pass through the typical microbiology 0.2 ?m filter used in cell culture and do not produce the characteristic turbid growth shown by other bacteria. Because they lack cell walls, they are unaffected by the standard antibiotics used in culture media (penicillin and streptomycin that act on bacterial cell walls). Serious infections can be detected in cultures by DAPI or Hoescht staining for DNA; stained cell nuclei will be surrounded by fluorescing structures in the cytoplasm.

Mycoplasma infections drastically affect cell metabolism, gene expression and antigenicity, and can be devastating to a hESC laboratory. Infections are difficult to get rid of once they take hold, and some tissue culture collections recommend that contaminated cells be destroyed as soon as mycoplasma are detected.

Mycoplasma are highly infectious and cross-contamination commonly occurs when new cells are introduced into laboratories. The ATCC (American Type Culture Collection) estimates that 16% of cell cultures are contaminated by mycoplasma. The bacteria can also come from tissue culture reagents such as serum and media supplements and from laboratory staff.

The best defense against mycoplasma contamination is good aseptic technique, and the laboratory should not allow inexperienced or careless workers to share cell lines, solutions, or equipment. As a precaution, the cell lines should be tested at least four times a year. Testing for mycoplasma can be done by enzymatic, polymerase chain reaction (PCR), fluorescent staining, or culture methods (see list below).

STATEMENT OF POLICY ON STEM CELL RESEARCH BY PRESIDENT GEORGE W. BUSH, AUGUST 9, 2001

2010-02-16T12:48:00.000-08:00

Good evening. I appreciate you giving me a few minutes of your time tonight so I can discuss with you a complex and difficult issue, an issue that is one of the most profound of our time.
The issue of research involving stem cells derived from human embryos is increasingly the subject of a national debate and dinner table discussions. The issue is confronted every day in laboratories as scientists ponder the ethical ramifications of their work. It is agonized over by parents and many couples as they try to have children, or to save children already born.

The issue is debated within the church, with people of different faiths, even many of the same faith coming to different conclusions. Many people are finding that the more they know about stem cell research, the less certain they are about the right ethical and moral conclusions.
My administration must decide whether to allow federal funds, your tax dollars, to be used for scientific research on stem cells derived from human embryos. A large number of these embryos already exist. They are the product of a process called in vitro fertilization, which helps so many couples conceive children. When doctors match sperm and egg to create life outside the womb, they usually produce more embryos than are planted in the mother. Once a couple successfully has children, or if they are unsuccessful, the additional embryos remain frozen in laboratories.
Some will not survive during long storage; others are destroyed. A number have been donated to science and used to create privately funded stem cell lines. And a few have been implanted in an adoptive mother and born, and are today healthy children.
Based on preliminary work that has been privately funded, scientists believe further research using stem cells offers great promise that could help improve the lives of those who suffer from many terrible diseases—from juvenile diabetes to Alzheimer’s, from Parkinson’s to spinal cord injuries. And while scientists admit they are not yet certain, they believe stem cells derived from embryos have unique potential.
You should also know that stem cells can be derived from sources other than embryos—from adult cells, from umbilical cords that are discarded after babies are born, from human placenta. And many scientists feel research on these type of stem cells is also promising. Many patients suffering from a range of diseases are already being helped with treatments developed from adult stem cells.
However, most scientists, at least today, believe that research on embryonic stem cells offer [sic] the most promise because these cells have the potential to develop in all of the tissues in the body. Scientists further believe that rapid progress in this research will come only with federal funds. Federal dollars help attract the best and brightest scientists. They ensure new discoveries are widely shared at the largest number of research facilities and that the research is directed toward the greatest public good.
The United States has a long and proud record of leading the world toward advances in science and medicine that improve human life. And the United States has a long and proud record of upholding the highest standards of ethics as we expand the limits of science and knowledge. Research on embryonic stem cells raises profound ethical questions, because extracting the stem cell destroys the embryo, and thus destroys its potential for life. Like a snowflake, each of these embryos is unique, with the unique genetic potential of an individual human being.
As I thought through this issue, I kept returning to two fundamental questions: First, are these frozen embryos human life, and therefore, something precious to be protected? And second, if they’re going to be destroyed anyway, shouldn’t they be used for a greater good, for research that has the potential to save and improve other lives?
I’ve asked those questions and others of scientists, scholars, bioethicists, religious leaders, doctors, researchers, members of Congress, my Cabinet, and my friends. I have read heartfelt letters from many Americans. I have given this issue a great deal of thought, prayer and considerable reflection. And I have found widespread disagreement.
On the first issue, are these embryos human life—well, one researcher told me he believes this five-day-old cluster of cells is not an embryo, not yet an individual, but a pre-embryo. He argued that it has the potential for life, but it is not a life because it cannot develop on its own.
An ethicist dismissed that as a callous attempt at rationalization. Make no mistake, he told me, that cluster of cells is the same way you and I, and all the rest of us, started our lives. One goes with a heavy heart if we use these, he said, because we are dealing with the seeds of the next generation. And to the other crucial question, if these are going to be destroyed anyway, why not use them for good purpose—I also found different answers.
Many argue these embryos are byproducts of a process that helps create life, and we should allow couples to donate them to science so they can be used for good purpose instead of wasting their potential. Others will argue there’s no such thing as excess life, and the fact that a living being is going to die does not justify experimenting on it or exploiting it as a natural resource. At its core, this issue forces us to confront fundamental questions about the beginnings of life and the ends of science. It lies at a difficult moral intersection, juxtaposing the need to protect life in all its phases with the prospect of saving and improving life in all its stages.
As the discoveries of modern science create tremendous hope, they also lay vast ethical mine fields. As the genius of science extends the horizons of what we can do, we increasingly confront complex questions about what we should do. We have arrived at that brave new world that seemed so distant in 1932, when Aldous Huxley wrote about human beings created in test tubes in what he called a “hatchery.”
In recent weeks, we learned that scientists have created human embryos in test tubes solely to experiment on them. This is deeply troubling, and a warning sign that should prompt all of us to think through these issues very carefully.
Embryonic stem cell research is at the leading edge of a series of moral hazards. The initial stem cell researcher was at first reluctant to begin his research, fearing it might be used for human cloning. Scientists have already cloned a sheep. Researchers are telling us the next step could be to clone human beings to create individual designer stem cells, essentially to grow another you, to be available in case you need another heart or lung or liver. I strongly oppose human cloning, as do most Americans. We recoil at the idea of growing human beings for spare body parts, or creating life for our convenience. And while we must devote enormous energy to conquering disease, it is equally important that we pay attention to the moral concerns raised by the new frontier of human embryo stem cell research. Even the most noble ends do not justify any means.
My position on these issues is shaped by deeply held beliefs. I’m a strong supporter of science and technology, and believe they have the potential for incredible good—to improve lives, to save life, to conquer disease. Research offers hope that millions of our loved ones may be cured of a disease and rid of their suffering. I have friends whose children suffer from juvenile diabetes. Nancy Reagan has written me about President Reagan’s struggle with Alzheimer’s. My own family has confronted the tragedy of childhood leukemia. And, like all Americans, I have great hope for cures.
I also believe human life is a sacred gift from our Creator. I worry about a culture that devalues life, and believe as your President I have an important obligation to foster and encourage respect for life in America and throughout the world. And while we’re all hopeful about the potential of this research, no one can be certain that the science will live up to the hope it has generated.
Eight years ago, scientists believed fetal tissue research offered great hope for cures and treatments—yet, the progress to date has not lived up to its initial expectations. Embryonic stem cell research offers both great promise and great peril. So I have decided we must proceed with great care. As a result of private research, more than 60 genetically diverse stem cell lines already exist. They were created from embryos that have already been destroyed, and they have the ability to regenerate themselves indefinitely, creating ongoing opportunities for research. I have concluded that we should allow federal funds to be used for research on these existing stem cell lines, where the life and death decision has already been made.
Leading scientists tell me research on these 60 lines has great promise that could lead to breakthrough therapies and cures. This allows us to explore the promise and potential of stem cell research without crossing a fundamental moral line, by providing taxpayer funding that would sanction or encourage further destruction of human embryos that have at least the potential for life.
I also believe that great scientific progress can be made through aggressive federal funding of research on umbilical cord placenta, adult and animal stem cells which do not involve the same moral dilemma. This year, your government will spend $250 million on this important research. I will also name a President’s council to monitor stem cell research, to recommend appropriate guidelines and regulations, and to consider all of the medical and ethical ramifications of biomedical innovation. This council will consist of leading scientists, doctors, ethicists, lawyers, theologians and others, and will be chaired by Dr. Leon Kass, a leading biomedical ethicist from the University of Chicago.
This council will keep us apprised of new developments and give our nation a forum to continue to discuss and evaluate these important issues. As we go forward, I hope we will always be guided by both intellect and heart, by both our capabilities and our conscience.
I have made this decision with great care, and I pray it is the right one. Thank you for listening. Good night, and God bless America.

ROE V. WADE ON THE LEGAL STATUS OF THE EMBRYO 410 U.S. 113, 1973

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Note: The first case in which the U.S. Supreme Court dealt specifically with the legal status of the embryo was Roe v. Wade, a case in which the primary issue was a woman’s right to have an abortion. In deciding that case, the Court first had to determine whether the embryo or fetus that was to be aborted was a person or not. If it were, it had all the legal rights of any citizen and could not be deprived of its life. If it were not, it did not have those legal rights. The Court began by reviewing the status of the embryo and fetus in English common law. (References are omitted from this extract.)

It is undisputed that at common law, abortion performed before “quickening”— the first recognizable movement of the fetus in utero, appearing usually from the 16th to the 18th week of pregnancy—was not an indictable offense. The absence of a common-law crime for pre-quickening abortion appears to have developed from a confluence of earlier philosophical, theological, and civil and canon law concepts of when life begins. These disciplines variously approached the question in terms of the point at which the embryo or fetus became “formed” or recognizably human, or in terms of when a “person” came into being, that is, infused with a “soul” or “animated.” A loose consensus evolved in early English law that these events occurred at some point between conception and live birth. This was “mediate animation.” Although Christian theology and the canon law came to fix the point of animation at 40 days for a male and 80 days for a female, a view that persisted until the 19th century, there was otherwise little agreement about the precise time of formation or animation. There was agreement, however, that prior to this point the fetus was to be regarded as part of the mother, and its destruction, therefore, was not homicide. Due to continued uncertainty about the precise time when animation occurred, to the lack of any empirical basis for the 40–80-day view, and perhaps to Aquinas’ definition of movement as one of the two first principles of life, Bracton focused upon quickening as the critical point. The significance of quickening was echoed by later common-law scholars and found its way into the received common law in this country.

[The Court then explored references made to “personhood” in the U.S. Constitution.]

The Constitution does not define “person” in so many words. Section 1 of the Fourteenth Amendment contains three references to “person.” The first, in defining “citizens,” speaks of “persons born or naturalized in the United States.” The word also appears both in the Due Process Clause and in the Equal Protection Clause. “Person” is used in other places in the Constitution: in the listing of qualifications for Representatives and Senators, Art. I, 2, cl. 2, and 3, cl. 3; in the Apportionment Clause, Art. I, 2, cl. 3; in the Migration and Importation provision, Art. I, 9, cl. 1; in the Emolument Clause, Art. I, 9, cl. 8; in the Electors provisions, Art. II, 1, cl. 2, and the superseded cl. 3; in the provision outlining qualifications for the office of President, Art. II, 1, cl. 5; in the Extradition provisions, Art. IV, 2, cl. 2, and the superseded Fugitive Slave Clause 3; and in the Fifth, Twelfth, and Twentysecond Amendments, as well as in 2 and 3 of the Fourteenth Amendment. But in nearly all these instances, the use of the word is such that it has application only postnatally. None indicates, with any assurance, that it has any possible pre-natal application.

[Based on this review, the Court then outlined its position on the legal status of the embryo and fetus.]

All this, together with our observation, supra, that throughout the major portion of the 19th century prevailing legal abortion practices were far freer than they are today, persuades us that the word “person,” as used in the Fourteenth Amendment, does not include the unborn. This is in accord with the results reached in those few cases where the issue has been squarely presented.

[The Court listed seven such cases.]

Indeed, our decision in United States v. Vuitch, 402 U.S. 62 (1971), inferentially is to the same effect, for we there would not have indulged in statutory interpretation favorable to abortion in specified circumstances if the necessary consequence was the termination of life entitled to Fourteenth Amendment protection.

Stem Cell Research Terms - Definitions - Glossary

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abortion An event in which a pregnancy is terminated. Some abortions occur naturally, while others are conducted for health, personal, or other reasons.

adult stem cell An undifferentiated cell found in some specific types of tissue (such as muscle or nerve tissue) with the ability to renew itself and develop into the type of tissue cell in which it is found. Adult stem cells are also called somatic stem cells. Evidence suggests that some types of adult stem cells may be able to differentiate into tissue cells different from themselves, nerve cells from blood stem cells, and muscle cells from nerve blood cells, for example.

allogeneic transplantation The process by which cells, tissues, or organs from one individual are transplanted into a second individual from the same species.

astrocyte A large cell found in nerve tissue.

autologous transplantation The process by which cells, tissues, or organs from an individual are transplanted back into the same person.

blastema A mass of undifferentiated cells from which an organ or body part develops.

blastocoel A cavity in the blastula of the developing embryo.

blastocyst An early stage of an embryo prior to its implantation into the uterine wall, usually made up of about 150 cells consisting of an inner cell wall and inner cavity, and an outer layer of cells, the trophoblast.

blastomere A cell formed in the first stages of embryonic development, after a fertilized egg has undergone division but before a blastocyst has formed.

blastula A hollow ball of cells one cell thick that appears in the early development of an embryo.

bone marrow stromal cell A stem cell that occurs in bone marrow that may develop into a bone, cartilage, fat, or fibrous connective tissue cell.

cell culture The process of growing cells in an artificial medium for the purpose of scientific research.

cell-based therapy A medical procedure in which stem cells are transplanted into a body with the expectation that they will develop into some specific type of cell that will repair damaged cells or augment the number of cells in some specific tissue.

cell division The process by which a single cell divides to produce two new cells.

cell line A collection of cells kept alive in an artificial environment that continues to reproduce itself essentially forever until its fate is changed by some external factor, such as through an experiment.

chimera An organism whose cells are derived from at least two different organisms, such as a mouse and a human. The term comes from an animal in Greek mythology with the head of a lion, the body of a goat, and the tail of a serpent.

clone An organism that is genetically identical to some original cell from which it was originally derived.

co-culture A group of two or more different kinds of cells that have been grown together.

conceptus A term used to describe an organism in its earliest stages of life, that is, as a zygote, an embryo, or a fetus. The term is sometimes used in an attempt to keep discussions “value-free,” and avoid talking about the organism as an “unborn child,” a “baby,” a “human being,” or some other term with philosophical or religious context.

cord blood Blood found in the umbilical cord and placenta.

cryopreservation The process of preserving an organic material by lowering its temperature to a very low point. In most in vitro fertilization facilities, fertilized eggs are cryopreserved by being suspended in liquid nitrogen at a temperature of 196°C.

dedifferentiation A process by which a mature cell with specialized structures and functions reverts to a simpler, more primitive state, as when a unipotent adult somatic cell reverts to a simpler pluripotent or totipotent stemlike cell.

differentiation The process by which a primitive unspecialized cell develops into a specialized cell, such as a muscle, heart, or nerve cell.

diploid cell A cell that has two sets of chromosomes, one set from the father and one from the mother.

directed differentiation The process by which a researcher establishes conditions so as to encourage a stem cell to develop into some specific type of cell.

DNA An acronym for deoxyribonucleic acid, a chemical compound found in the nucleus of all cells that carries instructions for making the proteins of which all cells are made.

ectoderm The outermost layer of the three layers of cells present in an embryo. The ectoderm eventually gives rise to the cells that make up the skin and the nervous system.

embryo A very early stage in the development of an organism. In humans, the term is used to describe the structure that exists from the time of fertilization until the end of the eighth week of gestation.

embryoid body A clump of cells that develops when stem cells aggregate with each other during the process of cell culturing.

embryonal carcinoma cell (EC cell) A cell derived from a teratoma.

embryonic germ cell (EG cell) A cell that occurs in the gonadal ridge portion of an embryo or fetus. Its properties are similar to those of an embryonic stem cell.

embryonic stem cell (ES cell) A primitive undifferentiated cell that occurs in an embryo with the potential for developing into any one of many kinds of tissue cells, such as heart, muscle, liver, nerve, or brain cells.

embryonic stem cell line A group of embryonic stem cells that have been cultured under in vitro conditions and that have been maintained without differentiation for long periods of time, ranging from a few months to many years.

endoderm The innermost layer of the three layers of cells present in an embryo. The endoderm eventually gives rise to the cells that make up the digestive and respiratory systems.

ex utero fertilization Fertilization that takes place outside of the body. Similar to and, in most cases, identical with in vitro fertilization.

feeder layer A group of cells used in a co-culture to maintain pluripotent stem cells.

fertilization The process by which male and female cells are joined to each other.

fetus A term used to describe an unborn young organism. In humans, the term is used to describe the unborn child from about two months after conception to birth.

gene A unit of heredity that consists of a specific segment of DNA that directs the formation of a protein.

germ cell An egg or sperm cell. Germ cells originate in the inner cell mass of the embryo before migrating outward and beginning to differentiate.

gonad An organ that produces germ cells. For example, the testis produces sperm cells and the ovary produces oocytes, or egg cells.

haploid cell A cell containing only a single set of chromosomes, half the number normally found in a somatic cell. Haploid cells are usually germ cells.

hematopoietic cell transplantation (HCT) Transplantation of cells with blood-forming potential. Such cells are most commonly removed from human bone marrow, but they may also be obtained from umbilical cord blood, the fetal liver, and a few other sources.

hematopoietic stem cell A stem cell found in bone marrow from which all kinds of red and white blood cells eventually develop.

human embryonic stem cell A pluripotent stem cell found in the inner cell mass of the blastocyst of the human embryo.

implantation The process by which the blastocyst is embedded into the endometrium, the lining of the uterine wall.

in vitro A Latin phrase that literally means “in glass,” referring to some type of procedure carried out in a test tube, on a laboratory dish, or in some other artificial environment.

in vitro fertilization (IVF) An artificial method of reproduction in which male and female cells are joined to each other outside the human body.

in vivo A Latin phrase that means “in life,” referring to a procedure that occurs within a living organism, as within a laboratory animal or a human.

informed consent Permission granted by a person to participate in a research decision, based on that person’s understanding of the potential risks and benefits associated with his or her participation.

inner cell mass (ICM) A cluster of cells within the blastocyst that eventually gives rise to the embryonic disk of the embryo and, ultimately, to the fetus.

long-term self-renewal The process by which stem cells replicate themselves without differentiating (that is, they remain stem cells) over long periods of times, ranging from a few months to many years.

mesenchymal stem cell According to one definition, “a multipotent cell found in embryonic connective tissue and, much more rarely, in adult bone marrow and connective tissue; capable of differentiating into bone, cartilage, and fat cells” (Dirckx, at http://www.hpisum.com/perspectives/ issue50/update.pdf). However, one authority in the field of stem cell research has reviewed the literature and concluded that “there is no accepted definition of a mesenchymal stem cell, not even an operational one”.

mesoderm The middle layer of the three layers of cells that make up the embryo. The mesoderm eventually gives rise to cells that make up connective tissue, muscles, bones, blood, the genital system, and some glands.

morula A ball of cells with the appearance of a mulberry (hence, the name) that forms three to four days after fertilization. The morula consists of 16, 32, or 64 cells.

multipotency The ability of a stem cell to differentiate into more than one type of tissue cell, although all of the cells into which it differentiates are of the same tissue type. For example, a blood stem cell may differentiate into any one of a variety of white blood cells or red blood cells, but does not typically develop into a nerve, muscle, skin, or nonblood type of cell.

murine Pertaining to mice or rats.

neural stem cell A type of stem cell found in human nerve (neural) tissue that develops into various kinds of nerve cells.

neuron A nerve cell.

niche A matrix of tissue cells and molecules in which one or more stem cells is embedded and which controls self-renewal and prevents the differentiation of those stem cells.

oligopotent progenitor cell A progenitor cell with the capability of differentiating into more than one, but only a limited number, of different kinds of cells.

oocyte (oocyte) A female gamete, or egg cell.

parthenogenesis The development of an unfertilized egg into a mature individual. The process occurs naturally in some animals and has been produced artificially in other animals. The process has been proposed as a method for producing embryonic stem cells without the intermediary step of fertilizing an egg.

plasticity The ability of a stem cell to grow and differentiate into a different kind of cell.

pluripotency The ability of a stem cell to develop into many different kinds of cells. The term often refers to the ability of a stem cell to differentiate into all kinds of cells found in the postimplantation embryo, fetus, or developed organism, but not in the trophoblast or placenta (so-called extra-embryonic entities).

preembryo A term sometimes used to describe the earliest stages of life, ranging from the fertilized egg to any larger entity in which cells have not yet begun to differentiate. The term is the subject of a great deal of dispute, with many experts in the field of embryology suggesting that it has no scientific meaning and is used only for political purposes.

preimplantation genetic diagnosis and screening (PGD) A set of procedures in which embryos that have been created by in vitro fertilization are tested for certain genetic traits so as to determine which of those embryos is to be implanted.

primitive streak A band of cells that develops about 14 days after fertilization along the longitudinal axis of the body that later becomes the fetal spinal cord.

progenitor cell A cell present in fetal or adult tissue that, like a stem cell, can differentiate into another kind of specialized cell. Unlike a stem cell, however, it is unable to continually renew itself by repeated cell division.

proliferation The multiplication of a single cell or small group of cells into a large population of identical cells through the process of cell division.

regeneration In medicine, the process by which an organism regrows tissue, organs, or some other body part.

regenerative medicine A field of medicine in which stem cells are introduced into a person’s body and induced to differentiate into some specific type of cell tissue in order to repair or replace damaged tissue.

reproductive cloning A process by which a complete new organism is created by somatic cell nuclear transfer (SCNT) beginning with a single body cell of another organism, to which the new organism is genetically identical; cloning of an embryo for transplantation into a uterus in order to produce a mature organism that is genetically identical to the nuclear donor.

somatic cell Any cell that is not a germ cell, that is, a sperm or egg cell.

somatic cell nuclear transfer (SCNT) The process by which the diploid nucleus of a somatic cell is transplanted into an unfertilized oocyte from which the nucleus has been removed. When this chimeric cell begins to divide, it produces totipotent stem cells that are genetically identical to the donor of the diploid nucleus.

stem cell A cell that is capable of dividing over some indefinite period of time and differentiating to produce one or more kinds of specialized cells.

“stemness” A term that is often applied to a stage in a cell’s life during which it has the properties of a stem cell (ability to divide over many generations and to eventually differentiate into a specialized cell) that can be characterized by certain biological and chemical characteristics of the cell. teratogeny The experimental study of teratomas.

teratoma A tumor that contains tissues from all three embryonic germ layers— endoderm, ectoderm, and mesoderm—most commonly found in the gonads (ovaries and testes).

therapeutic cloning The use of somatic cell nuclear transfer (SCNT) to produce an embryo that is allowed to develop to the blastocyst stage, at which point it is sacrificed for the purpose of harvesting embryonic stem cells contained within it.

totipotency The capacity of a stem cell to differentiate into any one of the 210 different types of cells normally found in the human body, or into all types of the cells found in some other organism.

transdifferentiation The process by which an adult stem cell from one kind of tissue differentiates into a cell of a different type of tissue.

trophoblast The outer layer of cells in the blastocyst that attaches to the inner wall of the uterus during implantation of the embryo, eventually becoming the placenta through which food passes to the embryo and, later, the fetus.

unipotency The ability of a stem cell to differentiate into a single type of mature somatic cell.

zygote Technically, a diploid cell formed by the fusion of two haploid cells during sexual reproduction. More commonly, a fertilized egg that has been formed by the fusion of a sperm cell and an egg cell.

BIOGRAPHICAL LISTING - STEM CELL RESEARCH

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Joseph Altman, currently professor emeritus at Purdue University, in Lafayette, Indiana. Altman carried out seminal studies on the production of new brain cells in the 1960s. Prior to Altman’s work, nearly all scientists believed that a person was born with all of the brain cells it would ever have. Altman demonstrated that he observed new neurons (brain cells) being created in the hippocampus portion of the brain and then migrating to other parts of the brain. Altman’s studies were largely ignored for at least a decade by many scientists, and for even longer by the majority of the scientific community.

Elizabeth Blackburn, Morris Herzstein professor of biology and physiology in the department of biochemistry and biophysics at the University of California–San Francisco. She is a widely respected authority in the field of stem cell research. Her field of expertise is telomeres, regions located at the ends of chromosomes that control the process of replication. She discovered an enzyme known as telomerase that controls this process. Blackburn served on the President’s Bioethics Advisory Commission from 2002 to 2004 before being asked to resign by President George W. Bush. The official reason offered for this action was that Blackburn had been unable to attend meetings of the commission. Blackburn and others, however, said that their views on embryonic stem cell research were regarded as too liberal for the administration. She has been critical of the operation and activities of the commission.

Rod Blagojevich, governor of Illinois. Blagojevich signed an executive order on July 12, 2005, committing $10 million in state funds over the coming year for research on stem cells. Blagojevish pointed out that the decision by the federal government not to fund embryonic stem cell research made it imperative for individual states to take over part of that funding responsibility. Prior to his election as governor, Blagojevich served as member of the state assembly from 1992 to 1996 and in the U.S. House of Representatives from 1996 to 2002.

Ariff Bongso, research professor in the department of obstetrics and gynaecology at the faculty of medicine of the National University of Singapore. He was the first person to isolate human embryonic stem cells from a five-day-old human embryo in 1994. Bongso was trained as a veterinarian in his native Sri Lanka but later became interested in the young science of in vitro fertilization (IVF), where he made a number of research breakthroughs while working in Singapore, to which he had moved in the early 1980s. He was a member of the IVF team that produced the first successful “test-tube baby” born in Asia in 1983. In addition to his isolation of the first human embryonic stem cells, Bongso has developed methods for maintaining human embryonic stem cells in a regenerating state outside the human body for essentially limitless periods of time, allowing the establishment of new stem cell lines.

Robert Briggs, former head of the embryology department at the Institute for Cancer Research and professor of zoology at Indiana University. With colleague Thomas J. King, he carried out some of the earliest research on somatic cell nuclear transfer, in which the nucleus of a cell from one organism is transplanted into an egg from which the nucleus has been removed of a second organism.

Ralph Brinster, currently Richard King Mellon Professor of Reproductive Physiology at the University of Pennsylvania’s college of veterinary medicine. He was responsible for some of the earliest research on the fate of nonembryonic stem cells transplanted into mouse blastocysts. He transplanted cells taken from teratocarcinomas and the bone marrow of mice into the blastocysts of host mice and found that those blastocysts grew into normal adult mice with the characteristics of both the host mouse blastocysts and the mice from which the transplanted cells had been taken.

Sam Brownback, Republican senator from Kansas. He has been one of the strongest opponents of stem cell research that results in the destruction of embryos or other early forms of life in the U.S. Congress. He has introduced bills to prevent cloning for either reproductive or therapeutic purposes in every session of Congress in recent years. Two such bills were S. 245 and S. 658, the Human Cloning Prohibition acts of 2003 and 2005, respectively.

George W. Bush, 43rd president of the United States. Bush announced his administration’s policy on stem cell research in an address to the nation on August 9, 2001. That policy allowed the use of certain existing stem cell lines for research on human embryonic stem cells, but prohibited federal funding of research in which new embryos would be created solely for the purpose of experimentation. Since his 2001 address, the president has maintained his opposition to any type of stem cell research in which embryos are created and then destroyed for any purpose whatsoever.

Lisa Sowle Cahill, J. Donald Monan professor of theology at Boston College. She has written extensively on ethical issues related to stem cell research, cloning, abortion, and other bioethical issues. Some of her most important work has involved the commercialization of new biotechnological procedures, such as those concerned with the production and use of human embryos for stem cell research.

Daniel Callahan, cofounder and director of the International Program at the Hastings Center, a bioethics research institution in upstate New York. He is a highly respected observer of and commentator on the development of stem cell research. He has been opposed to embryonic stem cell research because, as he has explained, he has “always felt a nagging uneasiness at trying to rationalize killing something for which I claim to have profound respect [a human embryo].” He also spoke out in opposition to California’s Proposition 71, establishing the California Institute of Regenerative Medicine to be funded at a level of $3 billion over a 10-year period. “Whether anything comes of this research,” he said, “it is sure to line the pockets of many scientists and biotechnology companies in the process.”

Mike Castle, Republican representative from the state of Delaware. Castle was cosponsor of House bill H.R. 810 in the 109th Congress. The bill authorized the Secretary of Health and Human Services to “conduct and support research that utilizes human embryonic stem cells.” The bill passed the House, was never acted on by the Senate. Castle was previously deputy attorney general, state legislator, lieutenant governor, and, for two terms, governor of Delaware. He was elected to the House of Representatives in 1993.

William Jefferson Clinton, 42nd president of the United States. Clinton was confronted with growing interest in and conflict about the use of embryonic stem cells in research throughout his presidency. He inherited a policy that banned the use of all federal funds for such research from his predecessor, President George H. W. Bush, but gradually liberalized that policy in order to permit the use of at least certain types of embryonic stem cells under certain conditions. By the time he left office in 2001, Clinton had set in motion a plan to permit the funding of such research with tax dollars, a policy that was reversed shortly after his successor, George W. Bush, took office in January 2001.

Diana DeGette, Democratic representative from the state of Colorado. She was cosponsor of House bill H.R. 810 in the 109th Congress, a bill authorizing the Secretary of Health and Human Services to conduct and support research using human embryonic stem cells. She served two terms in the Colorado legislature before being elected to the U.S. House of Representatives in 1997.

Jay Dickey, formerly a congressman from the 4th congressional district of Arkansas. He was author of an amendment to the annual appropriations bill for the Department of Health and Human Services in 1996, an amendment that prohibits the allocation or expenditure of any federal funds for research that involves the destruction of a human embryo or embryos. That amendment has generally become known as the Dickey amendment and has been reintroduced and adopted every year since it was first passed in 1996. Dickey was first elected to Congress in 1992, where he served for four terms until his defeat in 2000.

Richard M. Doerflinger, deputy director of the Secretariat for Pro-Life Activities of the United States Conference of Catholic Bishops. He is a prominent spokesperson in opposition to the use of embryos and materials obtained from embryos in research of any kind whatsoever. He is also adjunct fellow in bioethics and public policy at the National Catholic Bioethics Center in Boston. Doerflinger has written extensively for the Hastings Center Report, the Kennedy Institute of Ethics Journal, the Encyclopedia of Catholic Doctrine, the Dusquene Law Review, and a number of Catholic magazines and journals. He has also testified before the U.S. Congress, the National Bioethics Advisory Commission, and the National Institutes of Health on issues related to the use of human embryos in research.

Hans Driesch, German biologist and philosopher, carried out a series of classic experiments in the early 1890s in which he divided two- and fourcell sea urchin embryos and found that each of the individual cells produced was able to develop into a complete, normal adult sea urchin. In order to explain his results, Driesch later developed a theory of “entelechy” that attributed an organism’s growth and development to some sort of supernatural “unifying non-material mind-like something.” The effect observed by Driesch was later found to result from the proliferation and differentiation of embryonic stem cells.

Robert Edwards, professor emeritus at the University of Cambridge, England. With colleague Barry Bavister, he performed the first successful ex utero fertilization of a human egg in 1968. The experiment was one of the seminal steps in developing the procedure of in vitro fertilization, which has since been responsible for the birth of tens of thousands of children to otherwise infertile couples.

Sir Martin Evans, professor of mammalian genetics and director of the school of biosciences at Cardiff University, in Wales. Along with colleague Matthew Kaufman, he was the first person to successfully isolate embryonic stem cells from a mouse. He and Kaufman then developed a process by which those stem cells could be kept alive in a proliferative, nondifferentiating state for many generations, producing the first embryonic stem cell lines available for research. Because of this research, Evans has sometimes been called “the chief architect of stem cell research.” In addition to his work on stem cells, Evans has long been interested in the development of modern gene therapy, in which altered genes are introduced into organisms suffering from some sort of genetic disorder. During the 1990s, his research team performed the first successful experiments in curing a mouse with cystic fibrosis by means of gene therapy.

John D. Gearhart, C. Michael Armstrong professor of medicine and professor of gynecology and obstetrics, physiology, and comparative medicine at the Johns Hopkins School of Medicine and professor of biochemistry and molecular biology at the Johns Hopkins School of Public Health and Hygiene. Gearhart published an historic paper in stem cell research in 1998 when he reported on the derivation of human embryonic stem cells from primordial germ cells. Gearhart also serves as director of the Division of Development Genetics, director of Research for Gynecology and Obstetrics, and director of Preimplantation Genetic Diagnosis at the Johns Hopkins School of Medicine. In addition to his work on stem cells, Gearhart has published papers in the fields of genetics, development, and genetic counseling.

Howard Green, currently George Higginson Professor of Cell Biology at Harvard University. He invented a method for growing cells in vitro on irradiated mouse fibroblast cells. Green’s murine fibroblast mat was later to become the standard feeder layer on which stem cells are maintained in a proliferative, nondifferentiating state over periods of many months or years. In later research, Green found ways to embed epidermal stem cells in fibroblast mats for use as synthetic skin for patients who had been severely burned, earning him the accolade from some as Father of Skin Culture.

Jim Greenwood, president of the Biotechnology Industry Organization. He represented the 8th congressional district of Pennsylvania from 1993 to 2004. During his tenure in office, Greenwood was a strong supporter of embryonic stem cell research and introduced a number of bills authorizing the funding of such research with federal tax monies. As an example, he cosponsored (with representative Peter Deutsch) H.R. 2608 in the 107th Congress, a bill that would have permitted the production and use of human embryos for research and therapeutic purposes, but not for human reproduction. Greenwood’s bills consistently lost out to more restrictive legislation banning all forms of embryonic research.

John B. Gurdon, research scientist and group leader in the Institute of Cancer and Developmental Biology at Cambridge University. He was the first person to successfully clone an animal. During the 1960s, Gurdon carried out a number of experiments that destroyed the nucleus of the eggs of frogs, and transplanted into those eggs the nuclei from tadpoles. Some small number of those eggs eventually developed into new tadpoles, exact copies (clones) of the organisms from which the nuclei had originally been taken.

Gottlieb Haberlandt, Austrian botanist. A pioneer in modern studies of plant tissue culture, he hypothesized in 1902 that it should be possible to grow a complete mature plant beginning with no more than a single cell taken from that plant. He said that this experiment should be possible because each cell in a plant possesses a “totipotency” that allows it to develop into any one of the types of cells of which the mature plant consists and into which it can grow.

John Hearn, Australian reproductive and developmental biologist. He was formerly director of the Wisconsin Regional Primate Research Center, located at the University of Wisconsin–Madison, where he was responsible for recruiting James Thomson, the first person to isolate human embryonic stem cells in 1998, and to provide him and other researchers with unusually fine laboratory conditions in which to conduct their studies.

Konrad Hochedlinger, a postdoctoral researcher at the Whitehead Institute for Biomedical Research in Boston. He demonstrated in 2002 that a mouse can be cloned from mature, highly differentiated cells taken from an adult animal. Later in the same year, Hochedlinger and his advisorcollaborator Rudolf Jaenisch used embryonic stem cells to cure a mouse of an immune disorder, the first time in which a therapeutic application of stem cell research had been conclusively illustrated.

Robert Hooke, English physicist. He was the first person to explicitly recognize the existence of tiny pocketlike units within living organisms. He gave these units the name cell after the Latin word cella meaning “small room.”

Rudolf Jaenisch, one of the founding members of the Whitehead Institute for Biomedical Research, in Boston. He was joint author with one of his postdoctoral students, Konrad Hochedlinger, of an important paper in 2002 describing the cloning of mice using adult stem cells, the first time such a procedure had been conclusively shown to work. Jaenisch and Hochedlinger also authored a 2002 paper that described the use of stem cells to cure mice of an immune disorder, the first occasion on which the therapeutic value of embryonic stem cell transplantation had been conclusively demonstrated.

Leon R. Kass, chair of the President’s Council on Bioethics, on leave from his positions as Hertog Fellow in Social Thought at the American Enterprise Institute and Addie Clark Harding Professor in Social Thought at the University of Chicago. He earned his Ph.D. in biochemistry at Harvard University in 1967 and was a researcher in that field briefly before turning his attention to ethical and philosophical issues raised by advances in medical research and the biosciences. Since 1970, he has taught and written extensively on a variety of issues in the field of bioethics.

Thomas J. King, professor of embryology at Georgetown University, division director at the National Cancer Institute, director of the Kennedy Institute of Ethics, and deputy director of the Lombardi Cancer Research Center at Georgetown. King collaborated with Robert Briggs in the early 1950s to develop the procedure now known as somatic cell nuclear transfer (SCNT), in which the nuclei are removed from cells of one organism and transplanted into cells that have had their nuclei removed of a second organism. When done successfully, the host cell grows and develops normally, as Briggs and King discovered in their classic studies of the leopard frog (Rana pipiens).

Karen Lebacqz, professor emerita of theological ethics at the Pacific School of Religion, Berkeley, California. Lebacqz is coeditor, with Suzanne Holland and Laurie Zoloth, of The Human Embryonic Stem Cell Debate, a book of readings on the scientific, legal, and ethical issues related to stem cell research. She has also written, spoken, and taught extensively on other areas of bioethics, including bioethics of the Human Genome Project and ethical theory.

Gail Martin, professor of anatomy at the University of California at San Francisco. She is codiscoverer with Martin Evans and Matthew Kaufman of murine (mouse) embryonic stem cells in 1981. She is often credited with having invented the terminology now in common use for these cells. Her current research focuses on the roles played by specific molecules in the early differentiation of murine embryonic stem cells.

Alexander A. Maximow, a Russian military officer who first proposed the notion that all kinds of blood cells—white blood cells, red blood cells, and platelets—are all produced within bone marrow from a single precursor cell, which he called a Stammzelle. Some scholars believe that the modern term stem cell can be traced to Maximow’s use of the similar term Stammzelle. Maximow’s ideas were largely rejected or ignored throughout his lifetime and received experimental justification only with the work of Ernest McCulloch and James Edgar Till in the 1960s.

Richard McCormick, a moral theologian who taught at Georgetown University. He wrote extensively about the moral status of the human embryo, with special attention to its potential use in human embryonic stem cell research. After considerable research and deliberation, McCormick came to the conclusion, contrary to church doctrine, that the very early embryo was not truly a human and that, therefore, an argument could be made for its use in experimentation, provided the end results of such experimentation justified that use.

Ernest McCulloch, Canadian medical researcher. He carried out pioneering work on hematopoietic stem cells in the 1960s with James Edgar Till and Andrew Becker that provided experimental evidence for the existence of such cells, produced methods for the culturing of stem cells, and found methods for counting the number of hematopoietic cells in bone marrow and other body organs.

Douglas Melton, Thomas Dudley Cabot professor of the natural sciences at Harvard University and codirector of the Harvard Stem Cell Institute. His research focuses on the development of the pancreas, in general, and, more specifically, on the role played by stem cells in that process. One product of his research has been the development of 17 new stem cell lines, produced with private funding. Melton has testified before the U.S. Congress in support of federal funding for stem cell research and is active in a number of organizations promoting the conduct and funding of stem cell research.

Eva Mezey, Hungarian-born medical researcher, current head of the Adult Stem Cell Research Section at the National Institute of Dental and Craniofacial Research of the U.S. National Institutes of Health. In the late 1990s, Mezey discovered that some of the hematopoietic cells transplanted into mice migrated to the brain and transdifferentiated into neurons. Her discoveries were, at first, largely disbelieved, although many similar cases of transdifferentiation have since been discovered.

Beatrice Mintz, senior member of the basic science division of Fox Chase Cancer Center in Philadelphia. She carried out an elegant series of experiments in the early 1970s in which stem cells taken from a teratoma were transplanted into a normal blastocyst. The cells were incorporated into the blastocyst and became part of the normal embryo into which it grew. Mintz is perhaps best known for her work resulting in the production of the first transgenic mammals, organisms produced when the genes from one mouse have been transplanted into the body of a second mouse, resulting in an animal with two different genetic maps.

Thomas Okarma, current president and chief executive officer of Geron Corporation. After serving as a faculty member at the Stanford University school of medicine, Okarma joined the corporate world, where he was a founder, vice president for research, and president and chief executive officer of Applied Immune Sciences, Inc., and senior vice president at Rhone-Poulenc Rorer before joining Geron in 1997. At Geron, Okarma was vice president of cell therapies and vice president of research and development before being appointed to his present posts at the company. Geron funded Dr. James Thomson’s research on stem cells and has been awarded patents for nine stem cell lines developed at the University of Wisconsin–Madison.

Deborah Ortiz, state senator for California’s 6th state senate district. She has been an outspoken advocate for all forms of stem cell research and was the author of senate bill 253 in the 2002 legislative session, affirming the state’s support of human embryonic stem cell research. She was also active in the campaign for Proposition 71 in the November 2004 election, an initiative that created the California Institute for Regenerative Medicine, to be funded with $3 billion of taxpayer monies over a 10- year period.

Gordon Barry Pierce, former professor of pathology at the University of Colorado Health Sciences Center in Denver. He was codiscoverer with medical student Lewis Kleinsmith that teratomas are caused by the proliferation of individual stem cells known as embryonal cancer (EC) cells. Born in Canada, Pierce spent virtually all of his professional career in the United States. He is best known for his studies of the character of teratomas and the role of EC cells in their development.

Harriet Rabb, current vice president and general counsel to the Rockefeller University. She wrote a critical memorandum in 1995 on the legality of federal funding for human embryonic stem cell research while she was general counsel for the U.S. Department of Health and Human Services. Rabb ruled that, while federal money could not be used for the production of human embryos for research, it could be used for research on such embryos produced with private funds.

Nancy Reagan, wife and widow of former president Ronald Reagan. She became an outspoken advocate of all forms of stem cell research during the later stages of her husband’s battle with Alzheimer’s disease. At one point, she announced that “we have already waited too long” to find out how stem cells might be useful in treating diseases like the one from which her husband was suffering. During the 2005 congressional debate over federal funding of embryonic stem cell research, she encouraged both President George W. Bush and members of congress to act to approve federal funding for such research.

Ron Reagan, Jr., son of former president Ronald Reagan. He is a strong advocate for stem cell research, arguing that it has the potential for curing a broad range of debilitating diseases currently untreatable by other methods. He spoke at the Democratic National Convention in Boston in 2004, arguing that, although some people opposed embryonic stem cell research because they believed that embryos are live human beings, “[i]t does not follow that the theology of a few should be allowed to forestall the health and well-being of the many.”

Christopher Reeve, an actor with credits in stage, screen, and television productions. Reeve will perhaps always be best known for his portrayal of Superman in the film of that name and its sequels. In 1995, he was thrown by the horse he was riding in an equestrian competition and paralyzed as a result of an injury to his spine. During the next decade, he became very active in efforts to promote research for the treatment and cure of spinal cord injuries, including stem cell research. He died on October 10, 2004, although his work in the support of medical research is being carried on by the Christopher Reeve Paralysis Foundation.

Matthias Jakob Schleiden, 19th-century German botanist. He was the first to hypothesize that cells are the structural units of which all plants are made. His suggestion, along with a similar theory for the structure of animals by Theodor Schwann, constitute the basis of modern cell theory. At one point, Schwann makes the prescient observation that “any given cell may be separated from the plant, and then grown alone.”

Theodor Schwann, 19th-century German physiologist. He theorized that all living organisms are made of cells and that these cells grew out of preexisting cells. Schwann also discovered the enzyme pepsin, hypothesized that fermentation was a biological process, and identified yeast cells as plantlike organisms.

Patrick Steptoe, British obstetrician and gynecologist. With colleague Robert Edwards, he performed the first successful in vitro fertilization of a human egg that resulted in the birth of a normal child, so-called “test tube baby” Louise Brown, in 1978.

Leroy Stevens, long-time researcher at the Jackson Laboratory, in Bar Harbor, Maine. He has been called “the unsung hero of stem cell research” because of his early discovery of pluripotent cells in mice. Stevens’s research on pluripotent cells, a term he invented, began shortly after he joined the Jackson Laboratory in 1952 when he discovered the presence of teratomas, tumors consisting of many different kinds of cells, in the scrotums of a particular line of experimental mice. He spent the rest of his life studying these cells, retiring from the Jackson Laboratory in 1989.

Frederick Campion Steward (“Camp” Steward), former director of the laboratory of cell physiology, growth, and development at Cornell University. He became famous for a series of experiments he and his colleagues conducted in the 1950s during which they were able to regenerate a complete carrot plant beginning with a single carrot cell cultured in coconut milk. The experiment demonstrated the possibility of dedifferentiating a mature cell into a more primitive form that was then able to reproduce and form all of the mature cells needed to form a complete plant.

Tommy Thompson, 19th secretary of Health and Human Services. He was previously a member of the Wisconsin state assembly and governor of Wisconsin for four terms, from 1987 to 2001. He served in the George W. Bush administration from 2001 until his resignation in 2005. Prior to his appointment as secretary of Health and Human Services and during his first six months in office, he was an ardent supporter of stem cell research and encouraged Congress to support funding of SCR. After President Bush’s speech on August 9, 2001, outlining his opposition to most forms of stem cell research, however, Thompson changed his views and mounted a vigorous support of the president’s program.

James Thomson, professor of anatomy at the University of Wisconsin Medical School–Madison. He was leader of a research team that announced the first successful culturing of human embryonic stem cells in a paper published in the journal Science in 1998. Thomson received his doctoral degree in veterinary medicine from the University of Pennsylvania in 1985 and his Ph.D. in molecular biology from Penn in 1988. His current research studies factors that promote the self-renewal of stem cells, the maintenance of pluripotency, and the pathways leading to the differentiation to specific cell types.

James Edgar Till, Toronto-born specialist in radiation biology. With Ernest McCulloch and Andrew Becker, he cooperated to produce groundbreaking research on hematopoietic cells in the 1960s. Along with McCullouch, he was inducted into the Canadian Medical Hall of Fame for his work in this area in 2004.

Harold Varmus, current president and chief executive officer of Memorial Sloan-Kettering Cancer Center in New York City, was director of the National Institutes of Health from 1993 to 1999, a period during which much fundamental research on stem cells was being conducted. During that period and since, he was and has been an outspoken supporter of all types of stem cell research, suggesting on one occasion that “[i]t is not too unrealistic to say that this research has the potential to revolutionize the practice of medicine and improve the quality and length of life.” Varmus was awarded a share of the 1989 Nobel Prize in Medicine or Physiology for his studies of the cellular origins of genes that are responsible for certain types of cancer.

Catherine Verfaillie, a Belgian-born authority on hematology and oncology, and director of the University of Minnesota’s Stem Cell Institute. Her current research interests include the nature, development, and treatment of Fanconi and sickle cell anemia; the processes by which human hematopoietic stem cells develop, proliferate, and differentiate; and the properties of adult stem cells, in general. Verfaillie has written extensively on the scientific characteristics of stem cells and their potential therapeutic value, has appeared as an expert witness before the President’s Council on Bioethics, and is coeditor of Handbook of Stem Cells (2005).

Irving Weissman, Karel and Avice Beekhuis professor of cancer biology and professor of pathology and developmental biology at Stanford University’s School of Medicine. He was the first person to isolate any type of stem cell. In 1988, he demonstrated the existence of hematopoietic stem cells in mice and, four years later, repeated his success with human hematopoietic stem cells. In 2000, a research team led by Weissman became the first to find and isolate stem cells in the human nervous system. Weissman has founded two companies to promote research and development on stem cells, SyStemix and StemCells, Inc., and, in 2002, was named director of Stanford’s newly created Stanford Institute for Cancer/ Stem Cell Biology and Medicine. He has also served as chair of the Panel on Scientific and Medical Aspects of Human Cloning of the National Academy of Sciences.

Dave Weldon, Republican representative from the 15th congressional district of Florida. He has been an outspoken opponent of embryonic stem cell research. During the 109th Congress, he cosponsored with Representative Bart Stupak (D-Minn.) the Human Cloning Prohibition Act of 2005 (H.R. 1357) to prohibit the cloning of human embryos for any purposes whatsoever, either for purposes of reproduction or to obtain embryos for scientific research.

Michael D. West, current chairman of the board, president, and chief executive officer of Advanced Cell Technologies in Worcester, Massachusetts. He has been a major force in providing the funding necessary to conduct stem cell research in the United States without the expenditure of public tax dollars. In 1990, he founded the biotechnology firm Geron Corporation in 1990, where he served as director and senior executive officer until 1998. He then cofounded another biotechnology company, Origen Therapeutics, a company focused on the development of transgenic technologies. In 1999, he was a member of a group that took controlling interest in Advanced Cell Technologies, where he has remained ever since. In the early 1990s, West obtained funding from private sources for the research at James Thomson’s and John Gearhart’s laboratories that led to the first isolation of human embryonic and fetal stem cells.

Roger F. Wicker, U.S. representative from the 1st congressional district of Mississippi since 1995. He was cosponsor of the Dickey Amendment in 1996 that prohibited the Department of Health and Human Services from funding any type of research involving the production, purchase, or commerce in human embryos.

Ian Wilmut, professor and head of the department of gene expression and development at the Roslin Institute near Edinburgh, Scotland. He was leader of the research team that cloned the first mammal, a sheep, named Dolly, in 1996. Dolly was euthanized in 2003 because of lung problems believed related to her atypical method of conception and birth.

Laurie Zoloth, professor of medical ethics and humanities, and of religion, at Northwestern University’s Feinberg School of Medicine. She has written and spoken extensively on the ethical issues related to embryonic and adult stem cell research. Her current research interests involve ethical problems that have arisen as a result of advances in medical technology and research in genetics. She is a member of the National Advisory Council of the National Aeronautical and Space Administration (NASA), NASA’s Planetary Protection Advisory Committee, and the executive committee of the International Society for Stem Cell Research. She also serves as chair of the Bioethics Advisory Board of the Howard Hughes Medical Institute.

CHRONOLOGY STEM CELL RESEARCH 1665 - 2006

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This chapter presents a chronology of major events in the history of stem cell research, including scientific and technological advances, as well as political, social, ethical, and other events related to research in this field.

1665
English physicist Robert Hooke examines the structure of a piece of cork through a microscope and observes tiny pockets that he calls cells or pores, the first recognition of these structures as the basic unit of life.

1712
French physicist Rene Reaumur publishes his classic work on the ability of crawfish and crabs to regenerate claws and limbs in the Memoires de l’Academie Royale des Sciences.

1744
Abraham Trembley, Swiss scientist and philosopher, publishes a report of his extensive studies on the regeneration of hydra in his book Memoires pour servir a l’histoire d’un genre de polypes d’eau douce a bras en forme de cornes (Memoirs concerning the natural history of a type of freshwater polyp with arms shaped like horns).

1827
German naturalist Karl Ernst von Baer discovers that mammalian life originates with the fertilization of an egg cell.

1838
German botanist Matthias Jakob Schleiden suggests that the basic structural unit of all plants is the cell.

1839
German physiologist Theodor Schwann hypothesizes that cells are the basic structural unit of all animals. This hypothesis, along with Schleiden’s similar suggestion for plants in 1838, constitutes the basis of the cell theory.

1855
German pathologist Rudolf Virchow proposes a fundamental law of cell behavior, omnis cellula a cellula, “all cells arise from cells.”

1869
Pope Pius IX declares that the process of ensoulment begins at conception, establishing Roman Catholic doctrine that human life begins at that moment.

1878
Austrian physician and researcher S. L. Schenk attempts to fertilize a human egg outside the human body in an attempt to assist a woman unable to have children by normal coitus. The experiment fails, but it is the first recorded effort to achieve ex utero fertilization.

1888
German embryologist Wilhelm Roux carries out an experiment in which he destroys one of the two cells in a young frog embryo. The remaining cell develops into a deformed frog, convincing Roux that each of the two original cells contains only half of the frog’s complete genetic map. Roux’s results are later contradicted by experiments conducted by Hans Driesch.

1891
English biologist Walter Heape performs the first known case of embryo transfer when he flushes preimplanted embryos from the oviducts of one female rabbit and transplants them into the oviducts of a second female rabbit. The second rabbit produces a mixed litter of two angora and four Belgian rabbits.

1892
German embryologist Hans Driesch separates individual embryonic cells from two- and four-cell sea urchin embryos. He finds that each of the individual cells develops normally into a complete adult, contradicting the results of Roux’s experiments conducted only four years earlier.

1902
Austrian botanist Gottlieb Haberlandt proposes the theory of totipotency for plant cells, namely, that every cell in a mature plant has the capability to change back into an embryonic form that has the potential to then grow and differentiate into every type of cell from which the mature plant is made.

1903
Herbert J. Webber, at the U.S. Department of Agriculture, invents the term clon to describe “a colony of organisms derived asexually from a single progenitor.” Other biologists soon adopt the term and change its spelling to its modern form of clone.

1909
Russian physician and biologist Alexander A. Maximow hypothesizes the existence of immature cells within bone marrow—he calls them gemeinsame Stamzellen, or common stem cells—with the capability of generating new blood cells. Maximow’s hypothesis is largely rejected and ignored for nearly 50 years.

1928
Hans Spemann, German embryologist, performs the first somatic cell nuclear transfer experiment, transferring the nucleus from one salamander egg into the enucleated egg cell of a second salamander. For this research, Spemann was later awarded the 1935 Nobel Prize in Physiology or Medicine.

1953
Robert Briggs and Thomas J. King, American embryologists, remove the nuclei from frog embryos and insert them into unfertilized frog eggs from which the nuclei have been removed. They find that the eggs then begin to develop normally, growing through early embryonic stages, although seldom hatching into tadpoles. They further demonstrate that the effectiveness of the technique is a function of the age of the embryos used, with nuclei taken from younger embryo able to produce healthier eggs that develop more normally than with nuclei taken from older embryos.

1956
E. Donnall Thomas leads a surgical team that performs the first successful bone marrow transplant on a human. The patient achieves full remission after receiving a transplant from an identical twin.

1958
Cornell University botanist Frederick C. Steward demonstrates that it is possible to grow a complete new carrot plant starting with a single somatic cell removed from a mature carrot plant. The experiment shows the ability of a mature, completely differentiated somatic plant cell to revert to a similar, totipotent form that is then able to grow through the complete cycle of redifferentiation and development.

1961
Daniele Petrucci, an Italian embryologist, conducts one of the first successful in vitro fertilization experiments. He fertilizes an egg on a laboratory dish and embeds it in an “artificial womb.” The fertilized egg appears to develop normally for 29 days, at the end of which Petrucci claims to have detected a heartbeat. He then decides to destroy the embryo because of profound ethical questions raised by the research and because the embryo itself had become enlarged, deformed, and, according to Petrucci, “a monstrosity.”

1962
English biologist John B. Gurdon performs the first successful cloning experiments with animals by removing the nuclei of cells taken from stomach tissue of a tadpole and transplanting them into the enucleated eggs of frogs. Some small number of eggs eventually develop into young tadpoles, although they do not survive for more than a few days. American researcher Joseph Altman begins publishing a series of papers suggesting that neurogenesis (the formation of new nerve cells) occurs in the brain. Prior to this time—and, for many scientists, even following the publication of Altman’s papers—the prevailing wisdom among neuroscientists was that an organism is born with all the brain cells (neurons) it will ever have and that no new neurons are ever produced in the brain.

1963
Three Canadian researchers, Andrew Becker, Ernest McCulloch, and James Till, report on the existence of a group of cells in mouse spleens with the ability to regenerate rapidly and evolve into blood cells, the first direct evidence of the existence of hematopoietic blood cells in a mammal.

1964
Two American medical researchers, G. Barry Pierce and Lewis Kleinsmith, demonstrate that teratomas are caused by a specific type of stem cell, known as an embryonal cancer cell.

1968
British physiologists Robert Edwards and Barry Bavister successfully fertilize a human egg with human sperm in a petri dish. The procedure provides the fundamental technology later used for in vitro fertilization.

1973
January 22: The U.S. Supreme Court announces its decision in the case of Roe v. Wade, setting the nation’s policy on abortion that is essentially intact today. In that decision, the Court avoids a specific definition as to when human life begins but allows unfettered access to abortions by women during the first trimester of pregnancy. It permits some forms of restrictions on abortions during the second trimester, and even more restrictions by states during the third trimester, a pattern reflecting the Court’s view that the origin of human life is an evolutionary process, rather than an event that can be specified at a particular moment in an organism’s history.

1974
July 12: Congress passes the National Research Act, setting out the basic legal and ethical standards to be used in biomedical and behavioral research projects with human subjects. The act created the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research to oversee human experimentation in the United States and established a moratorium on the use of embryos and fetuses and embryonic and fetal tissue.

1975
American biologists Howard Green and James Rheinwald develop a method for growing cells on a culture called 3T3 made of irradiated mouse fibroblasts. The Green-Rheinwald method later becomes the standard procedure for maintaining stem cells in a proliferative, nondifferentiating state in vitro over long periods of time.

1977
September: Secretary of Health, Education, and Welfare (HEW) Joseph A. Califano appoints an Ethics Advisory Board (EAB) to provide the department with ethical advice about a number of scientific, medical, and health issues involving humans.

1978
July 25: Baby Louise Brown is born in England, the first child conceived by in vitro artificial insemination. She becomes popularly known as the world’s first “test-tube” baby.

1979
April 18: The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, authorized under the National Research Act of 1974, issues a report on the general principles and guidelines to be followed in research on human subjects. The report, commonly called the Belmont Report, is important because it is the first official document in U.S. history discussing the legal status of the embryo and its treatment in scientific research.
May 4: The Ethics Advisory Board issues its report on in vitro fertilization for the purposes of producing human embryos. The board recommends that federal funds be permitted for research involving embryos, provided that research takes during the first 14 days of embryonic development, and that all gamete donors are married couples.

1980
The original charter for the Ethics Advisory Board expires and is not renewed. Lacking any agency for approving the federal funding of research on embryos in the United States (the EAB’s original responsibility), a de facto moratorium on such research exists until 1993.

1981
Martin Evans and Matthew Kaufman in the United Kingdom and Gail Martin in the United States report the isolation and culturing of murine embryonic stem cells (stem cells obtained from mice), the first case in which embryonic stem cells are obtained from an animal.

1982
American ornithologists Steven Goldman and Fernando Nottebohm demonstrate the existence of neuronal stem cells in the brain of canaries that are responsible for the constant process of neurogenesis (the formation of new nerve cells). The experiment is particularly interesting because of the long-held belief that the brain is not capable of growing new nerve cells.

1984
The Committee of Inquiry into Human Fertilisation and Embryology, commonly known as the Warnock Committee, issues its report on infertility research and services in the United Kingdom. The report deals with a wide range of topics, including artificial insemination; sperm, ovum, and embryo donation; in vitro fertilization; use of frozen sperm, ova, and embryos; research with embryos; informed consent of participants; disposal of embryos; National Health Service planning; legal implications; and surrogate motherhood arrangements.

1987
A research team at the Walter and Eliza Hall Institute of Medical Research of the Royal Melbourne Hospital, in Australia, under the direction of David Paul Gearing, announces that they have been able to maintain murine embryonic stem cells in an undifferentiated state using a feeder layer consisting of leukemia inhibitory factor (LIF). The discovery is important because prior to this time, essentially the only effective feeder layer available was made of irradiated murine embryonic fibroblast cells.

1988
The first cord blood transplant is used to treat a five-year-old Parisian boy with Fanconi’s anemia, a genetic disorder in which a person’s bone marrow fails to produce adequate numbers of all types of blood cells. Cord blood is blood obtained from the umbilical cord of a newborn baby. The boy received a cord blood transplant from his sister and is still alive with no symptoms of the original disorder.
Irving Weissman and his colleagues at Stanford University develop a system for recognizing hematopoietic stem cells in mice, providing a reliable method for isolating such cells from other cells present in bone marrow. Four years later, Weissman’s team creates a similar system for the identification of human hematopoietic stem cells.
December: A committee of the National Institutes of Health (NIH) votes 19-2 to renew government funding of research on fetal tissue transplantation. The recommendation is never put into effect.

1990
The British Parliament passes the Human Fertilisation and Embryology Act permitting research on donated embryos for certain limited purposes, including studies on infertility and the detection of birth defects.

1993
The Canadian Royal Commission of New Reproductive Technologies, which had begun its work in 1983, issues a 14-volume report that includes 293 recommendations for national policy and procedures regarding the use of a variety of artificial reproductive technologies. For a variety of political reasons, the government does not begin to act on the most important of these provisions for over a decade.
The first cord blood transplant between unrelated adults with acute lymphoblastic leukemia (ALL) is conducted by Dr. Joanne Kurtzberg at Duke University Medical Center. Both transplants are successful and patients suffer remission from their disease for significant periods of time. January 22: Newly elected president Bill Clinton issues a memorandum revoking the existing ban on fetal and embryonic research.
June 10: Congress passes the NIH Revitalization Act of 1993 (later Public Law 103-43, Section 121(c)), effectively nullifying existing prohibitions against many forms of research on human embryos and fetuses. One provision of the act requires the National Institutes of Health (NIH) to establish a Human Embryo Research Panel (HERP) to assess the moral and ethical issues raised by in vitro fertilization and related research.

1994
September 27: The Human Embryo Research Panel (HERP), charged with developing standards for determining which fetal and embryonic research projects can ethically be supported with federal funds and which cannot, issues a report, listing areas of research that it regards as acceptable and unacceptable. Among the approved activities is the creation of human embryos specifically for the purpose of experimentation.
November: Sri Lankan gynecologist Ariff Bongso obtains stem cells from a five-day-old embryo and demonstrates that they are able to differentiate in vitro to produce other types of cells. Bongso is able to keep the embryonic stem cells alive and reproducing for two generations on the surface of fallopian tube tissue, but is unable to extend their lives beyond that point. Questions remain as to whether the cells obtained by Bongso were pure embryonic stem cells or whether they were cells that had already begun to differentiate.
December 2: President Bill Clinton issues an executive order directing the National Institutes of Health (NIH) not to provide funding for any research that would involve “the creation of human embryos for research purposes,” although the use of “spare embryos,” donated by individuals who give informed consent is permitted. Clinton’s order directs NIH to develop guidelines for the use of “spare embryos” in research.

1995
Representative Jay Dickey (R-Fla.) offers a rider to the appropriations bill for the Department of Health and Human Services (HHS). The Dickey amendment prohibits the use of federal funds for the creation of human embryos for research or for research that makes use of human embryos that have been destroyed.
October 3: President Bill Clinton issues Presidential Executive Order 12975 creating the National Bioethics Advisory Commission (NBAC) to provide guidance to federal agencies on the ethical conduct of current and future human biological and behavioral research. The commission has since been replaced by the President’s Council on Bioethics, created in connection with President George W. Bush’s address on human stem cell research of August 9, 2001.

1996
January 26: The Dickey amendment is passed by Congress. A similar rider is adopted in each subsequent year through 2001, after which President George W. Bush’s statement of policy on human embryo stem cell research makes the prohibition, to a large extent, moot.
July 5: A sheep named Dolly is born at the Roslin Institute in Scotland as the result of a cloning experiment conducted by a research team led by Ian Wilmut. Dolly is euthanized in 2003 as the result of a lung disorder believed to have been caused by her atypical method of conception.

1997
February 24: Researchers at the Roslyn Institute in Edinburgh, Scotland, led by Ian Wilmut, announce that they have used the process of somatic cell nuclear transfer to produce the first cloned mammal, a sheep named Dolly. The announcement comes many months after the sheep’s birth.
February 24: President Bill Clinton asks the National Bioethics Advisory Commission (NBAC) to review the ethical and legal issues associated with the use of cloning technology. The commission issues its report on June 9, 1997, recommending that the existing moratorium on the use of federal funding for somatic cell nuclear transfer research be continued and that researchers being funded by private funds be asked to comply voluntarily with the intent of the federal moratorium.
July: John Gearhart, from Johns Hopkins University, announces that he and his colleague Michael Shamblott have isolated germ cells from a human fetus. He also says that their research team is in the process of proving that the cells are pluripotent, an experiment that results in their historic paper of November 10, 1998, published in Proceedings of the National Academy of Sciences.

1998
January 7: Independent scientist Dr. Richard Seed announces his intention to clone a human being.
March 6: A group of Italian researchers led by Giuliana Ferrari discovers that hematopoietic stem cells are able to migrate to muscular tissue, where they differentiate and develop into normal muscle cells.
November 6: A research team at the University of Wisconsin–Madison led by James A. Thomson announces the first isolation of human embryonic stem cells from a group of 36 fertilized eggs that had been grown to the blastocyst stage before being sacrificed to permit removal of the inner cell masses. Five of the stem cells are cultured into cell lines that continue to grow for periods of four to five months.
November 10: In research closely related to that of Thomson, a research team from the Johns Hopkins University led by John Gearhart announces the isolation of human embryonic germ cells. The Johns Hopkins research differed from the Wisconsin research in two respects. First, the stem cells isolated were germ cells, which, after differentiation, become egg and sperm cells, not somatic cells, as in the case of the Thomson experiment. Second, the cells were obtained from gonadal ridges and mesenteries (membranous tissue that surrounds, supports, and carries blood to the intestines) of fetuses five to nine weeks old (postfertilization).

1999
January 15: Department of Health and Human Services General Counsel Harriet Rabb rules that the Dickey amendment restricting the funding of stem cell research on embryos does not prohibit the federal funding of research on stem cells obtained from embryos produced through private funding.
January 22: A research team of Canadian and Italian scientists headed by graduate student Christopher R. R. Bjornson announces that neural stem cells from mice are capable not only of generating new neural cells, but also of differentiating into certain types of blood cells, including myeloid, B, and T leukocytes.

2000
June 18: A committee headed by Sir Liam Donaldson, Chief Medical Officer of Great Britain, issues a report on recent developments in in vitro fertilization and other forms of assisted human reproduction. The committee essentially confirms the general policies established by the 1990 Human Fertilisation and Embryology Act.
December 1: The journal Science carries an article written by Eva Mezey, researcher at the U.S. National Institutes of Health (NIH) reporting the conversion of hematopoietic cells into neurons in rat brains. The study is so revolutionary and controversial that the magazine holds the report for over a year before allowing publication. The report accompanies a second paper by a group of researchers at Stanford University led by graduate student Timothy Brazelton reporting similar results with hematopoietic bone marrow cells that migrate to the brain and differentiate to produce new neuronal cells.

2001
May 4: A research team headed by Diane Krause, at the Yale University School of Medicine, reports that a single hematopoietic bone marrow cell is able to differentiate into many different types of specialized cells, such as those found in the digestive system, the liver, the lungs, and the skin.
July 31: The U.S. House of Representatives passes house bill H.R. 2505, the Human Cloning Prohibition Act of 2001, by a vote of 265-162. The bill bans the production of human embryos for any reason whatsoever, including both reproductive purposes and research on stem cells for therapeutic purposes. A companion bill, S. 1899, was introduced into the Senate, but never acted upon. Both bills became moot when President George W. Bush announced in August federal policy on stem cell research.
August 9: In a nationally televised address, President George W. Bush outlines his administration’s policy on stem cell research. He will permit research on certain preexisting human embryonic stem cell lines, on adult stem cells, and on cord blood stem cells, but will not permit the creation of new embryos for the purpose of either therapeutic research or human cloning experiments.
November 25: Advanced Cell Technology (ACT), a biotechnology research company located in Worcester, Massachusetts, announces two breakthroughs in studies of embryonic stem cells. In the first line of research, ACT scientists are successful in coaxing oocytes (egg cells) to begin reproduction without the addition of a male gamete (sperm cell), a process known as parthenogenesis. The activated oocyte is allowed to grow to the preimplantation stage. In the second line of research, DNA from both an oocyte and a sperm cell are removed, and the DNA from a somatic cell is transplanted into the oocyte nucleus. The oocyte is then stimulated to begin dividing and allowed to develop to the 16-cell stage before being sacrificed.
November 28: President George W. Bush establishes the President’s Council on Bioethics by Executive Order 13237.
December 4: The British Parliament passes the Human Reproductive Cloning Act, legislation that makes it a criminal offense to implant a cloned embryo into a woman. The act was made necessary when a British court ruled in 2000 that an earlier law, the Human Fertilisation and Embryology Act of 1990, did not specifically prohibit the cloning of a human being.
December 22: Scientists at Texas A&M College of Veterinary Medicine announce the birth of the first cloned cat, a kitten they name CC (for “carbon copy”).

2002
February: Two researchers from Boston’s Whitehead Institute for Biomedical Research, Konrad Hochedlinger and Rudolf Jaenisch, report on the cloning of mice using stem cells taken from the stomach of adult mice. The report is the first occasion on which the successful use of adult stem cells for cloning of an animal has been announced.
February: Researchers at Geron Corporation led by Chunhui Xu announced that they have developed a method for maintaining stem cell lines without the use of feeder layers of any kind. The discovery is of importance because of long-term concerns that contaminants in traditional kinds of feeder layers, either murine or human, might become incorporated into the stem cells on which they were maintained, adding a problem for the use of such cells in regenerative medicine.
April: A second research team at the Whitehead Institute reports on an experiment in which mice with an immune disease are cured by the transplantation of genetically engineered embryonic stem cells. The experiment was the first confirmed example of the successful therapeutic use of embryonic stem cell therapy.
July 4: A research team led by Belgian-born hematologist Catherine Verfaillie, now head of the University of Minnesota’s Stem Cell Institute, announces the discovery of a specialized kind of mesenchymal stem cell with the ability to differentiate into cells characteristics of all three blastocytal layers, epidermal, mesodermal, and endodermal. The researchers note that, if their findings are confirmed, these mesenchymal cells “May be an ideal cell source for therapy of inherited or degenerative diseases.”
September 22: Governor Gray Davis of California signs a bill passed by the state house and senate amending portions of the California Health and Safety Code authorizing the use of human embryonic stem cells, human embryonic germ cells, and human adult stem cells obtained from any source, as well as the process of somatic cell nuclear transplantation for research and therapeutic purposes, but not for the purpose of human reproductive cloning. The bill also requires health care providers to inform individuals whom they treat and advise of the possibility of donating surplus embryos produced in their treatment for stem cell research.

2003
May 15: Eleven Republican members of the U.S. House of Representative write to President George W. Bush, expressing their concerns over the progress (or lack of progress) of stem cell research at the National Institutes of Health (NIH) and asking that the president revisit his policy on stem cell research. The president declines to do so.

2004
January: The governments of both China and South Korea adopt legislation prohibiting reproductive cloning, but specifically allow human embryo cloning for the purpose of research.
January 4: New Jersey Governor James E. McGreevey signs into law senate bill 1909, the Stem Cell Research Bill, legalizing stem cell research in the state and providing mechanisms for its funding with state tax monies. New Jersey becomes the second state, after California, to legalize stem cell research.
February 12: South Korea researchers at Hanyang University under the direction of Hwang Woo Suk of Seoul National University announce that they have produced 30 human embryos by means of somatic cell nuclear transfer. The embryos were produced from 242 eggs taken from 16 volunteers and were allowed to reach the blastocyst stage before being sacrificed. (See May 2006.)
May 19: The British government announces the opening of the world’s first embryonic stem cell bank. The bank expects to receive, store, and supply tens of thousands of stem cell lines to be used in research for the treatment of chronic diseases, such as diabetes, Alzheimer’s disease, and Parkinson’s disease.
October: The Virginia Commonwealth University Life Sciences Survey releases the results of its most recent nationwide survey of public opinion on stem cell research. The survey finds that support for embryonic stem cell research has increased to 53 percent of those surveyed, compared to an approval rating of 47 percent only a year earlier. At the same time, opposition has dropped from 44 percent to 36 percent during the preceding year.
November 2: Voters in the state of California pass Proposition 71 by a margin of 59 percent to 41 percent. The proposition creates a new California Institute for Regenerative Medicine, empowered to regulate stem cell research and to provide funding for such research and necessary research facilities. The proposition also establishes a constitutional right for the conduct of stem cell research in the state and specifically prohibits state funding for any form of human reproductive cloning research. The proposition also authorizes the expenditure of $3 billion over a 10-year period for stem cell research in the state.

2005
February 16: Representatives Mike Castle (R-Del.) and Diana DeGette (D-Colo.) introduce a bill into the U.S. House of Representatives (H.R. 810) that authorizes the use of federal funds for stem cell research using materials taken from surplus embryos that have been donated for that purpose, provided that certain specific conditions have been met for the donation. Human reproductive cloning is specifically prohibited by the bill. The bill is passed by the full house on May 24, 2005.
March 4: The Brazilian Congress passes and President Luiz Inacio Lula da Silva later signs a Bio Safety Law that authorizes the conduct of embryonic stem cell research in the country.
March 17: Dave Weldon (R-Fla.) introduces a bill into the U.S. House of Representatives to prohibit all forms of human cloning, for whatever purpose, including both the reproduction of new humans and the production of embryos for stem cell research. It also prohibits the shipment, receipt, exportation, or importation of any human embryo or any product from a human embryo that has been produced by cloning. The bill is only one, but probably the best known, of similar bills seeking to reinforce the federal government’s ban on stem cell research using embryos or embryo products. The bill is never acted upon by the House, as is the case with almost all other such bills.
May 19: The Massachusetts state legislature overrides the veto of Governor Mitt Romney to a bill that would permit the creation of embryos for scientific research. Massachusetts becomes the third state, after California and New Jersey, to pass such a law. The bill passes 35-2 in the Massachusetts senate and 112-42 in the house.
May 20: South Korean scientists announce that they have, for the first time, produced 11 new stem cell lines tailored to match a specific individual (this later turns out not to be true). The scientists use the process of somatic cell nuclear transfer (SCNT) to insert the nuclei from skin cells taken from a person to be treated into an enucleated egg from a donor. (See May 2006.)
May 23: Representative Christopher H. Smith (R-N.J.) introduces a bill into the U.S. House of Representatives requiring the Secretary of Health and Human Services (HHS) to enter into agreements with organizations that collect and store cord blood stem cells, with the goal of collecting and maintaining 150,000 units of human cord blood. The blood is to be made available for transplantation through an existing program of HHS, the C. W. Bill Young Cell Transplantation Program. The bill is passed by a vote of 431-1 in the House on the next day and forwarded to the Senate.
June 15: Connecticut Governor Jodi Rell signs legislation passed by both houses of the Connecticut legislature authorizing the expenditure of $100 million in state funds to finance stem cell research in the state.
July 12: Illinois governor Rod Blagojevich sets aside $10 million in state funds to support stem cell research. The governor acted after a bill designed to achieve the same objective failed in the state legislature in fall
2004, largely as the result of vigorous opposition by religious groups. July 15: A CBS News poll of 632 adults nationwide finds that 58 percent of respondents approve of “medical research using embryonic stem cells,” while 30 percent disapprove.
October 13: A study conducted by researchers from the Genetics and Public Policy Center at Johns Hopkins University finds that 67 percent of those Americans interviewed either approve or strongly approve of stem cell research. Forty percent favor the expansion of government support for embryonic stem cell research. By contrast, 16 percent of respondents would ban all research using embryonic stem cells, and 22 percent support the Bush administration’s policy on stem cell research.
October 15: University of Minnesota Stem Cell Institute researcher Dan Kaufman announces that his research team has found a way to coax human embryonic stem cells to differentiate into natural killer (NK) cells that seek out and destroy cancer cells.
November: A group of business leaders, patient advocates, and researchers and Missouri file an initiative petition to permit cloning of human eggs for research purposes. The initiative is opposed by religious leaders who file suit to oppose the plan and, in some cases, preach against the proposal in local churches throughout the state.
November 25: South Korean researcher Hwang Woo Suk apologizes for ethical errors in the historic research on stem cells conducted in his laboratory. Some of the eggs used in the South Korean’s research were extracted from two junior scientists of the team. He announces that he will resign his post at Seoul National University.
December: Reaction to Hwang Woo Suk’s announcement of November 25 in South Korea is overwhelming, with government officials, scientists, and ordinary citizens announcing their support for the scientist, who has become a great hero for his work on stem cells. Supporters plead for his return to the research laboratory, and more than 700 women pledge to donate eggs for his future research.
December 14: Gerald Schatten, coauthor of the May 20, 2005, report on 11 new stem lines produced in South Korea, asks that the journal Science, publishers of the report, remove his name from the report because of ethical issues involved in conduct of the research about which he was not aware.

2006
February 3: New York City Mayor Michael Bloomberg donates $100 million of his own money to Johns Hopkins University, part of which is allocated to the funding of stem cell research.
February 10: The United Kingdom’s Human Fertilistion and Embryology Authority announces that it will change current rules to allow any woman who chooses to donate eggs for use in stem cell research. Currently, only women undergoing in vitro fertilization treatment are allowed to make such donations.
February 16: The National Academy of Sciences establishes a committee to provide informal oversight for stem cell research. Because of the Bush administration’s objections about stem cell research, the committee will be funded by private organization rather than with federal tax dollars.
February 27: Three organizations opposed to stem cell research— People’s Advocate, National Tax Limitation Foundation, and California Family Bioethics Council—file suit in Alameda (California) County Superior Court to prevent implementation of Proposition 71, adopted in the state’s November 2004 election. The suit is later dismissed.
March 10: Oregon Health Sciences University announces that it will use stem cells harvested from human fetuses to treat six children with Batten disease, a neurological disorder that is always fatal. It is the first research of its kind in the world.
March 13: The European Union fails to agree on a common stem cell research policy. Policies vary throughout the Union, ranging from total bans on such research in some nations to legal and/or financial support for stem cell research in others. Officials had hoped to reach a compromise that could be adopted throughout the Union but were unsuccessful in achieving that agreement.
May 12: Prosecutors in South Korea indict Hwang Woo Suk for faking most of his stem cell research.
July 18–19: By a vote of 63-37, the U.S. Senate passes S. 471, companion bill to H.R. 810, which was passed by the House of Representatives in 2005. A day after Senate passage, President George W. Bush vetoes the bill, exercising his veto power for the first time in nearly six years as president. Bush says the bill crosses “a moral boundary,” a move that he would not accept. The House of Representatives votes to override the veto, but there are not sufficient enough votes to do so, therefore the veto remains.

Decision Stem Cell

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In reaching its decision in Roe v. Wade, the Court reviewed in great detail ancient attitudes toward abortion; the Hippocratic Oath; common law, English statutory law, and American law on abortion; and the views of the American Medical Association, the American Public Health Association, and the American Bar Association. Based on its reading of these records, the Court made two fundamental decisions about the constitutional status of abortion in the United States. First, the Court decided that the Constitution did, in fact, confer a “right of privacy” to individuals and included within that right was the right for a woman to do with her own body as she chose to do, i.e., abort a child that she was carrying. Specifically, it said that “[t]his right of privacy, whether it be founded in the Fourteenth Amendment’s concept of personal liberty and restrictions upon state action, as we feel it is, or, as the District Court determined, in the Ninth Amendment’s reservation of rights to the people, is broad enough to encompass a woman’s decision whether or not to terminate her pregnancy.”

Second, the Court resolved the issue as to the point at which an embryo/ fetus becomes a person by separating pregnancy into three distinct trimesters, each representing a more advanced stage of development of the unborn child. It concluded that the chances of an embryo’s/fetus’s surviving outside the uterus during the first trimester was so small that it could not, at that point, legally be considered to be a “person” and that it had, therefore, no constitutional rights. Under the circumstances, a state could pass no laws prohibiting abortion during that first trimester.

By contrast, evidence suggested that a fetus might be able to survive during the second trimester and, hence, states could pass laws banning abortions in the interest of protecting a mother’s health or life. And, given the substantial likelihood that a third-trimester child could survive outside the uterus, states were allowed to pass any type of legislation restricting or banning abortion during this period of pregnancy.

Legal Issues - Stem Cell

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In its deliberations, the Court first had to contend with certain technical issues involving two other cases with which the original Texas case had been bracketed. The first involved a couple known only as “the Does” who, although not pregnant, were challenging the state of Georgia’s laws against abortions. The second concerned a Texas attorney named James Hallford who had already been convicted twice of performing illegal abortions, and also challenged the state of Texas’s antiabortion laws. The court decided that neither the Does nor Hallford had standing in this case, and dismissed their claims from further consideration.

The fundamental legal issue that remained, then, was simple in concept: Does a woman have a constitutional right to abort a child that she is carrying? In fact, the U.S. Constitution does not explicitly address the issue of abortion. But lawyers for Roe in this case argued that the right to abortion was inherent in a number of constitutional amendments, including the First, Fourth, Fifth, Ninth, and Fourteenth Amendments. The key amendments among these were the Fourteenth Amendment, the so-called due process amendment, and the Ninth Amendment, which reserves to the people all rights not specifically granted to the federal government in other parts of the Constitution. Roe’s attorneys argued that these two amendments guaranteed a right of privacy to citizens that, other constitutional restrictions notwithstanding, included the right to abortion.

The issue was complicated by the question as to whether, when, or under what circumstances the embryo, fetus, or unborn child being carried by a pregnant woman itself became a “person,” with all of the constitutional rights then due it. At that point, the constitutional rights of the pregnant woman alone could no longer be considered by themselves; they would have to be weighed against the constitutional rights of the new “person” she was carrying.

GENERAL LAWS OF THE COMMONWEALTH OF MASSACHUSETTS, CHAPTER 111L (2005) STEM CELL

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The state legislature of the Commonwealth of Massachusetts considered the question of stem cell research in 2005 and adopted legislation declaring that the state’s policy was to be one that would “actively foster research and therapies in the life sciences and regenerative medicine by permitting research and clinical applications involving the derivation and use of human embryonic stem cells, including research and clinical applications involving somatic cell nuclear transfer, placental and umbilical cord cells and human adult stem cells and other mechanisms to create embryonic stem cells which are consistent with this chapter.” The legislation also declared that it was the state’s further policy “to prohibit human reproductive cloning.” The Massachusetts legislation took a somewhat tortuous path through both houses of the legislature, beginning as SB 25, sponsored by senate president Robert E. Travaglini (D-First Suffolk and Middlesex), Cynthia Stone Creem (D-First Middlesex and Norfolk), and Harriette L. Chandler (D-First Worcester). That bill was reported out of the senate Committee on Economic Development and Emerging Technologies as SB 2027 and was later retitled SB 2032 (in connection with a comparable bill from the house, bill number 2792), and finally as SB 2039 after a conference committee between the two houses to resolve relatively minor differences in H 2792 and SB 2032.

SB 2039 was passed by both houses of the Massachusetts legislature, by the Senate on April 26, 2005, on a vote of 34-2, and by the House on May 4 on a vote of 119-38. Those votes set up a certain confrontation with Republican Governor Mitt Romney, who opposes stem cell research and had been threatening to veto the legislation. After the legislature defied the governor by passing SB 2039, Romney carried out on his threat, and vetoed the legislation, returning it to the legislature with four recommended changes that he said would make it acceptable to him. (Massachusetts is one of the few states in which governors can follow such a procedure.) The legislature was not convinced by the governor’s arguments, however, and both houses voted to override his veto on May 19, 2005, the House by a vote of 112-42, and the Senate by a vote of 34-2. Since the legislation had been given “emergency” status, it became law immediately upon the legislature’s vote.

STEM CELL THERAPEUTIC AND RESEARCH ACT OF 2005 P.L. 109-129

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The U.S. Congress has also considered a number of bills relating to forms of stem cell research discrete from studies that involve the use of human embryos. Such bills have generally met with greater success than those focusing on embryonic stem cell research. An example is the Stem Cell Therapeutic Research Act of 2005 (H.R. 2520), introduced by Representative Christopher H. Smith (R-N.J.) on May 23, 2005. The bill requires the Secretary of Health and Human Services to “enter into one-time contracts with qualified cord blood stem cell banks to assist in the collection and maintenance of 150,000 units of high-quality human cord blood to be made available for transplantation through the C. W. Bill Young Cell Transplantation Program.” (The C. W. Bill Young Cell Transplantation Program is a program named after U.S. Representative C. W. Bill Young (R-Fla.), who was instrumental in creating the U.S. bone marrow donor registry program in 1986. The program is described in section 379 of the Public Service Act, 42 U.S.C. 274l.)

H.R. 2520 passed the House of Representatives by a vote of 431-1 on May 24, 2005, and by the Senate on December 16, 2005. The bill was signed into law by President George W. Bush on December 20, 2005, and has become Public Law 109–129.

THE HUMAN CLONING PROHIBITION ACT OF 2005 (H.R. 1357) STEM CELL

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An example of the type of bills offered by opponents of stem cell research is H.R. 1357, introduced by Representative Dave Weldon (R-Fla.) on March 17, 2005, with 120 cosponsors, after which it was referred to the House Subcommittee on Crime, Terrorism, and Homeland Security. On the same date, a companion bill, S. 658, was introduced in the Senate by Senator Sam Brownback (R-Kans.), with 31 cosponsors. H.R. 1357 prohibited all forms of human cloning, for whatever purpose, including both the reproduction of new humans and the production of embryos for stem cell research. It also prohibited the shipping, receiving, exportation, or importation of any human embryo or any product from a human embryo that has been produced by cloning. No action was ever taken on H.R. 1357.

STEM CELL RESEARCH ENHANCEMENT ACT OF 2005 (H.R. 810) STEM CELL

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Following President George W. Bush’s decision in 2001 to limit the range of stem cell research that could be funded by the federal government, legislators from both parties offered a number of bills designed either to confirm and/or extend the president’s action or override his action by adopting laws that are more liberal in funding permitted for stem cell research. One of the most successful efforts was House bill 810 (H.R. 810), offered by representatives Mike Castle (R-Del.) and Diana DeGette (D-Colo.) and cosponsored by more than 150 other members of the House of Representatives.

H.R. 810 was introduced by Representative Castle on February 16, 2005. A comparable bill, S. 471, was also introduced into the U.S. Senate by senators Arlen Specter (R-Pa.) and Tom Harkin (D-Iowa). The House bill was reported out of the House Energy and Commerce Committee favorably and was passed by the House on a vote of 238-194 on May 24, 2005. S. 471 was passed by the Senate on a vote of 63-37 on July 18, 2006, but vetoed by President Bush on July 19. The House of Representatives was unable to override the veto.

Both bills took the form most commonly favored by supporters of stem cell research, permitting the funding of such research by the federal government, provided certain conditions are met. Those conditions are that the embryos to be used in research must come from the supply of surplus embryos supplied by in vitro fertilization clinics, but only if donors give their express, informed consent, and that donors not be paid for the embryos. No provision is made for the support of research on embryos that have been specifically produced for experimental use. To make the House’s position clear, the bill contains an explicit indication that the surplus embryos can be used “regardless of the date on which the stem cells were derived from a human embryo.” This explicit statement presumably was intended to contrast with President Bush’s limitation on the use of only those embryos in existence prior to his August 9, 2001, announcement.

NEW JERSEY PERMANENT STATUTES, TITLE 26, SECTION 2Z-1 (2004) STEM CELL

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In recent years, supporters and opponents of stem cell research have become increasingly active at the state level. At least one factor in this trend has been the feeling that President George W. Bush’s opposition to SCR is likely to prevent action at the federal level for the foreseeable future, so that the greatest hope for promoting such research may be actions taken within individual states. Thus, grassroots organizations and state legislators in California, Connecticut, Florida, Illinois, Maryland, Massachusetts, New Jersey, New York, Pennsylvania, Texas, and a number of other states have acted to codify approval of and/or governmental support for stem cell research within their own state boundaries. At the same time, organizations and legislators in Alabama, Arizona, Arkansas, Indiana, Iowa, Kansas, Michigan, Missouri, North Dakota, South Dakota, Tennessee, and other states have acted to ban such research and prevent its funding within their states. For example, the state of Nebraska’s statute 71-7606 prohibits the use of state funds for the support of human embryonic stem cell research or for any research using human fetal tissue “obtained in connection with the performance of an induced abortion,” as well as for “abortion, abortion counseling, referral for abortion, or school-based health clinics.”

A striking contrast to Nebraska’s law is one passed by the New Jersey legislature and signed by Governor James E. McGreevey in 2004. That law declared that “It is the public policy of this State that research involving the derivation and use of human embryonic stem cells, human embryonic germ cells and human adult stem cells, including somatic cell nuclear transplantation, shall: be permitted in this State; be conducted with full consideration for the ethical and medical implications of this research; and be reviewed, in each case, by an institutional review board operating in accordance with applicable federal regulations.” The bill specified certain conditions under which stem cell research had to be conducted and made it clear that cloning an embryo for the purpose of human reproduction was unacceptable and was declared to be a crime of the first degree.

The New Jersey act was passed with large majorities in both houses of the state legislature with the strong support of both Governor McGreevey and his successor, Governor Richard J. Codey. The first fruit of the bill’s passage was the announcement by Codey in 2004 that the state was establishing the first state-supported and -funded stem cell science research institute in the nation, an institute to be operated jointly by Rutgers University and the University of Medicine and Dentistry of New Jersey. Codey announced that the institute would be funded with an initial grant of $380 million, $150 million from state appropriations and $230 million from a state bond.

STATE OF CALIFORNIA PROPOSITION 71 (2004) STEM CELL RESEARCH

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Advocates of embryonic stem cell research in the United States have faced a two-pronged challenge since President George W. Bush laid out federal policy on SCR in August 2001. In the first place, they must ensure that individual state laws allow stem cell research, a situation that often does not exist in many areas of the country. Obtaining legal approval for stem cell research can be achieved by legislative action, by having state legislatures adopt laws that permit stem cell research, or by popular initiative, where such actions are permitted by state constitutions.

Getting stem cell research legalized is only the first step, however. Proponents of SCR must then find a way to obtain the funds needed to support such research. The types of research that are eligible for federal grants are limited by President Bush’s 2001 statement, and few states have sufficient funds in their own budget to offer funding for stem cell research.

One of the most popular options for obtaining funding is through bonds that are issued against a state’s financial credit, which was what stem cell research supporters in California did in 2004. Proposition 71 called for significant changes (running eight pages in length) to the state constitution and certain laws pertaining to medical research. It called for the establishment of a California Institute for Regenerative Medicine with the charge of regulating stem cell research and providing funding, through grants and loans, for such research and research facilities. The proposition also established a constitutional right for the conduct of stem cell research in the state and specifically prohibited the state’s funding of any form of human reproductive cloning research. To carry out the institute’s charge, the proposition provided $3 million from the state’s general fund to pay for start-up costs and authorized the issuance of general obligation bonds in the amount of $3 billion over a 10-year period, with a maximum expenditure of $350 million in any one year, the bonds to be paid for from the state’s general fund.

Proposition 71 was envisioned originally by state senator Deborah Ortiz (D-Sacramento) who had been working for two years to pass legislation in the California legislature to achieve the objectives of what was later to become Proposition 71. She was unsuccessful in her efforts in the legislature and decided to bring the issue to the general public in a proposition to be placed on the November 2004 ballot. The proposition passed handily in that election by a vote of 6,370,852 (59.1 percent) Yes to 4,419,373 (40.9 percent) No.

CALIFORNIA HEALTH AND SAFETY CODE §125300–125320 (2002) - STEM CELL

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The state of California has been at the forefront of efforts to encourage, promote, and support most kinds of stem cell research, including embryonic, adult, and fetal blood stem cell research. On February 15, 2001, state senator Deborah Ortiz (D-Sacramento) filed SB 1272 (later changed to SB 253 for technical reasons) outlining state policy and practice on stem cell research. The bill passed the State Senate on May 2, 2002, by a vote of 21-10; was sent to the State Assembly and passed that body on August 26, 2002, by a vote of 46-27; and was signed into law by Governor Gray Davis on September 22, 2002. The bill amended various parts of the California Health and Safety Code, including paragraphs 123440, 24185, 12115–12117, and 125300–125320, with the last of these sections containing the primary provisions of the act.

Senator Ortiz’s bill declared that California’s policy on stem cell research was to be that “research involving the derivation and use of human embryonic stem cells, human embryonic germ cells, and human adult stem cells from any source, including somatic cell nuclear transplantation, shall be permitted and that full consideration of the ethical and medical implications of this research be given.” To carry out this policy, the bill required that any health care provider involved in providing fertility treatments to supply his or her patients with information about the donation of surplus embryos produced in those treatments. The bill also outlined the specific conditions under which embryos could be donated for the purpose of research and prohibited the sale of embryos or embryonic tissue for research purposes.

GEORGE W. BUSH’S PRESIDENTIAL ADDRESS ON STEM CELL RESEARCH (AUGUST 9, 2001)

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During his campaign for the presidency leading up to the 2000 election, George W. Bush indicated that he was opposed to any form of stem cell research in which human embryos were created and then destroyed for the production of stem cells. Shortly after he took office in January 2001, Bush put a hold on a government program established to accept and review grant proposals for embryonic stem cell research until his own administration could review the state of affairs in the field. The review committee issued its report on July 13, 2001, recommending that the government support both embryonic and adult stem cell research. Only a month later, Bush made a televised speech from his ranch in Crawford, Texas, to announce his policy on stem cell research.

In that speech, Bush acknowledged the potential value of using embryonic stem cell in research for the cure of a number of disabling diseases and disorders. He also reviewed the ethical issues involved in conducting such research. One of the most powerful factors in the decision he eventually made, Bush said, was his belief that embryos are living human beings and should not be killed for any reason, including possible medical benefits that might arise from stem cell research.

His decision on this contentious issue was that the U.S. government under his administration would be allowed to fund research on stem cell lines that were already in existence, in most cases the “left-over” embryos produced for the purpose of in vitro fertilization, but that funding would not be permitted for the purpose of creating new embryos for research. Bush estimated that a total of 60 stem cell lines—later increased to 78 stem cell lines—met his criterion and were eligible for funding support. The actual number of embryonic lines in good enough condition for research has been something of a matter of debate ever since. In any case, recalling science fiction novelist Aldous Huxley’s description of baby “hatcheries” in his novel Brave New World, Bush rejected the possibility of allowing the federal funding of any new stem cell lines. In conclusion, he announced the creation of a new President’s Bioethical Advisory Commission “to recommend appropriate guidelines and regulations, and to consider all of the medical and ethical ramifications of biomedical innovation.”

The president’s decision on the funding of stem cell research turned out to satisfy only a relatively small fraction of the general public. Many of his supporters were disappointed that he had decided to permit any embryonic stem cell research, arguing that to do so was to make the government complicit in the deaths of the embryos. His detractors bemoaned the limited range of research his policy was to permit and warned that the nation would rapidly fall behind other countries in this important field of scientific research. Over the ensuing years, both sides worked to drive U.S. policy in one direction or another, proposing legislation that would ban all forms of embryonic research or that would permit all forms of therapeutic embryonic stem cell research, if not embryonic research for the purpose of human cloning.

THE LAW AND STEM CELL RESEARCH

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This chapter describes laws, legislative actions, administration rulings, and legal decisions relating to stem cell research conducted in the United States. Extracts from some of these laws, court decisions, and other legal documents appear in the appendices.

LAWS AND ADMINISTRATIVE ACTIONS

Stem cell research has been of public concern for only a relatively short period of time. Thus far, few laws at the federal or state level dealing specifically with stem cell research have been passed. Most SCR regulations occur in legislation passed for somewhat different purposes, such as rules covering human experimentation, or as administrative orders. The documents listed below are some of the most important of these laws and regulations.

PROTECTION OF HUMAN SUBJECTS, 45 CFR 46.208(A)(2) (1974) AND 42 USC §289-1 (1993)

Two of the very few federal laws that regulate stem cell research are to be found in the Code of Federal Regulations (CFR), Chapter 45, Section 46, Subsection 208(a)(2) and the United States Code (USC), Title 42, Chapter 6A, Subchapter III, Part H, paragraph 289g-1, both dealing with the protection of human fetuses. The former section permits research or experimentation on a human fetus under only two circumstances: (1) if the health of the fetus itself is at risk and (2) if experimentation on the fetus will cause minimal harm to it and will produce medical knowledge that cannot be obtained by any other means. The definition of a fetus used in this section of the code is “the product of conception from the time of implantation (as evidenced by any of the presumptive signs of pregnancy, such as missed menses, or a medically acceptable pregnancy test),” and thus includes the structure usually called an embryo also. A number of writers have commented on this somewhat unusual (or, as one observer has written, “rather bizarre”1) definition of fetus, which differs significantly from that used by almost all (if not all) embryologists as referring to the structure that develops after about the eighth week of pregnancy. In any case, the definition is still part of the U.S. Code in regulations dealing with experimentation on what most people would now call the embryo, as well as the fetus into which it later develops.

45 CFR 46.208(a)(2) grew out of the National Research Act, passed by the U.S. Congress in 1974 after a fairly lengthy debate over the ethics of experimentation on human subjects. One of the major motivations for that legislation was revelations of abuses that had occurred in the use of humans for experiments in the now infamous Tuskegee Syphilis Study, a project sponsored by the U.S. Public Health Service from 1932 to 1972 in Macon County, Alabama. In that study, African-American subjects were intentionally given syphilis to learn more about the nature and course of the disease. Treatment that would have cured the subjects was withheld, and many died or were permanently maimed as the disease progressed. During the congressional debate over various bills designed to deal with the ethical problems of human experimentation, information was released about the use of whole, live fetuses in various research projects funded by the National Institutes of Health. This information prompted calls for inclusion of protection for “unborn children” in any legislation on human experimentation. It was this concern that led to the eventual adoption of the language of 45 CFR 46.208(a)(2), which remains one of the few legislative guidelines for stem cell research in the United States.

The relevant section of the U.S. Code on embryonic stem cell research, 42 USC §289-1, is titled “Research on transplantation of fetal tissue.” It authorizes the secretary of Health and Human Services to use fetal tissue for therapeutic purposes “regardless of whether the tissue is obtained pursuant to a spontaneous or induced abortion or pursuant to a stillbirth.” The section defines human fetal tissue as “tissue or cells obtained from a dead human embryo or fetus after a spontaneous or induced abortion, or after a stillbirth.” It provides the conditions under which such tissue may be obtained (always with the consent of the donor) and prohibits the payment for any fetal tissue used in therapeutic procedures.

The legislative origin of 42 USC §289-1 is the NIH Revitalization Act enacted on June 10, 1993. The act was a far-reaching bill designed to increase federal support for research in virtually all of the fields in which NIH has research interest. New programs were outlined for the National Institute of Cancer; National Heart, Lung, and Blood Institute; National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Arthritis and Musculoskeletal and Skin Diseases; National Institute on Aging; National Institute of Allergy and Infectious Diseases; National Institute of Child Health and Human Development; National Institute of Neurological Disorders and Stroke; National Institute of Environmental Health Sciences; the National Eye Institute; and the National Library of Medicine. Interest in research using transplanted fetal tissue was emphasized by placement of that topic at the very beginning of the bill, following a section on general provisions governing proposals for biomedical and behavioral research. The purpose of the section, freely and openly recognized by the bill’s sponsors, was to remove the existing moratorium on fetal research that had been in place since March 22, 1988. Even the most staunch conservatives in the U.S. Senate expressed their support for the bill and its fetal transplantation section. For example, Senator Strom Thurmond (R-S.C.), one of Congress’s most conservative members, was quoted as saying that “This is not an abortion issue. It is a research issue. It is not about taking lives. It is about saving and improving lives.”2 Although the bill was eventually passed, signed by President Bill Clinton on June 10, 1993, and codified in the U.S. Code at 42 USC §289-1, its provisions have been largely ignored. Within 18 months of its adoption, a new president, George W. Bush, had taken office and announced a new and more restrictive approach to the use of fetal materials in research.

A final note about the NIH Revitalization Act: In section 121(c) of Public Law 103-43, the provisions of President Clinton’s Executive Order 12806 of May 19, 1992, establishing a human fetal tissue bank supported by the U.S. government, was nullified and declared not to have any legal effect.

DICKEY AMENDMENT OF 1995

The Dickey amendment was first proposed in 1995 by Representative Jay Dickey (R-Ark.) as a rider to the appropriations bill for the Department of Health and Human Services (HHS) for fiscal year 1997. It has been renewed in much the same form every year since. The amendment prohibits HHS from using appropriated funds for the creation of human embryos for research or for research that makes use of human embryos that have been destroyed. Although the language of the rider varies slightly from year to year, its overall intent remains essentially the same. The wording of the rider for fiscal year 2005, for example, prohibits:

The amendment also defines the terms human embryo or embryos as “any organism, not protected as a human subject under 45 CFR 46 as of the date of the enactment of this Act, that is derived by fertilization, parthenogenesis, cloning, or any other means from one or more human gametes or human diploid cells.”

The precise wording of the Dickey amendment for the fiscal years 1996 through 2005 can be found in the following public laws: 1996: 104-99; 1997: 104-208; 1998: 105-78; 1999: 105-277; 2000: 106-113; 2001: 106- 554; 2002: 107-116; 2003: 108-7, 2004: 108-199; and 2005: 108-447. Lacking any other federal regulation on embryonic stem cell research, the Dickey amendment has been one of the major cornerstones of federal policy on this subject. Although it was written to restrict the actions of one department only, Health and Human Services, President Bill Clinton extended the ban to all federal agencies in an executive order issued in March 1997.

One of the most serious challenges to the Dickey regulations occurred during the Clinton administration. In late 1998, officials at the National Institutes of Health asked Harriet Rabb, General Counsel for HHS, for a legal opinion as to precisely what the Dickey amendment meant for the funding of stem cell research. Rabb issued her opinion in January 1999, ruling that federal funds could not be used for the creation of human embryos for research, but they could be used for research on stem cells that had been removed from human embryos produced through private funding.

Rabb’s ruling became the basis of new guidelines on stem cell research published in August 2000, shortly before the presidential election in which George W. Bush defeated Al Gore. Given the change in presidential administrations and their philosophies on stem cell research, the newly published guidelines were never put into effect and were eventually supplanted by President Bush’s August 2001 speech on stem cell research.

THE EUROPEAN UNION AND OTHER NATIONS: STEM CELL RESEARCH

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Other nations of the world have taken positions on stem cell research ranging across the spectrum of permissiveness, from being highly supportive to strongly opposed to such research. In Europe, Belgium’s laws tend to mirror those in the United Kingdom, while Denmark, Finland, Greece, the Netherlands, and Sweden all tend to have fairly permissive policies that allow researchers to use spare embryos produced originally for in vitro fertilization. By contrast, Austria, France, Germany, Ireland, and Spain tend to have more restrictive laws and regulations, prohibiting the use of human embryos from in vitro fertilization and other sources. A few countries, including Czech Republic, Luxembourg, Malta, and Portugal, had no specific legislation dealing with stem cell research as of early 2006.

Most non-European nations currently have no laws or regulations about, or tend to take a permissive stand on, stem cell research. China, Japan, Singapore, South Korea, and Taiwan, for example, all have relatively permissive attitudes toward stem cell research, allowing experimentation for therapeutic purposes, but not for the purpose of human reproduction. In all of these countries, government has taken an active role in funding and encouraging such research. One observer from the United Kingdom described his impressions of visits to stem cell research laboratories in a number of Asian countries in 2005: “They are at, or approaching, the forefront of international stem cell research,” he said. “During our 14-day visit to China, Singapore and South Korea, we encountered some of the best equipped laboratories, most industrious research teams, and most adventurous clinical programmes that any of us had ever experienced.”

The status of stem cell research throughout the world is, to a certain extent, still in a state of flux. South Africa, for example, banned all forms of stem cell research until 2003. Its reason for this prohibition was not particularly concern about the destruction of early human life, but the fear that Western corporations would exploit poor South African women, offering them financial incentives to provide eggs and embryos for their research. The nation’s health bill for 2003 laid out a new policy for stem cell research, however. The government decided that the therapeutic advantages of stem cell research outweighed any risk to South African women and agreed to permit stem cell research from embryos up to the age of 14 days. The ban on cloning for human reproductive purposes was, however, reiterated.

No comprehensive legislation similar to the United Kingdom’s Fertilisation and Embryology Act of 1990 has ever been passed in the United States. The subject of research on fertilized eggs, embryos, stem cells, and related materials has, however, been the subject of considerable discussion for more than three decades. One of the key events motivating that debate was the Supreme Court’s 1973 decision on abortion, Roe v. Wade. That decision was important not only because it established policy in the United States on the termination of pregnancies, but also because it raised a number of fundamental questions about the beginning of human life and the rights, if any, possessed by prenatal organisms.

At about the same time that the Court was considering the issues involved in Roe v. Wade, a potentially revolutionary new scientific technique known as in vitro fertilization was being developed by scientists in Great Britain and other parts of the world. The potential for this new technology to radically change the way humans think about human reproduction was obvious, not only to scientists, but also to legislators, politicians, and the general public, all of whom recognized that new laws, regulations, and policies might be necessary to handle the new reproductive choices that humans might soon have.

A third factor feeding the debate over human experimentation in the 1970s was the disclosure of an experiment being conducted by the U.S. Public Health Service in Tuskegee, Alabama, on the effects of treatments for syphilis. In this study, a number of subjects had been left untreated while health problems attributed to syphilis grew steadily worse, even though effective treatments for the disease were available. Outrage at the lack of ethical consideration given to subjects led to calls for federal legislation to deal with this problem.

The confluence of these three forces led in 1974 to passage of the National Research Act (P.L. 93-348) which included, among other provisions, for the creation of a National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. One of the charges to the commission was the determination of basic ethical principles that should underlie the conduct of biomedical and behavioral research involving human subjects. That commission met for four days in February 1976 at the Smithsonian Institution’s Belmont Conference Center and then continued its research and analysis over a period of nearly four years. It issued its final report on April 18, 1979, a report that has since been called the Belmont Report. The Belmont Report laid out a relatively simple set of guidelines to be followed in research involving human subjects. It identified three basic ethical principles: respect for persons, beneficence (that is, protection of a person’s well-being), and justice (that is, a guarantee that a person is not treated unfairly because of his or her race, gender, class, or other personal characteristic). The report also proposed three fundamental guidelines that should direct human experimentation: informed consent, comprehension, and voluntariness. In connection with its studies, the Belmont committee also issued a number of documents on related topics, including reports on research on the fetus (1975), research involving prisoners (1976), research involving children (1977), psychosurgery (1977), research involving those institutionalized as mentally infirm (1978), ethical guidelines for the delivery of health services by the Department of Health, Education and Welfare (1978), and implications of advances in biomedical and behavioral research (1978).

Upon completion of its final report, the Belmont committee passed out of existence and was replaced by a new congressionally mandated committee, the President’s Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research. Like its predecessor, the President’s Committee devoted more than three years studying a variety of bioethical issues, issuing reports on topics such as defining death (1981), protecting human subjects (1981), whistle-blowing in biomedical research (1981), social and ethical issues of genetic engineering with human beings (1982), making health care decisions (1982), deciding to forego life-sustaining treatment (1983), implementing human research regulations (1983), screening and counseling for genetic conditions (1983), and securing access to health care (1983).

One of the recommendations of the Belmont committee was that the secretary of Health, Education and Welfare appoint an Ethics Advisory Board, whose responsibility it would be to review research proposals involving fetuses and children. Pursuant to that recommendation, Secretary Joseph Califano appointed such a board, called the Ethics Advisory Board (EAB), in 1977. One of the first issues with which the new EAB had to deal with was a research proposal for a study of in vitro fertilization procedures. The board agreed to consider that proposal at its May 1978 meeting, and later approved the research. At virtually the same time the board was considering this proposal, the birth of baby Louise Brown, the first child conceived by in vitro fertilization, was announced in England, and secretary Califano asked the EAB to undertake a more comprehensive review of the social, legal, and ethical issues surrounding IVF.

The board issued its report on this question, “HEW Support of Research Involving Human In Vitro Fertilization and Embryo Transfer,” on May 4, 1979.83 It reached conclusions similar to those adopted by various British commissions and the British Parliament, namely that research was acceptable from an ethical standpoint up to the 14th day of gestation, provided that a number of conditions were met. Those conditions included informed consent on the part of the embryo donors and evidence that the goals of the research could not be met by methods other than those that required use of an embryo.

The policy outlined by the EAB report and the recommendations it contained were never implemented. Within months of the report’s having been issued, a new president (Ronald Reagan) was elected, and a Republican administration opposed to embryo research replaced a Democratic administration that had been generally supportive of such research. Although the Reagan administration did not enact any new legislation on embryo research, by its refusal to continue support of the EAB and its recommendations, a de facto prohibition on embryo research was established. This restriction on embryo research remained in effect over the next 12 years, through the administrations of presidents Reagan and George H. W. Bush. Throughout this period, a number of scientists and politicians continued to push for an implementation of the EAB recommendations and a restoration of the board itself. In early 1988, for example, James Wyngaarden, director of the National Institutes of Health, appointed a committee to consider the ethical issues involved in research on the use of fetal tissue transplants for the treatment of certain diseases. The committee issued its report in December 1988 and, by a vote of 19-2, recommended that the federal government renew its funding of research on fetal tissue transplantation. The Bush administration decided not to implement that recommendation, and the de facto ban on research involving embryos and fetuses continued.

During the 12-year moratorium, opposition to the ban on fetal tissue research also continued to grow in Congress. By the last year of Bush’s term, in 1992, majorities in both the House and the Senate supported an overturn of the ban and legislation to that effect was passed in both bodies. Bush vetoed the legislation, however, and his veto was upheld in the House of Representatives. Only with the election of a new president, Bill Clinton, in 1992 was there once more a reversal of fortunes for embryonic and fetal research. One of Clinton’s first official acts was to issue a presidential memorandum on January 22, 1993, revoking the existing de facto ban on fetal and embryonic research. Simultaneously, Democratic leaders of the Senate and House of Representatives introduced legislation designed to codify the president’s action and to authorize the expenditure of federal funds for such research. These bills were eventually passed as the National Institutes of Health Revitalization Act of 1993 and signed by the president on June 10, 1993, after which they became codified as Public Law 103-43.

The treatment of fetal and embryonic research were handled in a somewhat indirect manner in the act. Rather than discussing the ethical issues of such research, S.1 and H.R. 4 simply eliminated provisions for the EAB, essentially removing any requirements for federal review of fetal and embryonic research. In response to this change, Harold Varmus, director of the NIH, appointed a new committee, the Human Embryo Research Panel (HERP), charged with developing standards for determining which fetal and embryonic research projects could ethically be supported with federal funds and which could not. HERP issued its report on September 27, 1994, listing areas of research that it regarded as acceptable and unacceptable. Among the approved activities was the creation of human embryos specifically for the purpose of experimentation. Two months later, on December 1 and 2, the Advisory Committee to the Director for NIH met to consider HERP’s recommendations. The committee approved all of those recommendations and forwarded them to Varmus.

Having heard about the NIH and HERP proposed actions, however, President Clinton intervened in the process on December 2 and issued an order prohibiting approval of the made-for-research embryo section of the committee’s report. “I do not believe,” he said, “that federal funds should be used to support the creation of human embryos for research purposes, and I have directed that NIH not allocate any resources for such research.” Except for the provision to which Clinton objected, Varmus accepted the HERP report and began the process of implementing its remaining provisions. He announced that, among other activities, the federal government would begin funding research involving so-called surplus blastocysts produced for in vitro fertilization.

Varmus’s announcement soon became moot. In 1995, Representative Jay Dickey (R-Ark.) proposed a rider (an amendment) to the appropriation bill for the Department of Health and Human Services. The rider prohibited the use of federal funds for “the creation of a human embryo or embryos for research purposes; or research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero . . .” That rider has been passed every year since 1995 as part of the appropriations act for the Department of Health and Human Services, under which the NIH is funded.

Current law and policy in the United States, unlike that in most other nations, has evolved not through the adoption of legislation developed through debates and discussions among scientists, politicians, and ordinary citizens within and outside federal legislative bodies, but as secondary actions (such as presidential memoranda and riders attached to appropriations bills) on an ad hoc basis in response to immediate problems. All of which is not to say that efforts have not been made along those lines, efforts to take the pulse of all interested observers in the stem cell de bate in an effort to understand the issues involved in stem cell research and the kinds of legislative and other actions that might be necessary to develop a coherent national policy. For example, on October 3, 1995, President Clinton issued Executive Order 12975, creating the National Bioethics Advisory Commission with two primary functions: to “provide advice and make recommendations to the National Science and Technology Council and to other appropriate government entities regarding the . . . appropriateness of departmental, agency, or other governmental programs, policies, assignments, missions, guidelines, and regulations as they relate to bioethical issues arising from research on human biology and behavior”; and to “identify broad principles to govern the ethical conduct of research, citing specific projects only as illustrations for such principles.” During its fiveyear existence, the commission issued a number of reports on bioethical issues, including the cloning of human beings, research involving persons with mental disorders, ethical issues and policy principles involving human biological materials, ethical issues in human stem cell research, and ethical and policy issues in research involving human participants. The report on stem cell research has long been one of the most comprehensive and thorough discussions of social, legal, ethical, and religious issues surrounding stem cell research yet produced. When the commission’s charter expired in 2001, it was not renewed by the incoming Bush administration.

The nearly simultaneous announcements in November 1998 by researchers at the University of Wisconsin and the Johns Hopkins University of the discovery of human embryonic stem cells raised an important new issue for federal regulators. The revolutionary promise of these discoveries was obvious to everyone concerned with stem cell research, but a question remained as to whether the federal government was allowed by existing legislation and regulations to fund research involving human embryos. That question was put to the general counsel of the Department of Health and Human Services, Harriet Rabb, shortly after the Wisconsin and Johns Hopkins announcements were made. On January 15, 1999, Rabb issued her opinion. She concluded that research on human embryonic stem cells was eligible for federal funding because the cells themselves “are not a human embryo within the statutory definition.” That opinion was later criticized by opponents of human embryonic stem cell research as overly legalistic and relying on the precise letter of existing law and regulations rather than recognizing the spirit of those rules. In any case, these objections were moot because the issuance of federal regulations is a long process, and regulations for the funding of the controversial research were not issued until August 23, 2000. Only two months later, the nation had a new president, George W. Bush, with a strong objection to the funding of human embryonic stem cell research.

Bush was, of course, aware of the promise and the controversies surrounding stem cell research. Early in his administration, therefore, he established his own bioethics committee, modeled to some extent on the Clinton commission that had just been dissolved. The President’s Council on Bioethics was created on November 28, 2001, by Executive Order 13237. Its charge was fivefold:

1. “To undertake fundamental inquiry into the human and moral significance of developments in biomedical and behavioral science and technology.

2. To explore specific ethical and policy questions related to these developments.
3. To provide a forum for a national discussion of bioethical issues.
4. To facilitate a greater understanding of bioethical issues.
5. To explore possibilities for useful international collaboration on bioethical issues.”

As with its predecessors, the council has carried out studies and issued reports on a number of subjects, with issues of stem cell research receiving perhaps greater attention than in earlier years. In July 2002, for example, it published Human Cloning and Human Dignity: An Ethical Inquiry, an effort to explore issues involved in cloning practices for the purposes of producing humans and for producing embryos for research purposes. In the conclusion to its report, the commission noted that its members had been unanimously opposed to the use of cloning for human reproductive purposes, but had been divided almost equally between the approval of and opposition to cloning for the purpose of medical therapeutic purposes (seven members favored the practice, seven opposed the practice, and three favored a moratorium on the practice).

In January 2004, the commission produced another major report on stem cell research, Monitoring Stem Cell Research, a report with three major foci: the science and technology of stem cell research, with a review of recent developments in the field; the legal and policy history and current status of stem cell research in the United States; and ethical issues related to the practice of stem cell research. The report makes no policy recommendations and is most useful for the summary it provides of the science, ethics, and legal status of stem cell research. Also of special interest are 10 appendices consisting of commissioned papers on a number of specific topics, such as current progress in human embryonic stem cell research, adult stem cells, the biology of nuclear cloning and the potential of embryonic stem cells for transplantation therapy, and stem cells and tissue regeneration.

The council’s most recent report, Alternative Sources of Pluripotent Stem Cells: A White Paper, was issued in May 2005. It was prepared in response to the growing debate over the use of embryonic stem cells in research as an attempt to suggest sources of pluripotent stem cells that do not involve the death of a fertilized egg, blastocyst, or embryo. The four sections of the report consider the collection of pluripotent stem cells from organismically dead embryos, via blastomere extraction from living embryos, from biological artifacts, and by somatic cell dedifferentiation.

LEGISLATION AND PUBLIC POLICY

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Disagreements over ethical issues what ought individuals and societies do with regard to certain practices often evolve into political and legal disputes what should individuals and societies be allowed to do. Such has been the case in the debate over stem cell research. Governments around the world are now trying to decide how, if at all, they should attempt to monitor and control research on stem cells.

GREAT BRITAIN

Probably the earliest attempt to deal with bioethical issues that would later arise in the stem cell debate took place in Great Britain in the early 1980s. Questions as to the status of the human embryo and how it should be treated in scientific research came about shortly after and as a result of the birth of the first baby born as a result of in vitro fertilization, Louise Brown, in 1978. Parliament appointed a committee in July 1982 Уto examine the social, ethical, and legal implication of recent, and potential developments in the field of human assisted reproduction.Ф The committee was chaired by Dame Warnock and is, therefore, generally known as the Warnock Committee, and its final report as the Warnock Report.

The major recommendation of the committeeТs 103-page report, issued in July 1984, was that research should be permitted on human embryos during a period up to 14 days after fertilization, provided a number of conditions were observed. The committee also recommended the establishment of a supervisory body to monitor all such research in the country. Parliament was somewhat slow in acting on the Warnock CommitteeТs recommendation, but eventually passed the Human Fertilisation and Embryology Act in 1990, essentially adopting the committeeТs major recommendations. The act also created the Human Fertilisation and Embryology Authority (HFEA) to regulate in vitro fertilization and experimentation on human embryos. HFEAТs charter restricted it to the approval of activities that fall into one of six categories:

1. promoting advances in the treatment of infertility;
2. increasing knowledge about the causes of congenital disease;
3. increasing knowledge about the causes of miscarriages;
4. developing more effective techniques for contraception; and
5. developing methods for detecting the presence of gene or chromosome abnormalities in embryos before implantation; or
6. for such other purposes as may be specified in regulations.

The Human Fertilisation and Embryology Act had been in force for only a relatively short period of time before scientific developments began to raise new issues regarding the cloning of human embryos. One of the most significant of those developments was the cloning of a sheep, named Dolly, in 1996, by scientists at the Roslin Institute in Scotland under the direction of Ian Wilmut. Dolly was conceived by somatic cell nuclear transfer (SCNT), a form of reproduction not envisioned by the Warnock committee, not mentioned in its report, and hence not specifically covered by the Human Fertilisation and Embryology Act.

The birth of Dolly prompted the British government to take two additional actions to handle new issues raised by SCNT. First, the HFEA and the governmentТs Human Genetics Advisory Commission (HGAC) undertook a review of the governmentТs position on human cloning, requesting input from the scientific community and the general public. As a result of this review, the HFEA and HGAC recommended, among other things, that the Secretary of State for Health should add two new fields of research for which the HFEA might issue licenses: the development of treatments for mitochondrial disease and the development of treatments for diseased or damaged tissues or organs.

The government also created a new advisory committee in September 1999 under the chairmanship of Sir Liam Donaldson, the nationТs Chief Medical Officer. The committee was charged with reviewing developments in the field of in vitro fertilization and other kinds of assisted reproduction, determining potential benefits and risks of these developments, and recommending changes in the Human Fertilisation and Embryology Act of 1990 to meet these new findings. The report of the Donaldson committee, issued in June 2000, is important, among other reasons, because of its specific and detailed consideration of the growing significance of stem cell research, a topic that earlier committees had largely not had to deal with.

The Donaldson report agreed in principle with the governmentТs existing position on embryonic research, as expressed in the Human Fertilisation and Embryology Act of 1990 and the policies adopted by the HFEA. It did make nine recommendations for changes in these policies, most notably the addition of three new fields in which embryonic research should be permitted and a firm restatement of the prohibition of cloning for the purposes of human reproduction. In response to the Donaldson report, the British Parliament enacted the Human Fertilisation and Embryology (Research Purposes) Regulations (HFER) of 2001 that added three categories of research for which the HFEA was responsible: increasing knowledge about the development of embryos, increasing knowledge about serious disease, and enabling any such knowledge to be applied in developing treatments for serious disease. The new regulations went into effect on January 31, 2001.

The deliberations within Parliament on the status of the human embryo were, of course, of considerable interest to a number of groups whose position was that human life begins at conception. For such groups, the Human Fertilisation and Embryology Act and related legislation and regulations were incorrect because they authorized the destruction of human life. Passage of the HFER of 2001 prompted one of those groups, the ProLife Alliance, to ask for a judicial review of the governmentТs position on embryonic research. On November 15, 2001, the British High Court ruled that embryos created by SCNT were not regulated by the new HFER because they were not created by fertilization of an egg by a sperm.

The courtТs ruling raised a host of new questions about the governmentТs system of regulating human embryo research. It meant that a possibility existed that the cloning of humans for reproductive purposes using SCNT might be permitted and beyond the reach of existing regulations. As a spokesperson for ProLife said, The Human Fertilisation and Embryology Act 1990 is now in tatters . . . the Human Fertilisation and Embryology Authority, which has become the mouthpiece of vested biotechnology interests, has been seriously undermined, and the GovernmentТs gross incompetence has been exposed.

The government responded immediately. It announced that legislation designed specifically to prohibit human cloning would be introduced and that the High CourtТs decision would be appealed to the Appeals Court. The promised legislation was rushed through Parliament, introduced on November 21, 2001, and passed into law on December 4, 2001. Only a month later, the Appeals Court overturned the High CourtТs decision and restored the original intent of the Human Fertilisation and Embryology acts.

Still, the story was not complete. At this point, ProLife appealed its case to the House of Lords, asking that it rule against existing policies. In response, the House of Lords appointed yet another committee to investigate the social, ethical, and legal implications of embryonic research. That committee issued its report on February 13, 2002, once more essentially upholding government policies and practices on cloning, in vitro fertilization, and embryonic stem cell research developed over the previous two decades. Among its 27 recommendations, perhaps the most important were:

- To ensure maximum medical benefit it is necessary to keep both routes to therapy [using adult and embryonic stem cells] open at present since neither alone is likely to meet all therapeutic needs.
- For the full therapeutic potential of stem cells, both adult and ES, to be realised, fundamental research on ES cells is necessary, particularly to understand the processes of cell differentiation and dedifferentiation.
- Whilst respecting the deeply held views of those who regard any research involving the destruction of a human embryo as wrong and having weighed the ethical arguments carefully, the Committee is not persuaded, especially in the context of the current law and social attitudes, that all research on early human embryos should be prohibited
- Fourteen days should remain the limit for research on early embryos
- Embryos should not be created specifically for research purposes unless there is a demonstrable and exceptional need which cannot be met by the use of surplus embryos
- Although there is a clear distinction between an IVF embryo and an embryo produced by CNR [SCNT] (or other methods) in their method of production, the Committee does not see any ethical difference in their use for research purposes up to the 14 days limit
- УThe Committee unreservedly endorses the legislative prohibition on reproductive cloning now contained in the Human Reproductive Cloning Act 2001

As is apparent from the preceding summary, British policies regarding in vitro fertilization, cloning, and stem cell research have remained consistent over the past two decades. They encourage and support, both financially and in terms of policy, the use of human embryos developed by a variety of methods in research up to about the 14th day following gestation, and they strongly and unequivocally oppose the cloning of embryos for the purpose of human reproduction.

Technical Concerns with Embryonic Stem Cell Research

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Research involving embryonic stem cells, like that with adult stem cells, has experienced some exciting breakthroughs in the past decade. Some of the most important of these discoveries have been in the area of technique and methodology. In March 2005, for example, researchers at the University of California-San Diego (UCSD) School of Medicine reported that they had developed a method for maintaining embryonic stem cells in a proliferative state without the use of feeder cells. The UCSD scientists added a human protein called activin A to an embryonic stem cell line culture and found that the culture survived and divided as efficiently as it had using more traditional (usually murine) feeder layers. This result is important because the use of either nonhuman or human feeder cells introduces the possibility that proliferating embryonic stem cells may become contaminated by those feeder cells, making them useless for transplantation into a human subject.

Only months earlier, a second group of UCSD researchers had described another methodological development in the use of embryonic stem cells. They reported initial progress in coaxing such cells to differentiate along one or more lines of specialization into liver, adipose, nerve, skin, or some other type of mature cell. The news was important because the use of embryonic stem cells in regenerative medicine and other applications depends on just this ability of directing the differentiation of cells in some specific direction.

Researchers have also reported success in using embryonic stem cells to treat certain medical conditions and disorders, usually in experimental animals In January 2005, for instance, a research team at the University of Wisconsin–Madison’s Stem Cell Research Program headed by Su-Chun Zhang reported that they had coaxed human embryonic stem cells to become spinal motor neurons, nerve cells that control movement.73 This accomplishment is one of the first steps needed for using embryonic stem cells in transplantations to treat diseases of the nervous system, such as amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease).

In a study that represents a potential second step in such treatments, researchers led by Hans Keirstead at the University of California–Irvine’s Reeve-Irvine Research Center reported in May 2005 that they had treated rats with spinal cord injuries with human embryonic stem cells and observed significant improvement in the rats’ motor skills. Rats who had been injured up to seven days before treatment experienced nearly complete recovery after treatment with ES cells, although treatment was not effective with animals who had been injured many months prior to treatment. The principal investigator later said that “We’re very excited with these results. They underscore the great potential that stem cells have for treating human disease and injury.”

That level of enthusiasm is not uncommon among scientists working with embryonic stem cells. However, most researchers acknowledge that the bright hopes for using ES cells in regenerative medicine and other applications depends on solving a number of difficult technological problems. One of the most serious of those problems is the possibility that embryonic stem cells implanted into a patient may begin to proliferate and grow out of control, as happens during the development of an immature teratoma. Of course, scientists already know that such events have occurred in experimental animals following the implantation of embryonic stem cells, and the question remains as to how it can be prevented in humans during a therapeutic application. Uncontrolled growth of cells would result in the development of a cancer, which could be as or more serious a medical problem as the one being cured.

A second major problem relating to the use of embryonic stem cells is the possibility of rejection by a patient’s body. Any time cells, tissue, or organs are transplanted from one person to another person, the recipient’s body is likely to initiate an immune reaction against the implanted material. Cells in the transplanted material carry chemical markers on their surface that provide them with a unique identity. A recipient’s immune system is able to detect those markers and recognize that they do not come from its own body. It then initiates an autoimmune response that can cause serious damage to, and even kill, the patient. Again, the cure in such a case is worse than the disease being treated.

A third problem relates to maintaining control over the destination of stem cells implanted into a patient’s body. Suppose that scientists are able to find a way of coaxing embryonic stem cells into a particular type of specialized cell that is then injected into a patient to treat a disease. Can a scientist be certain that, once injected, those cells then travel to the appropriate place in the body (nerve cells to the nervous system; muscle cells to muscle tissue; skin cells to skin; and so on), or is it possible that they would migrate to inappropriate locations (nerve cells to muscle tissue or skin cells to the nervous system, for example)? Thus far, scientists have been unable to answer that question.

Technical problems like these may exist for adult stem cells also, but are likely to be less serious than they are for embryonic stem cells. The reason is that the very quality that makes embryonic stem cells desirable for so many medical applications their pluripotency also contributes to some of the problems that may be associated with their use in medical situations.

Advancing Adult Stem Cell Research

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Since the discovery of murine and, more especially, human embryonic stem cells at the end of the 20th century, many scientists and nonscientists have held high hopes for their potential applications in a number of areas, particularly regenerative medicine. Similar hopes for adult stem cells have been somewhat more restrained. The comparatively less enthusiastic claims for adult stem cells have been based on some fundamentally difficult technical problems associated with adult stem cell research. First, as noted above, adult stem cells are relatively rare in tissues and organs, present on average to the extent of about one adult stem cell for every 100,000 differentiated cells. Indeed, some question remains as to whether stem cell counterparts even exist for all 200-plus cells present in the human body.

Second, adult stem cells tend to have physical and chemical characteristics similar to their mature counterparts, making identification and extraction quite difficult. Third, adult stem cells are usually difficult to maintain in an undifferentiated state than are embryonic stem cells. Finally, evidence suggests that adult stem cells may be less plastic then embryonic stem cells, demonstrating multipotency rather than the pluripotency observed in embryonic stem cells. Problems such as these have made many researchers leery of the potential of adult stem cells to achieve the same practical applications expected of embryonic stem cells.

That presumption may be changing. Over the past decade, a number of researchers have been reporting success in identifying and extracting adult stem cells from tissues and organs, maintaining those cells in a proliferative, undifferentiated state for long periods of time, and using them to treat a variety of medical conditions. One of the most exhaustive reviews of this research65 reports in a number of murine and human systems, including bone marrow, peripheral blood systems, the nervous system, muscles, liver, pancreas, the cornea, salivary glands, skin, synovial membranes of the knee, heart, cartilage, thymus, teeth, adipose tissue, and umbilical cord and amniotic fluid. The reviewer concludes his article on adult stem cells by saying that “results from both animal studies and early human clinical trials indicate that they have significant capabilities for growth, repair, and regeneration of damaged cells and tissues in the body, akin to a built-in repair kit or maintenance crew that only needs activation and stimulation to accomplish repair of damage. The potential of adult stem cells to impact medicine in this respect is enormous.”

Reports of successful adult stem cell transplants in the treatment of murine and human disorders have appeared not only in the scientific literature, but also in the popular media and in testimony before Congress and other legislative bodies. For example, scientists at the New York Medical College in Valhalla announced in September 2003 that they had successfully treated a patient’s cardiac disease by transplanting cardiac progenitor cells from rats into the patient’s heart. The transplanted cells apparently stimulated stem cells present in the patient’s heart muscle, causing regeneration of the damaged cardiac tissue. The team’s leader, Piero Anversa, explained that “We have already identified where the stem cells reside and are developing strategies to mobilize them to migrate to the damaged cardiac site. . . . In time we will be developing Phase I clinical trial protocols for submission to the FDA.”

A recent study of particular interest on adult stem cells is one reported at the Scripps Research Institute, in La Jolla, California, in 2003 under the direction of Sheng Ding. The Scripps team discovered a synthetic chemical that has the ability to induce dedifferentiation in somatic cells, restoring their multipotency. Ding’s team named the chemical reversine because of its ability to reverse the normal process of cell maturation. In announcing the discovery, Ding predicted that “this [type of approach] has the potential to make stem cell research more practical. . . . This will allow you to derive stem-like cells from your own mature cells, avoiding the technical and ethical issues associated with embryonic stem cells.”

One of the most intriguing features about adult stem cell research in the past decade has been the extent to which science, politics, and religion have become so intertwined with each other. The reporting of scientific developments that may have an impact on human life without personal or institutional bias is always difficult. Describing the invention of a new kind of nuclear weapon, for example, without revealing one’s own biases about nuclear warfare is always a challenge for a writer. And such has often been the case in reports of developments in adult stem cell research.

When such reports appear in journals, magazines, or newspapers, or on web sites sponsored by religious or political groups with strong feelings about stem cell research, they may become more than just reports. They may become part of the arsenal a particular individual or group uses in arguing against or in support of stem cell research, in general, or embryonic stem cell research in particular.

Adult stem cell research has been a topic of particular interest to groups opposing embryonic stem cell research because it provides a realistic technological option (adult stem cell therapy) to a practice that they abhor (embryonic stem cell therapy). In some cases, such groups may actually sponsor research on adult stem cells,69 but more often, they enthusiastically report on every new breakthrough in adult stem cell research as part of their campaign against embryonic stem cell research, even when such research has yet to be confirmed by outside researchers. For example, one physician who opposes embryonic stem cell research has provided a review of research on adult stem cell research and concludes that “whoever would cure, must use adult stem cells,” and has described some scientists’ cautions about the problems to be solved in pursuing embryonic stem cell research as “restrained language used by established science to describe a truly disastrous set of results.”

The lesson to be learned from these stories is that scientific developments in stem cell research are sometimes presented in the popular media with a twist that reflects an author’s or sponsor’s own particular prejudice about the morality of embryonic stem cell research. That statement applies equally to supporters of and opponents to embryonic stem cell research. Neither side is likely to miss an opportunity to point out how results of scientific research that, in and of itself are ethically neutral, confirm its own position on this controversial issue.

The power that political and religious organizations may have in influencing the direction of scientific research is illustrated by the stand of one major health advocacy organization on stem cell research. The American Heart Association (AHA) is listed in Chapter 8 of this book as an organization that supports stem cell research. But the organization itself funds only adult stem cell research, not embryonic stem cell research. This decision, according to the AHA web page on stem cell research, was based not on the relative merits of both types of research or the science and technology involved, but on concerns about “the impact of funding embryonic stem cell research on volunteer and staff retention . . . and the potential adverse financial impact which would restrict the ability to fund other lifesaving research and programs.” Without question, every individual and every organization has the absolute right to choose which forms of research to support and which to oppose. Anyone interested in learning more about the subject of stem cell research should be alert for the difference between objective reports of ethically neutral scientific developments and the “spins” placed on those reports by groups with vested interests in one side or the other of the stem cell research controversy.

CURRENT ISSUES IN THE DEBATE OVER STEM CELL RESEARCH

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While much of the controversy over stem cell research today focuses on a single fundamental question When does human life begin? a number of other points of dispute exist. These points include questions such as complicity of researchers and legislators in the death of potential human lives for research purposes, maintaining “respect” for an embryo and other early living entities before birth, pursuing the promise of adult stem cell research, and recognizing and solving technical and other problems related to embryonic stem cell research.

Complicity

In a radio address on August 9, 2001, President George W. Bush explained that he would allow federal funding for stem cell research on certain existing embryonic stem cell lines that were already in existence. By this decision, he argued that the U.S. government would not be responsible for the destruction of human life (early stage embryos) for the purpose of conducting research, but such research could still continue. A number of theologians and philosophers pointed out what they saw as the flaw in that argument. According to the doctrine of complicity, government officials were guilty of cooperating in the murder of these embryos, they said, even if they did not take part in the actual act of destruction. The doctrine of complicity says that persons or institutions are guilty of a crime, even if they do not actually participate in the crime provided that they aid or abet that crime in some way.

Scholars often list four types of complicity. In the first case, one may actively participate in an immoral act, such as the destruction of an embryo, an entity that some would regard as a living human being. President Bush was certainly innocent of this type of complicity since he directed that the federal government not fund any research in which embryos are destroyed. The second type of complicity is more indirect. It occurs when a person provides support for some kind of immoral act, gives approval to or benefits from the act, or ignores the act. From this perspective, President Bush and researchers would be considered to be complicit with the murder of embryos since they knew about the destruction of the embryos, gave tacit support to the act by making use of the embryos in research, and, in some cases, benefitted financially or in some other way in the use of the embryos. A third type of complicity arises when one knows that an immoral act is about to occur and does nothing to prevent the act. Finally, a fourth type of complicity occurs when someone protects the perpetrator of an immoral act from the legal, moral, or other consequences of the act.

Clearly, the doctrine of complicity casts a very wide net among stem cell researchers. For those who agree with all four aspects of the doctrine, anyone who knows about, takes part in, benefits from, and/or does not act to prevent the destruction of an embryo is as guilty of the murder of the embryo for research purposes as the perpetrator of the act himself or herself. In brief, there is no moral justification for the destruction of any embryo under any circumstances. As one opponent of embryonic stem cell research has written:

“Even if NIH [National Institutes of Health] doesn’t grant funds to destroy human embryos, it is encouraging those who do by providing a venue for use of the stem cells. Even without the exchange of money, NIH is producing a “market” for those cells. Furthermore, moral complicity moves with the cells. That is to say, when NIH is standing with its arms outreached to receive embryonic stem cells from those who have destroyed embryos to obtain them, the moral guilt passes from one hand to the next. Those who destroyed the embryos are guilty of homicide (there’s nothing else to call it), and that guilt passes to those who knowingly use in their research cells obtained at the expense of embryonic life. The NIH guidelines, to put it quite bluntly, do no less than encourage and sanction the destruction of human embryos.”

Those who would encourage the use of embryonic stem cell research see the issue of complicity somewhat differently. In the first place, they may regard the embryo as a nonliving entity, so that the issue of murder or any other type of immoral act is simply not relevant here. Even if they agree that the embryo is alive, they suggest that researchers are guilty of a different type of complicity, something that ethicist John A. Robertson has called beneficial complicity. That term refers to the fact that the benefits achieved by using the fruits of an immoral act may be sufficiently great to excuse the person’s complicity. That is, if a researcher can bring relief to many people as a result of using embryonic stem cells, then his or her complicity in the destruction of the embryo from which those stem cells came is of less importance than the progress made. As one observer has written, “At some point, the doctrine of implied complicity in immoral acts must be replaced with reasoned compassion for the living.”

Thus far, as with so many other issues in the area of embryonic stem cell research, little progress has been made in resolving the differences among those concerned about the question of complicity in the use of embryonic stem cells.

Respect for the Embryo

Some people involved in the debate as to when human life begins have a somewhat easier position to state and defend: They say that life begins at conception, and that from that point on, whatever you call the evolving organism, it is as much a living human being as anyone who has already been born. It must be treated in precisely the same way as any living human being. People who argue that life begins at some later date, however, are confronted with the more difficult question as to how to treat the living, but not-yet-human, entity that develops from the zygote to the preimplantation embryo to the embryo to the fetus. With what kind of regard should scientists, legislators, and others view this on-the-way-to-becoming human?

The most common answer given to that question is to say that the growing entity does have the potential for life and it does possess certain qualities of humanness, even though it is not an individual human being. For this reason, these people say, the zygote and preimplantation embryo deserve respect. The phrase “the embryo deserves respect” occurs frequently in reports on stem cell research and the writings of both those who support SCR and those who oppose it. For example, the National Bioethics Advisory Commission in its report on Ethical Issues in Human Stem Cell Research concluded that “the embryo merits respect as a form of human life, but not the same level of respect accorded persons.”

The question, then, becomes what does the phrase really mean in a practical sense? Various scholars have given differing answers to this question.

The most common view seems to be that the preimplantation embryo deserves more respect than an inanimate object or a clump of living cells, but less respect than a fully formed human being. One should not, for example, buy and sell an embryo, the way one might buy or sell a piece of laboratory equipment. But showing respect to an embryo is still more complex. One exhaustive examination of this issue suggests that an important factor in showing respect to an embryo is to ensure that the research in which it is used will really provide sufficient benefit to justify the termination of the embryo’s survival. Another factor may be the viability of alternative methods of carrying out the research. Should adult stem cells eventually demonstrate their value as a substitute for embryonic stem cell in the development of medical therapies, then it would show respect to an embryo for it not to be destroyed for research. Also, the possibility of using the embryo for some other purpose, such as being adopted by an infertile couple, may override its use in an experiment, and respecting the embryo in that case would mean choosing adoption over experimentation.

For some scholars, the bottom line in the debate over respecting the embryo becomes, to some extent, a matter of terminology. When one acknowledges that a fully developed human being “deserves respect,” the term refers to a whole set of moral privileges due that person. But when one refers to the “respect” one gives an embryo, that term may have a more symbolic meaning, one in which we recognize the solemn value of humanness, but are willing to withhold a number of protections routinely afforded the fully formed human.

Christian Views Stem Cell

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As was the case in much of scientific theory, AristotleТs views on the beginning of life had a strong influence on Western thought over the next 1,500 years. For example, the great Catholic scholar Thomas Aquinas essentially adopted AristotleТs views and taught that ensoulment began after 40 days of gestation for males and 80 days for females. Prior to those times, the unborn organism was not yet human and, under most circumstances, abortion would have been permitted.

Christian doctrine in the early church was also influenced strongly by Judaic traditions, especially as expressed in the Old Testament. Those teachings were not always clear, but appear to have defined humanness as the state at which a child is fully formed, probably at the time of birth. Because of a mistranslation of this portion of the Old Testament, early Christians adopted the notion of the state of humanness as occurring when the fetus (rather than the child) became fully formed, and centuries of debate ensued in which an attempt was made to decide the moment at which that event occurs. That debate was reflected in contrasting positions taken by the church over the beginning of life and, hence, the legitimacy of abortion. In 1140, for example, canon lawyer Gratian compiled the first collection of church laws to be accepted by the papacy. In this collection, he stated that abortion was not prohibited until the fetus was formed. Although he provides no definition for that term, it is generally assumed to mean the point at which the fetus actually looks like a human being.

Confusion about the moment at which life begins continued within the Catholic church for another seven centuries. In 1588, for example, Pope Sixtus V announced that all abortions were prohibited, and anyone who performed or received one would be excommunicated. That ruling lasted only a short time, however, as SixtusТs successor, Pope Gregory IX restored doctrine to its previous status, permitting abortion on unformed fetuses without penalty.

Current Catholic doctrine was established in 1854 when Pope Pius IX announced the dogma of the Immaculate Conception. That announcement made necessary the corresponding decision, contrary to teachings then in place, that human life begins at the moment of conception. This view has remained essentially unchanged with the church since that time.

The teachings on which many opponents of stem cell research in the Roman Catholic church rely for their arguments are drawn from a number of encyclicals and other statements issued by Pope John Paul II during his 27-year reign and by various Vatican offices. For example, in his encyclical Evangelium Vitae, John Paul used biblical passages to confirm the view that human life begins at the moment of conception. He quotes Jeremiah 1:5 as one piece of evidence: Before I formed thee in the belly I knew thee; and before thou camest forth out of the womb I sanctified thee. This passage shows, John Paul writes, that:

Human life is sacred and inviolable at every moment of existence, including the initial phase which precedes birth. All human beings, from their mothers womb, belong to God who searches them and knows them, who forms them and knits them together with his own hands, who gazes on them when they are tiny shapeless embryos and already sees in them the adults of tomorrow whose days are numbered and whose vocation is even now written in the book of life (cf. Ps 139: 1, 1316). There too, when they are still in their mothersТ wombas many passages of the Bible bear witness they are the personal objects of GodТs loving and fatherly providence.

The Pope also alludes to LukeТs story of MaryТs meeting with Elizabeth, at which time ElizabethТs fetus Уleap[ed] in her womb in recognition of the holy virgin. This story can be interpreted, John Paul says, as Уindisputable recognition of the value of life from its very beginning. Based on his studies of scripture and church teachings, John Paul concludes in this encyclical that Уthe use of human embryos or fetuses as an object of experimentation constitutes a crime against their dignity as human beings who have a right to the same respect owed to a child once born, just as to every person. John Paul II and other officials of the Roman Catholic church, including the current Pope, Benedict XVI, have been among the strongest opponents of embryonic stem cell research since its beginning. Still, some Catholic theologians, philosophers, and ethicists hold views somewhat at variance with those of official church teachings. For example, in a presentation before the National Bioethics Advisory Commission in June 2000, Margaret A. Farley, Gilbert L. Stark Professor of Christian Ethics at the Yale Divinity School, pointed out that:

A growing number of Catholic moral theologians . . . do not consider the human embryo in its earliest stages (prior to the development of the primitive streak or to implantation) to constitute an individualized human entity with the settled inherent potential to become a human person. The moral status of the embryo is, therefore (in this view), not that of a person, and its use for certain kinds of research can be justified.

Catholics who express this view often point to two factors to support their position. The first factor is the long tradition of the church itself, outlined above, in which the time at which life is said to begin has been set at some time other than the moment of conception. Because of this history, these scholars say, reasonable Catholics can disagree as to whether the early embryo is truly human and deserving of the full protection afforded the postnatal child. The second factor is scienceТs changing understanding of human embryology. As research advances, scientists realize that the process of becoming human is a developmental process that takes place over many weeks and months and does not occur all at once. Hence, the fertilized egg, the preimplantation embryo, the implanted embryo, and the fetus may all have different levels of physical and, presumably, spiritual development. At an early stage of development, the organism may certainly be due respect, without deserving the full respect and legal protection afforded a living human being after his or her birth.

The Roman Catholic scholar Thomas A. Shannon has integrated this notion of a developing human person into his own analysis of the embryonic stem cell research debate. In one article, he draws on the teachings of medieval Scottish philosopher Duns Scotus (ca. 12661308) who distinguished between a Уcommon nature shared by all humans and an individual nature that makes each person the distinct individual that he or she is. The early embryo certainly has the Уcommon nature qualities shared by all human beings, Shannon says, but it only develops its individual qualities that make it a unique human being over time. For this reason, he concludes that presentation of human nature in the blastomere is preindividual and prepersonal. And because this is human nature and not individualized human nature (the minimal definition of personhood), I argue that cells from this entity could be used in research to obtain stem cells.

Most Catholic scholars reject contrarian views on embryonic stem cell research like those of Farley and Shannon. For example, John J. Conley, S.J., in an essay for the University Faculty for Life web site says that:

Those supporting the view outlined above have indeed discovered a certain truth: that the Catholic Church has no definitive position on ensoulment . . . . However, they have distorted this ambiguity and drawn moral conclusions wrongly deduced from this ambiguity. They have operated a strange reading of Church history and have used this truncated reading to legitimize political movements which the Church clearly and rightly condemns.

The views of other Christian denominations on embryonic stem cell research range widely from one of full support of the Roman Catholic view (and opposition to all embryonic stem cell research) to one of support for such research. For example, in the question and answer section of its web site, Father John Matugiak of the Orthodox Church in America explains that since Christianity accepts the fact that human life begins at conception, the extraction of stem cells from embryos, which involves the willful taking of human lifeЧthe embryo is human life and not just a Сclump of cellsТЧis considered morally and ethically wrong in every instance. Father Matugiak then directs readers to a longer explication of the churchТs views presented before the National Bioethics Advisory Commission on May 7, 1999, by Father Demetrios Demopulos, of the Holy Trinity Greek Orthodox church, in Fitchburg, Massachusetts.

Positions taken by Protestant denominations differ as widely as do the denominations themselves. At one end of the spectrum, members of the Southern Baptist Church adopted a resolution at their annual convention on June 16, 2001, in opposition to stem cell research involving the use of human embryos. The statement said that, while acknowledging that as evangelical Christians, Southern Baptists Уapplaud the relief of human suffering and attempts to cure disease, they Уobject strongly to the notion that pursuing cures for some ever justifies intentionally destroying other human lives to achieve those cures. The Lutheran Church-Missouri Synod has taken a similar stance, adopting a resolution at its 2001 annual convention stating that У[e]mbryos are not, as some claim, Сpotential human beings,Т nor are they physical entities absent a soul, but are totally and fully human in every way although in the early process of physical development. Therefore, the church declares that Уstem cell research involving the destruction of embryos be rejected as sinful and morally objectionable.

At the other end of the spectrum are resolutions such as those adopted by the United Church of Christ and the Episcopal Church. The former body adopted a resolution supporting embryonic stem cell research at its 2001 general synod in October 2001, the first Christian denomination to take such a stand. The churchТs resolution called for a letter to the president supporting embryonic SCR, recommended lobbying congressional committees responsible for the field of stem cell research, and requested local churches, associations, and conferences to work in support of such legislation. The Episcopal churchТs action came at its 74th annual convention in 2003, at which a resolution was adopted supporting the Уwider availability of embryonic stem cells for medical researchФ and encouraging the U.S. Congress to Уpass legislation that would authorize federal funding for derivation of and medical research on human embryonic stem cells that were generated for IVF and remain after fertilization procedures have been concluded, with a number of provisions added to safeguard the proper use of those stem cells.

WHEN DOES LIFE BEGIN?

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Various cultures and various religions have attempted to answer the question as to when life begins at different times in history. Discovering those answers, however, is often difficult. Societies frequently have laws and customs that provide clues as to the way they think about the origin of life. But those laws and customs tend to deal with related issues, such as abortion and infanticide, rather than the beginning of life itself. Thus, scholars may be forced to make inferences about beginning-of-life beliefs from limited anthropological and ethnographic data.

Many cultures do have specific standards for determining the moment at which one becomes human. In northern Ghana, for example, a child is said to become a human being seven days after birth. In some parts of rural Japan, УhumannessФ is said to occur when a child utters its first cry after birth. Among some Native American tribes, the tradition was that the moment of becoming human did not occur until a child began to suckle at its motherТs breast. And, in a somewhat extreme case, the Ayatals of Taiwan do not grant personhood to a person until he or she is given a name at the age of about two or three years.

As might be expected, the ancient Greeks were very much interested in the question as to when life begins, partly as a matter of philosophical speculation and partly because of legal issues, such as the legitimacy of abortion. The natural philosopher Plato, for example, argued that the human soul does not enter a personТs body until birth. In such a case, abortion could not be considered as murder since the unborn organism was not yet truly a human. In contrast, PlatoТs student Aristotle believed that the beginning of life is an ongoing process that occurs up to the moment of birth. The embryo and fetus pass through various evolutionary stages until the organism is ensouled and becomes a human. Aristotle, with his usual misogynistic bent, dated this moment at about 40 days after conception for males and 90 days for females.

OPPOSITION TO STEM CELL RESEARCH

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Support for stem cell research tends to be widespread. Most people seem to understand and accept the proposition that stem cell technology may provide a significant breakthrough in the treatment of diseases for which there are currently no effective cures. In public opinion polls, a majority of respondents support even the most controversial field of stem cell research, that which involves the use of embryonic stem cells. Respondents in those polls also tended to support the use of federal funds to support research on embryonic stem cells and to a relaxation of federal restrictions on such research.

Opposition to stem cell research tends to focus not on the use of adult stem cells, a practice about which there is relatively little controversy, but on the use of embryonic and fetal stem cells, a procedure to which a number of people have very strong objections. These objections are based primarily on the belief that life begins at the moment of conception, the moment at which sperm and egg fuse to form a zygote. That zygote and the blastocyst, embryo, and fetus into which it grows are, according to this belief, as much alive as any person walking on the Earth today. It is entitled to the same respect and legal protection provided to any living human.

The irony of the present debate over stem cell research, then, is that one of the most contentious, complex, and difficult issues facing the modern world arises out of one basic question: When does life begin? If life begins at conception, then society as a whole and researchers in particular must answer the following question: Is it justifiable to destroy one life (the zygote, blastocyst, or embryo) given the possibility that such an act may bring relief to and, possibly, save other lives? On the other hand, if life does not begin at conception, but at some later point, what is that point, and how does that fact affect the conduct of research on embryonic stem cells?

This question when does life begin? is so critical that the terminology used in talking about stem cell research is important. Indeed, it seems possible that more words have been written and spoken about terminology in the debate over stem cell research than for any other social issue of the present day. For example, opponents of embryonic stem cell research talk about “the killing of the most defenseless and innocent of human beings” and “destroying a living human embryo,” the first statement by Senator Sam Brownback of Kansas and the second, by President George W. Bush. By contrast, proponents of embryonic stem cell research and others who hope to conduct the debate in a different context may use scientific terms, such as zygote, blastocyst, or embryo, or quasi-scientific words, such as entity or preembryo.

The latter term has an especially interesting history. It was apparently first used in 1979 by American embryologist Clifford Grobstein, who apparently meant for the term to designate the entity that develops from a fertilized egg up to the point at which it implants, about 14 days after conception. Some later observers have suggested that Grobstein introduced the term, shortly after the birth of the first baby conceived by in vitro fertilization, in anticipation of the problems that were to come about in the use of such entities in future scientific research. The term has upset and frustrated opponents of embryonic stem cell research ever since, who argue that it represents a way of hiding from the general public the murder of unborn children for scientific research. One critic, for example, has written that:

The so-called preembryo is a false stage (period) of human development invented by an amphibian embryologist for political reasons, only. It has no credible scientific justification. Thus, the inclusion of this term into the language of Human Embryology has become a hoax of gigantic proportion. Adolph Hitler said: “The great masses of people . . . will more easily fall victims to a big lie than to a small one.”

And yet, the term preembryo has apparently received wide acceptance in the scientific and legal fields. Many of the court decisions discussed in Chapter 2, for example, make use of the term, as do a number of scholarly articles that discuss the legal and ethical issues associated with stem cell research. The lesson to be learned, then, is that the choice of words that one uses in talking about stem cell research is hardly an esoteric or academic matter. The language used in the debate can as easily be used to inflame passions and attempt to influence opinion as it is to develop ideas and explain positions.

APPLICATIONS OF STEM CELL RESEARCH

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The discoveries of murine and human embryonic stem cells by Evans and Kaufman in 1981 and by Thomson and Gearhart, independently in 1998, were greeted with great hope and optimism by scientists for a number of reasons. In the first place, these and related discoveries opened up the possibility that embryologists would have a much better opportunity to study one of the fundamental unsolved problems in their science: How does an organism grow, develop, and differentiate? The question as to the mechanism by which a single fertilized egg evolves into a complex complete organism has long been one of the most fundamental questions in biology. These discoveries provide scientists with the opportunity of examining the very earliest stages of a living entity, the stem cells from which the rest of the organism would eventually grow, and observing the changes that take place in those cells over time. By studying the differentiation of stem cells, biologists now have the opportunity of discovering those factors inherent within an organism itself and those from its surrounding environment that make possible and direct the evolution of a fertilized cell.

A second possible application of stem cell research is its use in drug development. Many steps must be completed before a new chemical compound can be approved as a drug. After the chemical itself has been invented and synthesized, it must be tested both for toxicity and for effectiveness. The testing process often takes many years, beginning with laboratory animals, such as rats and mice, before advancing to human tests. Stem cells can be used more easily and at less expense in place of rats, mice, and other experimental animals for the early stages of drug testing. Compounds found to be toxic to stem cells would then not be advanced to other stages of testing, saving significant amounts of time and money for a pharmaceutical firm.

The applications of stem cell research most often discussed are those in the field of regenerative medicine. The term regenerative medicine has been defined by one medical institution as “the regeneration or remodeling of tissue and organs for the purpose of repairing, replacing, maintaining, or enhancing organ function, as well as the engineering and growing of functional tissue substitutes in vitro for implantation in vivo as a biological replacement for damaged or diseased tissues and organs.” The use of stem cells in regenerative medicine is based on the fact that many medical problems arise because essential cells, tissues, or organs in the body are damaged or destroyed as the result of disease or injury.

For example, diabetes is a condition that develops when the body is no longer able to metabolize glucose properly. Glucose is a simple sugar, one of the essential fuels used by the body to produce energy. In a healthy body, cells called ?-cells are produced in the pancreas. ?-cells produce the hormone insulin, which is responsible for the regulation of glucose levels in the body. If ?-cells are damaged or destroyed, they are no longer able to produce insulin, or they produce insulin in insufficient quantities. Damage to ?-cells may occur at an early age, when the body’s immune system attacks and destroys those cells, a condition known as Type 1, or juvenile, diabetes. ?-cells may also begin to die off or lose their efficacy later in life, often as a result of a genetic error, a condition known as Type 2 diabetes. Spinal cord injuries are another example of damage that may be susceptible to treatment with stem cells. Spinal cord injuries occur as the result of automobile accidents or falls in which a person’s body is twisted or contorted in such a way that nerve cells are damaged or destroyed and connections among cells are interrupted. Such injuries present three kinds of problems. First, the damaged neurons (nerve cells) must themselves be repaired or replaced. Second, correct connections between neurons must be restored. Third, the protective coating that nerve cells normally have, called myelin sheaths, must also be reconstructed. These coatings act like the insulation on electrical wiring, preventing neural messages from flowing away from the nerve cells.

The hope among stem cell researchers is that embryonic and/or adult stem cells may be used in treating a variety of medical conditions caused by damage to cells, tissues, and organs such as those that occur in diabetes and spinal cord injuries. A general outline as to how such treatments would be conducted is as follows: First, workers would obtain a source of stem cells, such as the blastocyst of a mouse, rat, or human. Then, stem cells would be retrieved from the inner cell mass of the blastocyst. Those embryonic stem cells would then be cultured in vitro, allowing them to proliferate to some stage. At some point, the stem cells would be coaxed into differentiating into some specific type of cell, such as those in the pancreas responsible for the production of ?-cells or the cells responsible for the production of new neurons or myelin sheaths. These differentiated cells would then be injected into the “patient,” whether it be a rat, mouse, or human with some type of medical problem. Those cells would then, presumably, migrate to the proper location in the host animal and begin carrying out the function for which they are programmed (making ?-cells, neurons, or myelin sheath material) or stimulate existing cells in the tissue or organ to initiate the body’s own repair of its damaged part.

This brief description ignores the numerous difficult technical problems still to be solved in carrying out such a procedure. For example, scientists currently have very little idea as to how to make an embryonic stem cell begin differentiating into some desired type of mature cell. Also, they are uncertain as to how likely it is that a transplanted cell will find its way to the precise location in the body and begin operating the way a natural cell in that tissue or organ operates. Also, a number of questions remain as to possible undesirable side effects that might result from such a procedure. What is to prevent a transplanted cell, for example, from wandering away from the injection site to a totally inappropriate organ, such as a ?-cell to the brain? The fact that numerous problems remain to be solved does not mean that scientists have no hope for this procedure, however. Indeed, many new areas of science, like stem cell research, pose apparently limitless and, sometimes, unsolvable problems for researchers. Given enough time, however, many if not all of those problems may eventually be solved.

At this point in history, then, the question about stem cell research might be whether there is any evidence that transplantation of stem cells shows any promise in regenerative medicine. The answer to that question is probably yes. In June 2001, the National Institutes of Health (NIH) issued a report, Stem Cells: Scientific Progress and Future Research Directions, describing the use of stem cells in regenerative medicine and outlining some of the progress that had been made thus far in the field. Individual chapters discussed progress in the treatment of autoimmune disorders, diabetes, disorders of the nervous system, and cardiac problems. In each case, authors of the report were able to find examples of preliminary research that might eventually lead to the use of stem cells in regenerative problems.

In one experiment, for example, a team of researchers led by Ron McKay at the National Institutes of Health’s National Institute of Neurological Disorders and Stroke, in Bethesda, Maryland, attempted to cure diabetes in mice with murine embryonic stem cells. They cultured those cells until they formed embryoid bodies, clumps of cells that develop when stem cells aggregate with each other during the process of cell culturing. They then selected from the embryoid bodies a certain subset of cells that had some of the characteristics of ?-cells. They passed those cells through five culturings until they began to show some of the physical characteristics of the portion of the pancreas where ?-cells are produced, the islet of Langerhans. When treated with a solution of glucose, those cells secreted insulin, the way a normal pancreas would. When injected into diabetic mice, however, they appeared to have no effects on the symptoms of the disease. Typical of research on any new problem in science, this study was able to demonstrate one small step forward, in spite of its falling short of a complete success.

A similar report was issued by a group of scientists led by Douglas Kerr at Johns Hopkins University in 2001. In this case, researchers studied the effect of injecting barely differentiated embryonic germ cells into rats with spinal cord injury. The injury was induced in the rats by injecting them with the Sinabis virus, which attacks and destroys motor neurons in the spinal cord. Rats that survive the treatment display greater or lesser restrictions on their ability to walk and move around. Kerr’s team began with a culture of embryonic germ cells that they allowed to proliferate until it just began to differentiate. At that point, they identified and extracted a subset of cells that showed chemical markers characteristic of nerve cells. They allowed those cells to develop to embryoid bodies and then injected them into the spinal fluid of the injured rats. Three months after the treatment, rats that had been injected with stem cells were moving significantly better than those who had not received the treatment.

The problem with these results was that researchers were not able to identify the reason for the rats’ improvement. On the one hand, the injected cells may have behaved as expected, reproduced, and generated enough new neurons to make up for those killed by the Sinabis virus. On the other hand, the cells may have in some way stimulated existing cells remaining in the rats’ spines, causing them to take over the job of repair. In either case, the results were the same and were apparently the result of the cell transplantation procedure. Authors of the NIH study concluded their report with the observation that “[i]nvestigators have shown that differentiated cells generated from both adult and embryonic stem cells can repair or replace damaged cells and tissues in animal studies.” The problem thus far has been, however, that “there have been very few studies that compare various stem cell lines with each other,” making it unclear as to the precise reason that certain results are observed in each experiment. “Predicting the future of stem cell applications,” they conclude, “is impossible, particularly given the very early stage of the science of stem cell biology. To date, it is impossible to predict which stem cells those derived from the embryo, the fetus, or the adult or which methods for manipulating the cells, will best meet the needs of basic research and clinical applications. The answers clearly lie in conducting more research.”

A REVIEW OF STEM CELL SCIENCE

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A great deal still needs to be learned about stem cells before scientists can start achieving the hopes and expectations expressed for them. Still, scientists have learned a great deal about stem cells since they became a major focus of research attention only two decades ago.

First, as pointed out earlier, it is known that a stem cell is characterized by two essential properties: its ability to proliferate repeatedly, over many months or years, without differentiating into a specialized cell; and its tendency to begin differentiation when provided with the proper environment and/or stimulus. Stem cells are also characterized by their plasticity, that is, their ability to differentiate into other kinds of cells. A fertilized egg and a blastomere (a cell formed after a fertilized egg has undergone division, but before a blastocyst has formed) are said to be totipotent because they can differentiate to produce any cell in the body. An embryonic stem cell, by contrast, is classified as pluripotent because it can make nearly all kinds of specialized cells. Adult stem cells, like those found in bone marrow, tend to be multipotent because they can make more than one, but not a large number, of specialized cells.

Stem cells are apparently unique among cells in respect to the way in which they can undergo mitosis (cell division). They apparently can following any one of three mitotic paths:

1. The parent cell can divide to produce two daughter stem cells.
2. The parent cell can divide to produce one daughter stem cell and one differentiated cell.
3. The parent cell can divide to produce two differentiated cells.

Stem cells are named and classified according to their origin:

- Embryonic stem (ES) cells are found in the inner cell mass (ICM) of the blastocyst.
- Embryonal carcinoma (EC) cells are obtained from immature teratomas.
- Embryonic germ (EG) cells are derived from primordial germ cells, present in the gonadal ridge of a fetus. EG cells normally develop into mature gametes (eggs and sperm).
- Adult stem cells are undifferentiated cells present in mature tissue and organs, dispersed among the differentiated cells that make up that tissue or organ. Adult stem cells are uncommon, usually present in a concentration of about one stem cell for every 100,000 specialized cells. Evidence suggests that adult stem cells tend to be somewhat less plastic, less capable of differentiating, than are embryonic stem cells or embryonal carcinoma cells. In recent years, scientists have begun to use the term somatic stem cell as a preferred synonym for adult stem cell.
- In addition to these, there are stem cells with characteristics similar to those of adult stem cells that are found in blood remaining in the umbilical cord that is usually discarded after the birth of a baby.

The behavior of stem cells is highly dependent on the environment in which they are placed. One major line of stem cell research has been the search for environments that discourage stem cells from differentiating, while encouraging them to proliferate. Experimental environments that meet this criterion are called feeder cells or feeder layers. Historically, the most common feeder layers have consisted of mouse cells (specifically, embryonic fibroblasts, an early form of connective tissue) irradiated to prevent them from growing or developing. In recent years, irradiated human cells have also proved to be successful as the basis for feeder layers. A feeder layer provides a surface to which stem cells can attach, and cells in the feeder layer release nutrients for the stem cells.

A second major line of research has had the opposite goal: to find environments that coax stem cells to differentiate into one or another kind of specialized cell. Some substances that have been found to be effective in directing the differentiation of stem cells are listed in the table found in Appendix A.

Stem cells are also seldom easy to identify by visual examination alone. They tend to look like other kinds of differentiated cells and are, therefore, usually identified by indirect means, such as by chemical markers that exist on their outside surfaces or by chemicals that they secrete. This has important practical significance because researchers often have a problem distinguishing stem cells from specialized cells that occur together within a tissue or an organ, making it difficult to decide what role, if any, the putative stem cell plays in the tissue or organ.

In addition, the plasticity of adult stem cells has become a very important line of research in recent years. Researchers are eager to find out how much potential for use in regenerative medicine and other applications adult stem cells have. From a practical standpoint, most (if not all) of the ethical objections to embryonic stem cell research can be avoided if researchers use only adult stem cells, and not embryonic stem cells. A growing body of evidence suggests that, in contrast to prevailing wisdom, adult stem cells may have some of the plasticity once thought to be a property of embryonic stem cells only.

For example, some researchers now believe that some adult stem cells may have the ability to dedifferentiate. Dedifferentiation is the process by which a unipotent, mature cell somehow reverts to a more primitive multipotent or pluripotent form. Questions as to whether an adult stem cell can actually dedifferentiate; if so, which ones can do so; and how the conversion occurs are currently the subject of intense study. If adult stem cells can be made to dedifferentiate into stemlike cells, researchers will have a whole new source of such cells to use in their studies instead of embryonic stem cells.

Another line of research related to dedifferentiation is transdifferentiation, the process by which an adult stem cell from one kind of tissue differentiates into a cell of a different type of tissue. Some examples of transdifferentiation that have been reported include the conversion of hematopoietic stem cells into three kinds of brain cells, neurons, oligodendrocytes, and astrocytes; into skeletal muscle cells, cardiac muscle cells, and liver cells; and the differentiation of brain stem cells into blood cells and skeletal muscle cells. Although the process of coaxing an adult stem cell to transdifferentiate into a cell of another type appears to be very difficult, it does in theory provide another option to the use of embryonic stem cells for use in regenerative medicine and other applications.

SOMATIC CELL NUCLEAR TRANSFER

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For that reason, a discovery announced in 2005 by a team of researchers working at South Korea’s Hanyang University under the direction of Hwang Woo Suk was all the more startling and thought provoking. The South Korean team reported that they had created a number of stem cell lines carrying the DNA of a patient with a specific disease or injury capable of treatment by stem cell therapy. The announcement was important for two reasons. First, the stem cell lines were created without the use of a human embryo. Instead, researchers used a process known as somatic cell nuclear transfer (SCNT). That process involves the removal of the nucleus from one cell (the host cell) and the insertion of the nucleus that has been removed from a second cell (the donor cell). In the South Korean experiment, the donor cells were skin cells taken from a patient to be treated, and the host cell was an egg cell from which the nucleus had been removed.

The SCNT process may, for reasons that are not clear, stimulate the host cell to begin reproducing asexually in a process known as parthenogenesis. Parthenogenesis occurs naturally among certain types of animals and can be induced experimentally in a wider number of species. It results in the growth and development of the host cell (the egg) in a pattern indistinguishable from that associated with sexual reproduction that involves a sperm and egg. In the South Korean experiment, host cells were allowed to develop to about day six of gestation, at which point embryonic stem cells from the inner cell mass of the blastocysts were extracted. The second important point about the experiment was that the stem cells harvested by this process were clones—precise genetic matches—of individuals for whom treatment was needed. Should any of these stem cells have been transplanted into one of the patients, there would be no problem of rejection by the patient’s immune system because the stem cells would be identical to cells already present in the patient’s body. The problem of rejection of stem cells transplanted for medical therapy has been one of the technical questions troubling researchers.

Any hopes that the South Korean experiment would diminish the debate over the use of embryonic stem cells were, however, dashed when comments on the research began to appear. On the one hand, many people saw great promise in the breakthrough that had been made. For example, Robert Schenken, president of the American Society for Reproductive Medicine, said: We applaud Professor Hwang and his colleagues on this stunning scientific advance. Research of this quality serves to motivate and excite us as researchers and as clinicians.

With this announcement the fulfillment of the incredible promise that stem cell research will lead to treatments for some of the most disabling illnesses is that much closer.

Critics of embryonic stem cell research felt differently, however. For example, Richard Land, president of the Southern Baptist Convention’s commission on ethics and religious liberty said that “A cloned embryo is a human being. We should not be the kind of society that kills our tiniest human beings in order to seek a treatment for older and bigger human beings.” 26 The method of research developed by the South Korean research team has obviously not proved to be a solution to the debate over embryonic stem cell use.

ISOLATION OF STEM CELLS

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Even as Brinster and Mintz were completing their research on stem cell plasticity, other researchers were following another line of inquiry, perhaps the most fundamental problem of all: the isolation of an actual embryonic stem cell. Recall that Stevens had not discussed embryonic stem cells, per se. The cells he first found and worked with for most of his life were stem cells taken from immature teratomas, stem cells more precisely called embryonic carcinoma (EC) cells. Their name reflects the fact that EC cells are collected from a cancerous tumor, a teratocarcinoma, not from an actual embryo. Stevens had never been able to carry his research that far back into the embryonic development of mice. And although EC stem cells have all the characteristics of any other embryonic stem cell (the ability to proliferate endlessly and to differentiate at some point), they were not the “Holy Grail” for research for which scientists soon began searching after hearing of Stevens’s accomplishments.

The first researchers to achieve that goal were British geneticist Martin Evans and British anatomist Matthew Kaufman, both at the University of Cambridge, in the United Kingdom. In 1981, Evans and Kaufman announced that they had extracted murine (mouse) embryonic stem cells from the inner cell mass of blastocysts extracted from mice and suspended on culture in a petri dish.17 They then coaxed the stem cells to proliferate and differentiate both in vitro and in vivo. The term in vitro (literally “in glass”) refers to any process that takes place in an artificial environment, such as a test tube or a petri dish, while the term in vivo refers to any process that takes place within a living organism. Only five months later, a similar result was reported by Evans’s first postdoctoral student, Gail Martin, then professor of anatomy at the University of California at San Francisco School of Medicine. Martin had extracted stem cells from the inner cell mass of a blastocyst and then cultured them in a medium obtained from a preexisting teratoma culture. Like Evans and Kaufman, she was able to demonstrate the ability of her stem cells to proliferate and to differentiate. In her paper, she used the term embryonic stem cell to describe the cells with which she worked, apparently the first time that term was formally used in a scientific paper.18 The isolation of embryonic stem cells from mice was a huge step forward in the field of stem cell research. Perhaps most important, it set the stage for the breakthrough for which researchers were really waiting: the isolation of human embryonic stem cells (HSCs). Although that next step was conceptually easy the process of removing stem cells from a human embryo was technically not all that different from that of removing stem cells from a mouse—the challenges facing researchers were enormous.

In the first place, the technology for isolating stem cells from any type of mammal is staggering. The blastocyst is very small, no more than about 0.1 mm in diameter, considerably smaller than the period at the end of this sentence. Extraordinary skill and special equipment are needed to dissect a blastocyst and remove the stem cells contained within the inner cell mass. Other technical skills are required to transfer and work with the stem cells once they are removed and to maintain the cells in a nondifferentiating, proliferative culture.

Also, working with human embryos presents a host of moral and ethical questions not involved in research on mice, rats, and other animals. As long as a researcher wants to study murine embryos and stem cells, few people are likely to object or withhold research funding. But moving to a study of human embryos and stem cells is a very different matter. Many people believe that human life begins at the moment of conception, the moment a sperm cell penetrates an egg. In that context, the resulting zygote and the blastocyst into which it develops are both living humans on which one should not experiment.

Finally, even if one does not subscribe to that view and accepts research on the blastocyst as morally and ethically legitimate, there remains the problem of finding enough human embryos on which to conduct one’s research. Until the mid-1970s, there was virtually no way that a scientist could obtain a human blastocyst short of extracting it from a pregnant woman, which was virtually impossible to do. No matter the technical or ethical restrictions, then, human blastocyst research was nearly impossible, for all practical purposes, because of the limited supply of blastocysts. An important technological breakthrough in a field that was, at the time, totally unrelated to stem cell research, occurred in the late 1960s and early 1970s that dramatically changed this picture. In 1968, British physiologists Robert Edwards and Barry Bavister successfully fertilized a human egg with human sperm in a petri dish. The experiment provided, for the first time, the basic technology needed for in vitro fertilization (IVF), the combining of egg and sperm outside the human body in order to achieve reproduction of an organism. The successful application of that technology was not reported for another decade when, in 1978, Louise Brown, the world’s first “test-tube baby,” was born in Great Britain. Before long, interest in IVF among infertile couples began to grow rapidly and by the year 2002, the last year for which data are available, the Centers for Disease Control and Prevention reported that 45,751 children conceived by IVF technology were born in the United States in the preceding year.19 That trend was common in many parts of the world. In 2005, for example, the Japanese government reported that more than 100,000 babies had been born by means of IVF technology in that nation.

One side effect of the growing interest in the use of IVF among infertile couples has been the availability of “extra” (often known as “spare”) fertilized eggs. Typically, an IVF clinic attempts to produce more fertilized eggs than a couple is likely to need to achieve pregnancy. These fertilized eggs can then be stored in case the couple decides to attempt another pregnancy at a later date. Since the number of fertilized eggs is often greater than the number a couple needs and since many couples never choose to attempt a second pregnancy, IVF clinics often have large stocks of fertilized eggs that will never be used. According to one survey, more than 400,000 fertilized eggs are currently in storage in IVF clinics in the United States alone.21 In many cases, these fertilized eggs are simply stored until they are no longer viable (capable of developing into an embryo upon implantation), and then discarded.

For researchers who had developed the necessary technical skills for working with human blastocysts and who had no moral or ethical reluctance to working with such entities, the spread of IVF technology removed the last hurdle in the search for human embryonic stem cells. It had become only a matter of time before someone would match Evans and Kaufman’s and Martin’s success with mouse embryonic stem cells. When that breakthrough came in 1998, it was announced not by one, but by two research teams almost simultaneously.

The first report came from a team of researchers at the University of Wisconsin Madison Regional Primate Research Center under the direction of James A. Thomson.22 For their studies, the team used early stage embryos obtained by in vitro fertilization and donated to the researchers by the owners of the embryos. The embryos were cultured to the blastocyst stage, and then a total of 14 inner cell masses (ICMs) were isolated from those embryos. Of the 14 ICMs, five distinct embryos were produced, each of which was used to create a new stem cell line. Four of the five stem cell lines were maintained in a proliferative state for five to six months without change, and the fifth had been maintained for more than eight months at the time the report was issued. In their report, Thomson’s team describes the criteria and tests used to prove that the stem cell lines were indisputably human embryonic stem cell lines.

Less than a week later, the second report appeared in the Proceedings of the National Academy of Sciences, authored by a research team headed by John Gearhart at the Johns Hopkins University School of Medicine, in Baltimore, Maryland.23 Although the Wisconsin and Johns Hopkins teams used similar research protocols, they began with different materials. While Thomson had used fertilized eggs produced by IVF procedures, Gearhart obtained his stem cells from the immature gonads in fetuses that had been aborted. One difference in these two approaches was that Thomson’s research did not fall within the criteria that would have allowed federal funding of his project, while Gearhart’s did. In spite of that fact, both researchers chose to carry out their projects entirely with private funding, obtained in both cases from the Geron Corporation, in Menlo Park, California, to whom commercial rights for the discoveries were awarded.

News of the Thomson and Gearhart discoveries met with mixed responses. A number of people were delighted that this important breakthrough in stem cell research had been made. The chances of advancing stem cell therapy to the point where it could be used to treat a number of human diseases seemed, almost overnight, much more promising. Other people were less enthusiastic about Thomson’s and Gearhart’s achievements. They feared the pressure for manufacturing human embryos to be destroyed for research and medical therapy had also increased substantially. Enough politicians, religious leaders, scientists, and other people including President George W. Bush and a substantial number of federal and state legislators held this view to guarantee that, in the United States at least, questions would continue to be raised about and limitations placed on further stem cell research that used human embryos.

THE PLASTICITY OF STEM CELLS

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Over the next four decades, many of these questions (but by no means all of them) were answered by a relentless groups of researchers. Some clues to the amazing plasticity of embryonic stem cells first began to emerge in the 1970s, for example, with research by Beatrice Mintz, at the Institute for Cancer Research in Philadelphia, and Ralph L. Brinster, at the University of Pennsylvania, among others. Both researchers were interested in the fate of embryonic stem cells removed from teratomas and then transplanted into normal blastocysts.

Leroy Stevens had established the model for such experiments by transplanting embryonic stem cells into “unnatural” environments (environments where embryonic stem cells do not normally exist), such as the kidney or spleen of a mouse. He discovered that the transplanted stem cells seemed to go out of control, developing and differentiating rapidly and forming a mature teratoma. He concluded that the transplanted stem cells were reacting abnormally to having been placed into an environment that was somehow “wrong” for them.

Mintz and Brinster were interested in a somewhat different question: What happens when one removes embryonic stem cells from an immature teratoma and places them into a blastocyst, an environment that might be regarded as a “natural” location for embryonic stem cells? Both researchers reached a similar conclusion. In such circumstances, the embryonic stem cells were incorporated into the blastocyst and began to grow, develop, and differentiate naturally, migrating to various parts of the embryo and developing into normal tissues and organs. In everyday terminology, “cancerous” cells (cells taken from a cancerous tumor) had, when transplanted into the proper environment, grown and developed along with and just like healthy ordinary cells.

These experiments confirmed one of the key facts about stem cells. A stem cell, like all cells, consists of DNA molecules that carry instructions as to how that cell is to behave, what kind of cell it is eventually to become. Those instructions are stored in sections of the DNA molecule known as genes. But all of the genes in a DNA molecule are not “turned on” at the same time. In fact, in the first few days of a fertilized egg’s existence and in the stem cells that make up an immature teratoma, the only genes that are working are those that tell the cell to reproduce: divide, divide, divide are the only messages the cell receives from the genes in its DNA molecules. At some point, something happens to the DNA molecules in stem cells. In some cells, the genes responsible for making a fat molecule “wake up” and the cell begins acting like a fat cell. In other cells, the “neuron genes” get turned on and the cell starts to look like a nerve cell. In still other cells, the “skin genes” becomes activated, and the cell turns into a skin cell. What Brinster, Mintz, and their colleagues showed was that the DNA in stem cells was just waiting for the correct signals to activate genes. Once implanted into a normal embryo, they started receiving the messages they needed as to how they were supposed to differentiate, and they all ended up producing normal cells that migrated to the correct parts of the embryo’s growing body doing what they were supposed to do. They were never “bad” cells (even though they came from cancerous tumors). They had just never been given the correct environment or received the correct stimuli.

SOLVING THE STEM CELL PUZZLE

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Anyone familiar with the history of stem cell research must recognize the striking connections between Leroy Stevens’s research on teratomas and the work on monstrosities of his 19th-century predecessors. As one historian has said, “ES stem cells, in other words, were discovered as experimental counterparts of the teratocarcinoma—monstrosities, according to the language of nineteenth century biology.”

Still, Stevens’s research provided no more than an introduction to the study of stem cells. He had become convinced that such cells do exist, showed where they could be found in mice, discovered how they behaved in various kinds of tissues, and successfully established the first stem cell lines, populations of cells that continue to proliferate for very long periods of time without differentiating.

But Stevens’s successes only emphasized the research challenges that remained. After all, he (nor anyone else) had never actually seen a stem cell; a great deal more needed to be learned about maintaining stem cell lines; and no one knew very much about the conditions that caused stem cells to differentiate. The search for answers to those and related questions was to drive stem cell research through the 1960s to the present day.

TERATOMAS

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The third line of scientific inquiry that has led to the modern field of stem cell research is the study of teratomas. The term teratoma comes from two Greek words meaning “monstrous tumor.” Teratomas consist of large clumps of cells typically found in the ovaries or testes, although they may also occur in other parts of the body. They are classified as immature (malignant) and mature (benign) teratomas. The vast majority of cells that make up an immature teratoma are undifferentiated, with a virtually unlimited capacity for growth. Left untreated, an immature teratoma grows so large that it eventually kills its host. This characterization is the classical description of a cancerous tumor, and, for that reason, immature teratomas are also known as teratocarcinomas.

Immature teratomas are quite uncommon, found almost exclusively in humans and especially in adolescent boys and girls between the ages of 10 and 20. One of the most prominent cases of teratocarcinomas in recent years has been that of seven-time winner of the Tour de France bicycle race, American Lance Armstrong, whose condition was diagnosed early enough to permit a successful treatment of his teratocarcinoma.

Mature teratomas, also known as dermoid cysts, are benign tumors consisting of cells that have differentiated into a mixture of cells and tissues, including lipoid (fatty) tissue, neurons (nerve cells), bone, teeth, primitive eyes, hair, and other body structures derived from all three germ lines—ectoderm, endoderm, and mesoderm. Researchers have reported finding virtually all types of cells and tissues in mature teratomas, including partially formed eye sockets, groups of pulsating cardiac cells, and clumps of hair. The bizarre appearance of a mature teratoma under the microscope accounts for its having been named a “monstrous” tumor.

Teratomas have been known and studied for many centuries. The 19th century was a period of particularly intense research, as biologists gradually developed the skills to dissect and analyze teratomas. One of the premier teratologists of the early 19th century, French biologist Isidore Geoffroy Saint-Hilaire (1805–61), clearly understood the biological importance of teratomas or, as they were then known, “monstrosities,” beyond their basic appeal as biological peculiarities. Teratomas, he wrote, were a vital key to understanding the process of biological development itself. They are, in some respects, he wrote,

permanent embryos; they show us the emergence of simple organs just as in the first days of their formation; as if nature had halted its course in order to provide our too slow observation with the time and means to apprehend it. In the future, therefore, the science of monstrosity cannot be separated from embryogenesis; it will usefully contribute to its progress and will receive no less considerable services in return.

Unfortunately, the technical skills needed to follow up on this prediction were not available to Saint-Hilaire and his colleagues in the early 19th century. Indeed, it was more than half a century before the technology for the careful study of teratomas became possible and scientists began to develop a more thorough understanding of the composition, genesis, and development of teratomas.

Probably the leading exponent of the experimental study of teratomas at the end of the 19th century was French biologist Camille Dareste (1822–99), sometimes called “the founder of the experimental science of teratogeny.” Dareste posed the question as to whether the forms of development normally encountered—normal development, arrested growth, and excessive growth—are the only ones possible for an organism. Or, he asked, are there other ways an embryo might develop, provided the correct environment and stimuli are provided to direct that development? His research objective was to produce embryos with those environments and those stimuli to see how their development would be affected or, as he said, to make teratogeny “a science of all possible bodies” that might have an “unlimited variability” of forms. Saint-Hilaire and Dareste’s views of the role that could be placed by the study of teratomas in promoting the understanding of human embryology were remarkably prescient, even though little was to come of their musings for well over a half century.

In fact, the next episode in the scientific study of teratomas relating to the understanding of embryological development did not occur until the 1950s. Then, a series of quite remarkable discoveries began to emanate from the Roscoe B. Jackson Memorial Laboratory in Bar Harbor, Maine. The author of these discoveries was Leroy Stevens, a young researcher who had earned his Ph.D. in embryology at the University of Rochester in 1952 and then taken a job as a junior researcher at the Jackson lab.

Stevens’s initial assignment at Jackson was to explore the relationship between cigarette smoking and cancer, using a large population of mice bred at the Jackson laboratory specifically for research purposes. On one occasion, Stevens noticed that one of the mice in his laboratory had developed an enlarged scrotum. Assuming that a tumor might be present, Stevens sacrificed the mouse and dissected the scrotum. He was amazed to find a mature teratoma, one that contained both skeletal and cardiac cells, the latter beating in unison, as they would in a mature heart.

The remarkable point about Stevens’s discovery was that it was one of the few times that a teratoma had been found in a nonhuman organism, and the first time it had ever been seen in a male mouse. The fascination of studying teratomas struck Stevens in much the same way that it had many of his 19th-century predecessors, and he was to spend the rest of his working career (37 years) learning more about these intriguing structures.

The first challenge facing Stevens after finding the unexpected teratoma was to determine its source. After all, his primary research project was on locating the possible causes of cancer, one of which might well be a genetic defect in an organism. Stevens decided to look for a genetic defect in the strain of mice with which he was working (the now-famous strain 129) that might have led to the development of the teratoma. He followed this line by looking at male mice in the 19th day of gestation (19th day after fertilization; the day on which a mouse is born), then the 18th day, the 17th day, the 16th day, and so on. With each dissection, he hoped to find the earliest point at which evidence of a teratoma could be found.

Stevens was faced with a daunting task. The teratoma he had found, he discovered, was a rare occurrence among mice, even within strain 129, which appeared to have an unusually high propensity for the development of teratomas. In fact, he eventually discovered that only one in about 1,000 male mice developed such tumors, meaning that he was going to have to be very patient to work his way backward through the developmental history of many mice before uncovering his aberrant cell, provided it existed! Finally, 12 years after beginning his quest, Stevens was successful. In 1964, he found the earliest example of an aberrant cell leading to the development of a teratoma. He located an abnormal sperm cell growing along the genital ridge of a mouse embryo that eventually developed into a teratoma. Stevens named the aberrant cell a pluripotent embryonic stem cell, that is, a stem cell present in the embryo of the organism with the capability of developing into a wide range of other cells, including the fat, muscle, cardiac, nerve, skeletal, and hair cells and tissues that he had seen in the first teratoma dissected in 1952.

Over the next two decades, Stevens extended his research on teratomas and embryonic stem cells in an effort to learn as much as possible about them. His next step, after identifying pluripotent embryonic stem cells, was to demonstrate that they were the actual culprits in the formation of teratomas. He did so by transplanting tissue taken from the genital ridge of a young fetal mouse into the testis of an adult mouse. When he did so, he made two discoveries. First, he found that the transplanted tissue began to grow and eventually formed a teratoma, confirming the genital ridge as the source of the abnormal stem cells he had found. Second, he found that some of those cells did not differentiate into teeth, hair, muscle cells, and other specialized cells, but simply divided over and over again, remaining in their undifferentiated stem cell form. Stevens summarized these two conclusions in what has become one of the most famous papers in developmental biology: “Some of these cells continued to proliferate indefinitely and served as stem cells of transplantable teratocarcinomas composed of many kinds of tissues. Teratomas originate from a disorganized population of undifferentiated embryonic cells.”

REGENERATION IN THE HUMAN BODY

2010-02-16T07:43:00.000-08:00

The regeneration of human cells and tissues is also a familiar phenomenon to most people. Each time a person cuts a finger, for example, he or she knows that the cut will eventually heal as new skin grows over the wound. A few weeks after the cut, there is usually no evidence that injury ever occurred. Regeneration has occurred spontaneously to replace an essential body part: skin.

The regenerative process of perhaps greatest interest to biologists has long been the production of new blood cells in the body. Blood cells tend to have a very rapid turnover rate in the human body. Some kinds of white blood cells live no more than a few hours (although other types of white blood cells live much longer), while red blood cells die after about 120 days, and platelets survive only seven to 10 days. At these turnover rates, the average human body loses more than a billion blood cells a day. This loss means that the body must have a very active blood-making system, a system that has been found to lie in bone marrow, the spongy tissue that makes up the innermost core of bones.

Long before stem cells were hypothesized or found in any other part of the body, scientists were hypothesizing the existence of something like them in bone marrow, primitive cells that were capable of changing into red and white blood cells and platelets. For example, Russian hematologist Alexander Maximow (1874–1928) carried out a series of precise experiments during the first decade of the 20th century that led him to conclude that there exists a “common stem cell of different blood elements.” He thought that this stem cell was the lymphocyte, which, he wrote, “may look different according to the site of residence as well as to the local conditions and which can produce different products of cellular differentiation. The lymphocytes are ‘ubiquitar,’ of equal value everywhere.”

Maximow was wrong about the identity of the blood stem cell, but he was certainly correct in recognizing that some primitive type of blood-making cell existed. The fact that he had picked the wrong stem cell did not make much difference, however, as his work was largely ignored for more than 50 years. In fact, it was not until 1960 that the first real evidence for the existence of a hematopoietic stem cell was obtained. That evidence came from a research team working at the Ontario Cancer Institute in Toronto led by Ernest Armstrong McCulloch and James Edgar Till. McCulloch and Till were studying the effects produced by injecting mice with irradiated and nonirradiated bone marrow. They found no differences in the mice being treated but, incidentally, noted that some mice in both groups tended to develop nodules on their spleens. (Blood formation in mice occurs in both the spleen and in bone marrow.) These nodules were unexpected and, with further analysis, turned out to correspond in size and number with the amount of bone marrow transplanted into the mice. McCulloch and Till eventually concluded that the nodules were produced by the undifferentiated replication of a single “colony forming unit.” Before long, they recognized that this “colony forming unit” was, in fact, the hematopoietic stem cell for which hematologists had been looking for so long.

Not only had McCulloch, Till, and their colleagues actually found a stem cell for the first time, but they were also able to provide a definition for the term that has survived to the present day. In a 1963 paper on their discovery, they defined a stem cell as a cell that “should be capable of producing new stem cells like itself; otherwise, it would be on a dead-end street. And it should have the potential to produce different kinds of differentiated cells.”

At long last, a real breakthrough in stem cell research had occurred. The first cell to fit that definition a hematopoietic stem cell had been discovered. For at least some observers, then, “that, basically, is how stem cell science began.”

REGENERATION IN LOWER ANIMALS

2010-02-16T07:39:00.000-08:00

Many people who have grown up in contact with the natural world have learned that many kinds of animals salamanders and newts, starfish, segmented worms, zebrafish, hydras and other polyps, and tadpoles among them are capable of regenerating body parts lost in accidents or, in some cases, removed by curious children or inquisitive scientists. Some of the earliest controlled experiments on regeneration were conducted by French physicist Rene Antoine Ferchault de Reaumur (1683–1757), who is probably better known for his invention of the alcohol thermometer and a temperature scale named in his honor.

Reaumur’s interest in regeneration appears to have started as he walked through the marketplaces of Paris and noticed that many of the crayfish and crabs on sale there had claws of different sizes. He hypothesized that this phenomenon was due to the loss of one claw by the animals, which then regrew a substitute claw that had not yet reached its mate’s size when captured for the marketplace. In a paper presented to the French Academy of Sciences in 1712, Reaumur expressed regret that humans had not been granted the ability to regenerate limbs like that possessed by crayfish and crabs, but that they should be glad that nature had given them “a beautiful opportunity to admire her foresight” in providing the lower animals with this ability. Reaumur was hardly the only researcher to become interested in the process of regeneration. In fact, one modern scholar has observed that “almost everything that moved in Europe was amputated” by one scientist or another in order to learn more about the process. Reaumur’s work, for example, became an inspiration for Swiss scientist Abraham Trembley (1710–84), who took on the daunting task of dissecting a freshwater polyp now known as the hydra. Trembley’s careful experiments on the tiny hydra conducted well over two centuries ago have become classics in their field, providing some of the most exhaustive and detailed information about regeneration available for more than a century.

Most researchers were not satisfied simply to experiment on animals and observe regeneration. They also were eager to find an explanation for the phenomenon. By what mechanism did an organism as simple as a hydra “know” how to reconstruct an arm that had been removed? How did a small part of its body “know” how to regenerate all the other parts from which it had been removed? What “knowledge,” in particular, did these so-called “lower” animals have that, compared to the far more complex human bodies, to a considerable extent lacked?

These questions intrigued scientists for two primary reasons. First, they hoped that by finding answers to such questions, they might learn to help human bodies regenerate also. Ann Parson speculates that one motivation for Swiss biologist Charles Bonnet’s research on regeneration in salamanders— their abilities to regrow an eye, for example—may have been his own failing eyesight. Certainly other researchers of the time who were losing their sight, their hearing, or other mental faculties might have been excused for fantasizing about the possible applications of the process of regeneration not only to the advancement of science, but also to their own well-being, a dream often expressed in today’s world by those who see stem cell research as a way of solving medical problems.

Second, regeneration researchers also hoped to answer perhaps the most profound question in all of human biology: How does development occur? What are the processes by which a single fertilized egg grows and develops into all the different cells, tissues, and organism that make up a mature organism?

Most early researchers on regeneration attempted to use their experimental results to support one philosophical view or another about the mechanisms of human development. For example, German biologist Johann Friedrich Blumenbach (1752–1840) attributed to the freshwater polyps on which he experimented a life force that he called Bildungstrieb, or “formative drive.” Like many scientists of the time, Blumenbach believed that many natural phenomena could be explained only by the presence in plants, animals, minerals, and other materials of a “vital force,” which he himself admitted was an “occult quality” that could not be defined in scientific terms but served merely to “designate a peculiar power” to explain the phenomenon’s occurrence.

Interestingly enough, modern-day stem cell researchers interested in the process of regeneration find themselves in a situation similar to that of their 19th-century colleagues. They have accumulated large amounts of data about the role of stem cells in the regeneration of cells, tissues, and organs. But they are still largely ignorant of the mechanisms by which that process occurs. The terminology of the problem has changed, surely. Instead of trying to understand “vital forces,” researchers are attempting to learn how stem cells “know” how to make new cells and which ones to make. But discovering basic causes eludes modern scientists nearly to the extent that it did Reaumur, Trembley, Blumenbach, Bonnet, and their colleagues.

STEM CELLS: THE ALMOST CERTAIN UNCERTAINTY

2010-02-16T07:35:00.000-08:00

One of the fundamental problems in discussions of stem cell research is deciding precisely what the term stem cell means. According to one writer for the American Bioethics Advisory Committee, “the phrase ‘stem cell’ or ‘stem cells’ actually came from histologists [and] . . . has been in the histology texts for a long, long time.” Other writers have traced the use of the term to texts dating to the late 19th century, such as E. B. Wilson’s classic study, The Cell in Development and Heredity (republished by Garland Publishing in 1987), although with a variety of different meanings. Still, other researchers believe that they invented the term, or some variation of it. For example, a publicity release by the Jackson Laboratory in Bar Harbor, Maine, claims that one of its preeminent researchers, Leroy Stevens, was responsible for “the origin of the term ‘stem cell’” in 1970, while the writer Ann B. Parson thinks that credit for a modern definition of the term may go to researchers Ernest McCulloch and James Till who spoke and wrote about “stem cells” in their 1960s research on blood-forming cells found in the bone marrow.

Whatever the history of the term, many biologists have long been convinced that cells with the special properties now attributed to stem cells must exist, even if no one had ever seen one. The evidence for the existence of these types of cells falls into three major categories: the ability of many plants and animals to regenerate one or more body parts, the ability, in particular, of the human body to continually regenerate cells, tissues, and organs, such as blood cells, skin, and the lining of the gut, and the existence of teratomas, peculiar tumors that contain tissue and organs from all three embryonic germ layers, endoderm, mesoderm, and ectoderm.

AN INTRODUCTION TO STEM CELL RESEARCH AND ISSUES OVER ITS USE IN THE UNITED STATES

2010-02-16T07:28:00.000-08:00

Stem cell research has burst on the national political scene in the United States only within the last decade. Stem cells are special types of cells with the potential for developing into any one of the 200 or more different types of cells found in the human body: skin cells, nerve cells, muscle cells, liver cells, blood cells, and heart cells, for example. This potential raises the possibility that stem cells may be used in bringing about revolutionary changes in medicine, drug testing, and a number of other fields of research.

Although research on stem cells is not new—scientists have been studying them in one form or another for more than two decades—they have become a topic of political, scientific, and general interest only very recently, with the discovery of human embryonic stem cells in 1998. Some people saw these discoveries as heralding a new day in biological and medical research, foreshadowing the development of new materials and procedures that could bring relief to millions of people around the world suffering from diseases that are currently intractable of treatment by any other means. Other people viewed these discoveries with apprehension, recognizing that human embryonic stem cell research can proceed only with the destruction of human embryos, a practice they equated with the murder of human beings.

The debate over stem cell research has been waged on many fronts. Scientists, for example, disagree as to the relative potential value offered by different types of stem cells, those obtained from embryos, from fetuses, and from adult humans. By far the most acrimonious debates, however, have focused on ethical questions. Some people view the fertilized egg and the entity into which it develops over a period of days and weeks as a living human being, alive from the moment of conception. Other people acknowledge the “potential for life” in such entities, but do not accept their designation as fully and completely human. These differences define profound distinctions in the way both groups view research in which these very young entities are used.

The question as to how fertilized eggs, embryos, and fetuses are to be treated by researchers has been resolved in various ways at different times in varying parts of the world. The British Parliament, for example, appointed a committee to study issues surrounding embryonic research in 1985, a committee later to be called the Warnock committee after its chair, Dame Warnock. By 1990, Parliament had adopted legislation defining the conditions under which research on human embryos and fetuses could be conducted and had appointed a national agency, the Human Fertilisation and Embryology Agency, to oversee such research. While many other countries followed the British lead in adopting legislation on embryonic and fetal research over succeeding years, other countries, including the United States, did not. In those countries, policies on human research are often set by a combination of laws on related issues, administrative orders, regulations, and other quasi-legal directives.

Perhaps the most difficult problem about stem cell research is that it raises questions about fundamental beliefs about life and death, about what constitutes a human being. People tend not to be very flexible on such basic aspects of their personal system of ethics and morality, making compromises among competing arguments difficult. And that is the position of the debate over stem cell research in the United States today. The country operates under a general set of regulations set by President George W. Bush in a public address on August 9, 2001, restricting the types of stem cell research that can be funded by the federal government. Yet, much of the research prohibited by the president’s policies continues in the United States and other parts of the world, financed here by private funds and overseas by both private and public funds. And individual states, such as California, New Jersey, and Connecticut, have largely rejected the president’s arguments in support of his ban on research funding and have adopted legislation that allows state funds to be used for such research.

Thus, stem cell research of all kinds is occurring in the United States and other parts of the world, but under different systems of funding and different types of governmental control. It seems likely that the dispute over stem cell research will continue for the foreseeable future and that diverse approaches to the conduct of such research will also endure.

Stem cells are cells with two essential characteristics: the ability to proliferate—to continue reproducing without changing—for weeks, months, or years, and the tendency to undergo changes during which they differentiate into one or another of the more than 200 different kinds of cells of which the human body is made. The excitement about stem cell research (SCR) arises because of the seemingly endless number of applications it may have in biological research on the fundamental character of cells, in drug research and development, in medical therapeutics, and in other fields. The controversies that have developed in regard to some forms of stem cell research are based on the necessity of destroying entities that are regarded by many people as living organisms—fertilized eggs, embryos, and fetuses—in order to conduct such studies.

What is stem cell research? On what scientific principles are its potential applications based? What scientific challenges face researchers in the field? What ethical, legal, economic, and other questions are raised by stem cell research? These and related questions are the subject of this chapter.

Graft Versus Host Disease Prophylaxis And Acute

2010-02-16T02:41:00.000-08:00

Overview

Graft-versus-host disease (GVHD) is one of the classical complications of allogeneic stem cell transplantation. It is dependent on the presence of histocompatibility differences between the host and the donor. These can be minor antigens in the case of matched transplantation or major histocompatibility complex (MHC) antigens if there is some human leukocyte antigen (HLA) incompatibility. Minor antigens are presented to the T cells presumably in the same way that bacterial or viral antigens are presented to T cells. Thus, in essence, the graft is functioning as if there were a severe infection, and the graft tries to eradicate antigens that are intrinsic to the host. This results in the tissue damage that we clinically recognize as GVHD.

There are two main categories of GVHD, acute and chronic, each with two subcategories:

Classic acute GVHD
Persistent, recurrent, or late-onset acute GVHD
Chronic GVHD
Classic chronic GVHD

Previously, acute GVHD (aGVHD) was arbitrarily assigned to all allogeneic manifestations that occurred before day 100. Similarly cGVHD was the manifestations occurring after day 100. This distinction is no longer considered useful. It is now recognized that there can be late-onset aGVHD (frequently but not exclusively after reduced intensity transplantation) that looks clinically like aGVHD.

Moreover, there can be cGVHD that occurs early after transplantation, which is considered classic without features of aGVHD, and an overlap syndrome in which features of chronic and acute GVHD appear together.

Prophylactic Regimens

Effective prophylaxis is limited by an incomplete understanding of the pathophysiology of the disease. Traditionally, aGVHD is thought to be the most critical risk factor; therefore, the focus has been to prolong or intensify aGVHD prophylaxis to reduce the incidence of cGVHD. Prolonging the immune suppression has mixed results. Drugs are often used in combination in an attempt to block several pathways thought to cause GVHD. Use of calcineurin inhibitors in conjunction with methotrexate is the most common combined regimen. Institutions use a variety of combinations. See section on each agent for dose and toxicity.

1. Tacrolimus/methotrexate (MTX) or cyclosporine/MTX superior to single-agent prophylaxis. When calcineurin inhibitors are used with lower doses of MTX, so-called mini methotrexate may attenuate mucositis caused by MTX.

2. Tacrolimus/mycophenolate mofetil (MMF) or cyclosporine/ MMF. MMF is an alternative for patients who are unable to take MTX.

3. Sirolimus/tacrolimus/MTX or sirolimus/tacrolimus may allow for lower doses or the elimination of MTX.

Agents Used for Prophylaxis

Methotrexate

Mechanism of Action
Blocks the enzyme dihydrofolate reductase, which inhibits the conversion of folic acid to tetrahydrofolic acid, resulting in an inhibition of the key precursors of DNA, RNA, and cellular protein.

Metabolism
It is bound to serum albumin. Hepatic and intracellular metabolism. Half-life is 2 hours; 50 to 100% is renally excreted. Peak concentration is 3 to 12 hours after administration.

Dose
For GVHD prophylaxis (when combined with tacrolimus only): MTX 15 mg/m2 on day + 1, followed by 10 mg/m2 on days 3, 6, and 11. At Dana-Farber, if used with tacrolimus and rapamycin, it is given on days 1, 3, and 6 only (not day 11) and the dose is decreased to 5 mg/m2 (mini-MTX dosing).

These doses are too low for MTX levels to be of use. Studies demonstrate that skipping doses is associated with an increased risk of GVHD. Therefore, efforts to administer MTX while minimizing toxicity are warranted. Recent studies suggest that doses as low as 5 mg/m2 are effective. Redundant

Toxicity
It is important to assess patient for pleura or pericardial effusions, ascites, third-spacing or renal failure before each dose. MTX will exacerbate chemotherapy-induced mucositis and it may be necessary to hold or reduce the dose of MTX if there is airway compromise secondary to mucositis.

Methotrexate/Leucovorin Rescue Recommendations

It has been noted that certain patients receiving MTX for GVHD prophylaxis may be at higher risk for MTX-related toxicities including oral mucositis and possibly hepatic veno-occlusive disease (VOD). Neville et al. (1992)1 suggest that the use of leucovorin rescue may reduce these toxicities attributable to MTX and might allow accelerated engraftment as well without compromise in the MTX immunoprophylaxis against GVHD. (Note: This small study may have missed any compromise in effectiveness of GVHD prophylaxis.)

Patients believed to be at greatest risk for MTX-related toxicities (i.e., late engraftment, hepatic dysfunction, severe mucositis) are those with either decreased renal function (serum creatinine > 1.5 X baseline or > 2.0 mg/dL) or significant fluid collections, that is, ascites, pleural effusions, and so on where MTX can accumulate and thus delay MTX clearance. Hyperbilirubinemia is not an indication for leucovorin therapy, and leucovorin therapy is not indicated if the risk has resolved (e.g., creatinine level has improved or fluid collection has resolved).
Leucovorin rescue schema for patients with the high-risk features mentioned are as follows:

-- Twelve hours after day +1 MTX (15 mg/m2 ), give leucovorin 15 mg/m2 intravenous (IV) every 6 hours for three doses (max single dose = 30 mg)
-- Twelve hours after day +3 MTX (10 mg/m2 ), give leucovorin 10 mg/m2 IV every 6 hours for six doses (max dose = 25 mg/m2 )
-- Twenty four hours after subsequent doses of MTX, give leucovorin 10 mg/m2 IV every 6 hours for eight doses (max dose = 25 mg/m2 )

Note: If leucovorin is given, MTX levels should not be measured.

Calcineurin Inhibitors Tacrolimus and Cyclosporine
Both tacrolimus and cyclosporine (CSA) have a similar mechanism of action and toxicity. Tacrolimus is slightly more potent and has a more reliable area under the curve (AUC).

Mechanism of Action
Cyclosporine binds to cyclophyllin and tacrolimus binds to FKBP12. In either case,the complex inhibits calcineurin phosphatase. Calcineurin is a protein necessary for a number of cellular processes and calcium-dependent signal transduction pathways. These drugs inhibit activation and proliferation of T-lymphocytes by interfering with interleukin (IL)-2 production, and expression of IL-2 receptor. They interfere with cell cycle in G0 phase.

Tacrolimus (Prograf, FK-506)

-- Metabolism: CYP450–3A4 system in the liver and intestine. Ninety-two percent is excreted in bile. Absorption is decreased with food. Peak level varies when the drug is administered orally (1.5 to 3.5 hours), IV 1 to 2 hours. Half- life is 21 to 61 hours in healthy volunteers. Average half-life is 18 hours in HSCT patients. Half-life is prolonged with hepatic dysfunction.
-- Dose: 0.02 to 0.05 mg/kg IV (continuous over 24 hours). It is convenient to start on day –3 and continue until the patient is able to take oral medication. The IV to oral conversion can be approximated as the IVCI daily dose ? 3, divided into two doses/day. Levels need to be followed daily until they are stable. Standard oral dose used is 0.05 mg/kg every 12 hours rounded to the nearest 0.5 mg. Patients may require a lesser dose to maintain therapeutic levels depending on albumin levels and hematocrit. Drugs that alter P450 metabolism may increase or decrease levels of tacrolimus. Schedule and therapeutic levels will vary by protocol and institution. Levels of 5 to 10 ng/mL seem to be better tolerated in HSCT patients than higher levels. Note: Spuriously high levels will often be obtained if drawn from the line used for infusion of the drug. Confirm unsuspected or abnormally high levels with the level drawn from periphery.

-- Dose adjustments are based on clinical toxicity, blood levels, and GVHD. For supratherapeutic levels (between 10 and 15), the dose is decreased by 25% every 2 days. If level is >15 hold the dose until level drops to <10> male recipient, older patient age, older donor age, multiparous female donor, herpes simplex virus (HSV) or cytomegalovirus (CMV) seropositivity. Patients who develop aGVHD are at higher risk for developing cGVHD.

As noted earlier, aGVHD can be classic, acute, or persistent/ recurrent/late. Acute GVHD is characterized by skin, alimentary tract, and/or hepatic involvement.

Classic symptoms include maculopapular rash, nausea, vomiting, anorexia, profuse diarrhea, abdominal cramps, and ileus or cholestatic hepatitis and occur within 100 days of transplantation or DLI. Persistent/recurrent/late aGVHD include features of classic aGVHD (without cGVHD) occurring beyond 100 days post transplantation or DLI. Biopsy is often obtained to support the diagnosis.

Timing
Skin rash typically appears from day +5 to day +50 (or later) with a median time onset at day 20. In nonmyeloablative transplants, onset may occur later. Liver abnormalities often follow within several days. GI involvement is usually seen in the context of skin and liver GVHD but not always. Each organ can be involved in isolation or with any combination of the other two organs.

Histopathology

-- Skin – biopsy is useful when unclear as to whether etiology of the skin rash is due to GVHD or drug eruption. Epidermal abnormalities are most common, with basal cell vacuolar degeneration, eosinophilic “mummified” keratinocytes, and separation of epidermal/dermal junction in severest cases.

-- GI – upper endoscopy is useful in patients with persistent nausea, vomiting, bloating, and/or anorexia. Diagnosis is made by the presence of apoptotic cells in crypts of GI mucosa. Colonoscopy/rectal biopsy is useful in patients with diarrhea, and/or who are unresponsive to antidiarrheal agents. It is important to send samples for culture, especially viral (e.g., CMV, adenovirus, HSV). In addition to biopsy, stool studies should be done to further rule out infection.

-- Liver – biopsy may be useful when the etiology is unclear, that is, whether liver function test abnormalities are because of GVHD or other etiologies: hepatic veno-occlusive disease (VOD)/sinusoidal obstructive syndrome (SOS), infection or drug reaction. Biopsy is often performed via transjugular route especially if the patient has low platelet count or other bleeding risk. It is important to request multiple cores to be sent for evaluation to ensure that portal triad/ducts are present in pathology specimens. Biopsy can show focal portal inflammation with bile ductile obliterations. If VOD/SOS is in the differential, transhepatic wedge pressure measurements should be requested as part of the transjugular biopsy procedure (see VOD/SOS).

Staging

Acute GVHD is usually graded according to the modified Glucksberg Scale in which the affected organ is staged individually and an overall grade is assigned on the basis of the severity of each organ system. Most published studies have utilized the Glucksberg scale (or variant thereof ) to report aGVHD. Less commonly, the severity of GVHD may also be assessed using the IBMTR Severity Index, which assigns severity on an A–D scale. This scale is similar in its organ staging. Some researchers feel this staging system shows better predictive ability for outcomes. Each system has some advantages. The scheme encompasses all possible combinations of organ stages without overlap.

Staging of Acute GVHD “Modified Glucksberg Scale”

Relative risk of transplant-related mortality and risk of treatment failure (includes nonrelapse mortality and relapse) appears to correlate with GVHD staging.

Treatment Guidelines for Acute Graft-versus-Host Disease

Primary Therapy

Steroids + / – calcineurin inhibitor All other therapies are investigational at this time and there is no comparative data available.

-- Therapy is effective 30 to 60% of the time.
-- Typically, therapy needs to be maintained until manifestations are completely resolved; however, be careful of the risk of opportunistic infections and EBV lymphoproliferative disorders that are associated with increased immunosuppression.
-- If GVHD develops after TCD CSA or tacrolimus are useful additions.

Steroids
Methylprednisolone 0.5 to 2 mg/kg/day. Most common dose is 1 to 2 mg/kg day in grade II to IV GVHD.

-- Conversion: Hydrocortisone (1?), Prednisone (4?), Solumedrol (5?), Decadron (20?). In other words, solumedrol to prednisone conversion is 5/4 solumedrol dose.
-- There is no standard method to taper steroids once symptoms improve. A reasonable approach is to taper steroids by 10% of original dose per week in the absence of GVHD symptoms and after GVHD has been controlled for about 1 month.

Side effects of steroid therapy include new onset hyperglycemia or exacerbation in diabetic patients, insomnia, increased appetite, psychosis/mania (rare), fluid retension (less likely when using decadron – less minerlocorticoid effect), cushingoid changes, and muscle wasting/weakness.

Topical therapy such as steroids and/or calcineurin inhibitor preparations may be helpful in mild skin GVHD and may allow for lower doses or avoidance of systemic steroid therapy. These medications can be used in combination with antipruritic agents.

Topical steroids

-- Desonide 0.05% applied BID to affected area.
-- Triamcinolone 0.1% applied BID to affected area. Cannot be used on the face, groin, or axilla.
-- Fluocinonide 0.5% applied BID to affected area. Cannot be used on face, groin, or axilla.
-- Clobetasol 0.05% applied BID to affected area. Cannot be used on face, groin, or axilla. Most potent and is not recommended for use for longer than 2 weeks.

Topical calcineurin inhibitors
-- Tacrolimus 0.03% or 0.1% applied BID to affected area.
-- Pimecrolimus 1% applied BID to affected area.

Secondary Therapy – Steroid Refractory

Mycophenolate mofetil (CellCept)(described in the section on prophylactic regimen). Mycophenolate mofetil is a very useful adjunct to steroids. Typical dose is 15 mg/kg every 12 hours; may need to start low and work up the dose, for example, 250 mg every 12 hours orally > 1,000 mg every 12 hours orally to manage GI side effects. An advantage is that is can be given orally or IV.

May depress counts, cause GI upset (abdominal discomfort or diarrhea), and cause herpes virus flares. It may interfere with the interpretation of a rectal biopsy. MMF-induced injury mimics GVHD injury.

Tacrolimus or Cyclosporine (Described in Prophylactic Regimen Section)
Tacrolimus or Cyclosporine is used alone or in combination.

ATG
-- Horse antithymocyte globulin (ATGAM, Upjohn).
-- Dose for GVHD: 15 to 20 mg/kg/day for 5 to 7 days (after test dose, see subsequent text).
-- Rabbit antithymocyte globulin (Thymoglobulin, Sangstat).
-- Reasonable dose is 1.5 mg/kg per day ? 4 days. May be repeated once at 7 to 14 days.

-- Specific ATG toxicities:
-- Anaphylaxis
-- Fevers, chills
-- Rash
-- Joint pain (serum sickness)
-- Renal damage
-- May decrease counts
-- Reversible hepatic dysfunction

Denileukin diftitox Ontak (Diphtheria Toxin Conjugated IL-2)
This is a recombinant fusion protein with selective cytotoxicity against activated T lymphocytes based on preferential binding to IL-2 receptors. It has been found to have significant activity against steroid refractory GVHD.

-- Phase II data – 70% response rate, 40% CR.
-- Phase II dose – 9 ?g/kg QD IV days 1, 3, 5, 15, 17, 19.
-- Dose-limiting toxicity is transaminitis. Usually transient and peaks about 1 week after administration. There is no dose adjustment needed for renal or hepatic dysfunction.
-- Common side effects include the following:
-- Infusional reactions that may be prevented with appropriate premedication using steroids (can use daily steroid dose for GVHD 30 to 60 minutes before infusion), Tylenol, and diphenhydramine. First dose should be administered over 60 minutes, subsequent doses over 30 minutes if there is no reaction.

-- Vascular leak syndrome that may present as hypotension (usually during the first 24 hours after infusion), shortness of breath (pulmonary edema), and increasing peripheral edema. Studies suggest that incidence of vascular leak syndrome is reduced when corticosteroids are given as premedication. Leak syndrome is usually associated with low serum albumin. Salt-poor albumin (SPA) infusions should be considered in patients with severe hypoalbuminemia (albumin <2/5)>1L)
-- nausea
-- emesis

Diet
-- NPO for bowel rest
-- TPN with increased calorie and protein provision (35 kcal/kg and 1.8 g/kg) to meet caloric, protein, and vitamin needs.

Phase II
Symptoms
-- diarrhea volume decreasing
-- forming of stool
-- significantly less to no cramping
-- no emesis

Diet
-- Continue TPN.
-- Start clear, isotonic, low residue beverages (Gatorade, diluted juices, Pedialyte, decaffeinated tea, chicken broth, sugar free Jell-O).

Diet must be tolerated for a minimum of 2 to 5 days before progressing to Phase III. If liquids are not tolerated (increased cramping, diarrhea, or vomiting), return to Phase I for bowel rest. Do not advance from Phase I until stool is formed and cramps markedly improved.

Phase III
Symptoms
-- stool is formed (pudding like)
-- no emesis
-- decreasing diarrhea frequency and volume
-- tolerating Phase II diet without complications

Diet

-- Continue TPN.
-- Progress to solid foods (GVHD stage I diet).
-- Limit fat intake (~20 gm/day), lactose free, low insoluble fiber, free of gastric irritants, high soluble fiber (white bread, potato, saltines, cream of wheat, pasta, white rice, canned fruits, Lactaid milk, plain egg, plain tuna and chicken, cooked carrots).

If tolerating all foods with minimal changes in symptoms, progress to Phase IV.

Phase III diet is limited in protein due to the limitation of fiber, lactose, and fat. Patients going home tolerating the GVHD stage I diet require supplements or TPN to meet caloric and protein needs.

Preventative Care

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Preventative care will vary on the basis of transplant type, protocol, medications, and pretransplant viral studies.

Infection Prophylaxis

Please see Chapter 15 on infectious diseases.

Hospital Infection Control
Hospital personnel should follow standard infection control and Center for Disease Control (CDC) guidelines to prevent nosocomial infections. The efficacy of different transplant-specific precautions in preventing nosocomial infections has not been studied. Hand washing continues to be the single most critical and effective procedure for preventing infection. If gloves are utilized, they should be changed between patients. All equipment should be sterilized or disinfected and Environment Protection Agency (EPA) registered.

Allogeneic hematopoietic stem cell transplantation (HSCT) patients should be placed in private rooms that have >12 air exchanges per hour and high-efficiency particulate air filters (HEPA) that are capable of removing particles ?0.3 ?m in diameter. It is generally considered good practice to treat all immunocompromised people in HEPA-filtered rooms although the data are less clear than for allogeneic HSCT.

Gut Decontamination

Gut decontamination is used in some centers in all myeloablative and cord allogeneic procedures. Its use is most important when the risk of GVHD is higher (i.e., mismatched and URDs). In autologous and T-cell depleted patients an alternative is to use levofloxacin 500 mg PO starting on day 1 and continue through engraftment.

Nonabsorbable antibiotic regimen may include nystatin 2 MU, bacitracin 500 mg, and polymixin 106 units orally every 8 hours. Many patients are not able to tolerate these oral medications during transplantation, but if they can take it early in conditioning,
the intestinal track tends to stay sterile as long as they are treated in a HEPA environment.

Skin and Oral Care

HSCT patients should take daily showers or baths. Skin sites that have the potential to be portal sites for infection should be inspected daily. Good dental hygiene before and after HSCT (at least 1 year) is important. All patients should receive a dental evaluation as part of the pretransplant assessment. Dental work including eliminating sources of infection should take place before conditioning therapy.

In addition to gut decontamination, patients should receive mouth care 4 to 6 times/day. Mouth care should start with conditioning and continue through engraftment. Mouth care and daily oral hygiene is encouraged to prevent infection secondary to mucosal breakdown, it does not reduce the incidence of mucositis. Oral rinses should be administered 4 to 6 times/day. Teeth should be brushed with an ultrasoft toothbrush or Toothette twice daily. When platelets are >50 K and ANC >500 patients can resume flossing. It is important to note that nystatin is often prepared in a sugar- based suspension, which can, over time, promote tooth decay and exacerbate diabetes. It is possible to use nystatin in a water-based formula if desired.
Clotrimazole troches and other azole antifungal medications will affect the levels of cyclosporine, tacrolimus, and sirolimus by increasing the bioavailability.

At the time of discharge, patients should continue to use mouth rinses for at least 2 to 4 weeks. They can resume routine dental cleanings once counts have fully recovered.

Dental Prophylaxis

Standard regimens include:
Amoxicillin 2 g, 1 hour preprocedure Ampicillin 2 g IV, 30 minutes before procedure PCN allergy:

Clindamycin 600 mg PO, 1 hour preprocedure Cephalexin or cefdroxil 2 g PO, 1 hour preprocedure Azithromycin 500 mg PO, 1 hour pre procedure Clarithromycin 500 mg PO, 1 hour pre procedure

Common Oral Conditions

Hairy Tongue
Have patients try to increa se moisture in their mouth during waking hours. Encourage patients to vigorously brush their tongue with a toothbrush as far back as possible. If no mucositis and ANC >500, it is okay to use firm toothbrush or tongue scraper. Oxygel scrub (glyoxide) or a 1:1 peroxide and water mix can be tried. Oxygel is 11% urea peroxide in a glycerine gel made by Block Drug Co. Jersey City, NJ. Peroxide has a drying effect so it is important to keep the mouth moist.

Xerostomia

Use approaches are as follows:
¦¦ Mouthwashes without alcohol only.
¦¦ Artificial saliva products such as MouthKote.
¦¦ Sugarless gum or hard sour candies (patients should be instructed to use caution since they are prone to caries). ¦¦ Biotene mouth products.
¦¦ Ensure that patient is getting adequate fluoride. Most patients are drinking bottled water and therefore may not be getting enough.
¦¦ Pilocarpine (Salagen) 5 mg PO BID.
¦¦ Pilocarpine 1% 15 gtts in water QID. Results are variable.
¦¦ Evoxac (Cevilemine) 30 mg TID.
Preventing Ba cterial Intravascular Ca theter-Related Infections Catheter care should follow published guidelines. Caps and dressings should be maintained on a routine basis.

Post-Hsct Restrictions, Reducing the Risk of Infection While At Home

Most transplant centers will have specific guidelines for patients to follow. Patients should follow transplant center–specific instructions. The following is a general overview of post-HSCT care.
¦¦ Avoid environmental exposures by hand washing frequently. Exposures can occur when preparing food, changing diapers, touching plants or dirt, pet contact, secretion contact.
¦¦ Respiratory infections may be prevented if patients observe the following precautions: hand washing, avoid close contact with persons with respiratory illness (if unavoidable the person with illness should wear a mask), avoid crowded/public areas, avoid high-risk occupations, avoid construction sites, damp basements, attics, travel, tobacco exposure, marijuana (associated with invasive fungal infections), gardening, yard work, wood-burning fire places.
¦¦ Infections that occur because of direct contact can be minimized by advising patients to avoid the following: contact with soil, plants, animal feces/urine, diapers and others.
¦¦ Water safety: HSCT recipients should avoid contact with recreational water to avoid Cryptosporidium, Escherichia. coli O157:H7, sewage, or animal/human waste. Well water should be avoided if possible. If the well is from a municipal community where the water is tested frequently it may be regarded as safe. HSCT recipients who drink tap water should check their local source often for increase in bacterial levels. Other precautions include avoiding fountain beverages, frozen/fruit drinks, iced tea/coffee and other products made with tap water. Bottled water should be from a distributor that sterilizes the water. Examples of sterile water are Dasani and Aquafina. All carbonated drinks are considered sterile.

¦¦ Travel: In general, travel should be avoided for the first 6 to 12 months post HSCT. Any travel in this period should be discussed with the transplant physician.
¦¦ Safe sexual precautions: Having intimate contact with a partner may increase the risk for infections but a patient may resume sexual activity when he or she feels ready. Nonintercourse sexual expression (i.e., hugging and kissing) with the significant other is encouraged after transplant. The following are guidelines to help minimize the risk of infections in patients who are in nonmonogamous relationships or seeking relationships with new individuals after transplantation. The risk of acquiring disease is very low in longstanding monogamous relationships.

Single partners – partner should not have a cold, flu, or cold sores; platelets should be greater than 50,000; latex condom should be used to reduce the risk of acquisition of cytomegalovirus, herpes simplex virus, and human papilloma virus, as well as other sexually transmitted pathogens. Condom use will also, theoretically, reduce the risk of acquisition of human herpes virus. Data regarding the use and efficacy of “female condoms” are incomplete, but these devices should be considered as a risk-reduction strategy. Patients may receive, but not administer oral sex. Oral exposure to feces should be avoided at all costs to reduce the risk of intestinal infections (e.g., cryptosporidiosis, shigellosis, campylobacteriosis, amebiasis, giardiasis, and hepatitis A and B, E.coli). Anal intercourse should be avoided since the risk of infection is high. Radiation and GVHD cause an increase in dryness in the lining of the vagina. To make intercourse comfortable and to help prevent tearing of the mucosal membrane, water soluble lubricant or gel, which contains no perfume or coloring, is recommended. Some brands include Replens, Astroglide, Lubrin, K-Y Jelly, Surgilube, Today Personal Lubricant, and Ortho Personal Lubricant. Do not use a Vaseline or oil-based lubricant as these may cause an increase in yeast infections.

Pet Safety: Patients should not routinely part with their pets but in general, minimal contact is recommended. HSCT recipients should avoid contact with animal feces to reduce the risk for toxoplasmosis, cryptosporidiosis, salmonellosis, and campylobacteriosis. HSCT recipients should not clean cages, litter boxes, or dispose of animal waste. Any pet that is ill or has diarrhea should be evaluated by a veterinarian. Contact with reptiles (food or anything the reptile has touched) should be avoided because of the risk of salmonellosis. Direct contact with birds, exotic pets, and fish should be avoided. If avoiding direct contact with pets is not possible, then the HSCT recipient should be advised to wear gloves and wash hands thoroughly.

Diet, Early Posttransplant,Reduced Bacteria

In general, the HSCT diet consists of foods considered to be low in pathogenic bacteria. Dietary restrictions and food safety practices should be reviewed before conditioning begins to ensure that the patient and his/her care givers understand the importance of adherence to the special diet. HSCT patients should not consume any raw or undercooked meat, including beef, poultry, lamb, wild game, or seafood (including oysters and clams). Raw eggs or foods that contain raw eggs should not be consumed. HSCT patients should avoid thin-skinned fruits and vegetables unless cooked until well done.

Following is a general list of foods allowed and not allowed. This may vary per transplant center and geographic location.

Long Term Prevention of Infections After Hsct Vaccinations

Cellular and humoral immunity are severely depressed after transplantation. Antibody titers to diseases such as tetanus, polio, and others decline, often to levels that may not be protective. Moreover, within the first year after transplantation there may be a suboptimal response to vaccines. Therefore, it is important to administer vaccines after the patient has reconstituted his or her immune system. B- and T-cell function can take 12 months or longer to restore if the patient has GVHD or is still on immunosuppressive medications. Live vaccines should not be utilized, until at least 2 years after HSCT and then only if the patient is no longer on immunosuppression and has no chronic GVHD. The use of varicella vaccine has not been studied after HSCT and is generally not administered.

It is also important to monitor immunoglobulin (Ig) G levels after HSCT and replace gammaglobulin if the patients become hypogammaglobulinemic. The duration of replacement varies pending recovery of active immunity. A convenient schedule is replacement for 6 months followed by reevalation. If IgG levels are maintained, replacement can be stopped. Otherwise, continue monthly intravenous gammaglobulin reevaluating approximately every 6 months. Vaccine or toxoid

Abo Compatibility

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Whenever possible, donors who are ABO compatible with the recipient should be selected. However, since human leukocyte antigen (HLA) and ABO types are unrelated, it is common to have HLA compatible donors who are ABO incompatible. This disparity often requires special attention. Transfusion problems may occur immediately or after a delay.

Soon after birth, antibodies form against bacterial polysaccharides that cross-react with ABO substance. Thus, type A people have anti-B antibodies without ever being exposed to type B blood. Similarly, type B people have anti-A antibodies and type O people have antibodies to both A and B, while type AB people have no antibodies. Immediate transfusion risk is due to this preformed antibody reacting with infused red blood cells (RBCs) during the stem cell product infusion. Delayed reactions reflect slow turnover of plasma cells or antibody weeks or months after the transplant, resulting in delayed erythroid recovery. When considering transfusion risks, both the red cell type and the associated antibodies must be considered. Donor recipient pairs are defined as follows:

Major Mismatch

As shown in the following table, if the recipient is type O and the donor is type A, B, or AB, there is a major ABO incompatibility. In this situation, there are preformed antibodies in the type O recipient against A and/or B substance on the donor’s RBCs.

Transfusion of the unmodified stem cell product can result in an acute hemolytic transfusion reactions (AHTR). This type of reaction can manifest in multiple ways, pain at the IV sight, hypo/ hypertension, fever, back or flank pain, gross hematuria.

After an otherwise successful transplant, some patients will have prolonged failure of erythropoiesis. If the recipient is a high-titer antibody producer (primarily anti-A), he or she may maintain high titers of antibody long after otherwise successful engraftment. This appears functionally to be a form of pure red cell aplasia since the antibody destroys red cells or red cell precursors in the marrow before they enter the circulation.

Minor Mismatch

In this setting the donor is type O and the recipient is A, B, or AB. Normally this does not result in any immediate problems since the type O red cells will not react with preformed antibody in the recipient. However, there are some data that the preformed antibody in the type O donor’s plasma can cause problems1 – including nonspecific organ injury (veno-occlusive disease [VOD] etc.) due to complement activation and endothelial damage. Removing the plasma from the product before infusion can eliminate this risk. A second concern is a delayed hemolytic transfusion reaction. The type O donor has B cells that are capable of producing antibody to A or B substance. When these B cells are infused with the graft, they may be stimulated by A or B substance on the recipient’s red cells, which can result in a prompt increase in antibody production. A severe hemolytic anemia that can be life-threatening can result 2 to 10 days later. This usually occurs with T-cell depletion of type O donors. Finally, use of plasma from type O donors will add unwanted antibodies (see also major–minor mismatch). In these circumstances one must be careful about the use of both the plasma infusions and the plasma that comes with cellular products.

MAJOR–MINOR MISMATCH

Donor is either A or B and the recipient is the opposite. In this case, there is a risk of both immediate transfusion and infusion of preformed antibody as noted earlier. Thus the product needs to be both RBC and plasma depleted.

Rh and Minor Red Cell Antigens

For the most part, these incompatibilities are not associated with acute problems and do not need to be addressed. Rh-negative women receiving Rh-positive cells can be at risk of hemolytic disease of the newborn if their fertility is spared by the transplant. If sparing of fertility in a woman is anticipated, use of red celldepleted products followed by WinRho is preferred.