Thanks to Stuart from the news thread. I'm going to cut and paste so that I can add emphasis.......
PROFILE
Origin of Species
Stem cell research holds endless possibilities
By Bob Sinclair
Just last year Science hailed stem cell isolation and culture as the "breakthrough of the year."1 Much of the
excitement over stem cells derives from their potential to differentiate into any cell type in the body. A fertilized egg,
for example, is a single cell that is capable of eventually creating all the different cells that make up the mature
organism. As such, a fertilized egg and the daughter cells of the first few divisions are often referred to as
"totipotent."
After a few days of division, a human fertilized egg reaches the blastocyst stage. The inner cell mass of the
blastocyst eventually forms the mature adult body. This inner cell mass cannot form an organism because it cannot
form a placenta, which is derived from the outer cells of the blastula. But cells from the inner cell mass can form all
other cell types and are consequently described as "pluripotent." As development proceeds, these pluripotent cells
further differentiate into more specialized cell types referred to as "multipotent." Multipotent cells are typified by the
stem cells found in adult bone marrow, which can give rise to all the different types of blood cells but not, for
example, nerve cells.
Stem cell research intensified at the close of 1998, when two separate groups of researchers almost simultaneously
announced the successful culturing of stem cells from human embryos for many generations without loss of cell
pluripotency. James Thomson of the University of Wisconsin, Madison, expanded on his longtime work with
monkey stem cells to develop a method for growing human embryonic stem cells, derived from the inner cell mass,
on a layer of cultured mouse fibroblast feeder cells.2 Through careful control of the cell environment, not only were
the cells able to grow and divide without apparent chromosomal aging, but they also were held in a
nondifferentiated pluripotent state. Embryonic stem cells are now known to express constitutively high levels of
telomerase, an enzyme that helps immortalize cells by preventing or repairing damage to the ends of their
chromosomes. John Gearhart's laboratory at the Johns Hopkins University School of Medicine also reported
successful maintenance of human embryonic cells in culture.3 These cells, derived from the primordial germ cells
that are the embryonic precursors of sperm and eggs, could be cultured for several months without differentiation
or significant loss of viability.
Just one year later, Margaret Goodell and colleagues at the Baylor College of Medicine published evidence that
stem cells isolated from adult tissue can also exhibit much of the flexibility of their embryonic cousins. Stem cells
isolated from adult muscle demonstrated an unexpected ability to differentiate into all the different cell types of adult
blood.4 These data, combined with results such as Dolly, the now-famous cloned sheep,5 demonstrate that
non-stem cell adult nuclei can be reprogrammed to a totipotent state when injected into enucleated egg cells. Thus
reprogrammable pluripotent and multipotent cells derived from adult tissue hold research potential similar to that of
embryonic stem cells.
Prior to these discoveries, stem cells were primarily used for studies of gene function in transgenic mice using
so-called knockout technology; Thompson's and Gearhart's work, closely followed by publications from several
other researchers, opened up the possibility of applying much of what had been learned in mice to problems in
humans. New therapeutic interventions and possible cures for numerous diseases very suddenly seemed realistic,
but the euphoria that stem cells' potential caused was immediately matched with harsh and vocal criticisms.
Traditionally--if "tradition" can be applied to an approach that in humans is not yet two years old--pluripotent stem
cells are prepared from the inner cell mass of the embryo. It is necessary to destroy a living embryo to create a
pluripotent stem cell line, a practice that draws controversy regardless of the benefits that may be derived from it.
In fact, the use of human pluripotent stem cells was prohibited in federally funded research until Aug. 25, when the
National Institutes of Health published final guidelines allowing human embryo cell research.6
Private Lives
Though private companies have been considerably less fettered by regulations on how federal research funds may
be used, potentially patentable or patent-breaking academic research was essentially grounded while ethics were
debated. Thompson's work, for example, was extensively funded by Geron Corp. of Menlo Park, Calif., and was
performed in a "duplicate lab" that was carefully separated from his NIH-funded lab and work. The Wisconsin
Alumni Research Foundation owns the patent on Thomson's stem cell lines, and Geron has "exclusive license for
potentially profitable uses of the cells."7
Geron, which also funded much of Gearhart's work, is one of the leaders of a dozen or so companies that intend to
take advantage of stem cells' enormous versatility. The company focuses on embryonic and fetal stem cells. Geron
plans to use stem cells to generate cell lines appropriate for screening new drugs and therapeutics; current
approaches that involve animal cells or aberrant human cells, such as those from tumors, have obvious flaws. It also
intends to use stem cells to study the function of cells in currently inaccessible stages of human development. And
the big payoff is probably through the third approach: modifying stem cell lines to evade attack by the immune
system and using those lines to engineer replacement cells and tissues. Other companies working primarily with fetal
cells include Layton BioScience of Atherton, Calif., NeuralSTEM Biopharmaceuticals of Bethesda, Md., and Stem
Cell Sciences of Melbourne, Australia.
Osiris Therapeutics of Baltimore has developed methods to increase a population of approximately 100,000
mesenchymal stem cells aspirated from a patient's bone marrow to a therapeutically useful dose of some 500
million cells. Mesenchymal stem cells have been shown in animals to reconstitute bone marrow after chemotherapy,
to rebuild cardiac muscle after acute myocardial infarction, to repair bone breaks and defects, and even perhaps to
assist with repair of articular cartilage and menisci. Nexell Therapeutics Inc. of Irvine, Calif., has developed cell
selection techniques to improve hematopoietic stem cells' clinical usefulness. Aastrom Biosciences of Ann Arbor,
Mich., also works with hematopoietic stem cells for transplantation therapy. Its Replicell System contains all the
growth media, cytokines, and controls for automating the on-site, single-shot production of therapeutically valuable
cells from a patient. Other companies, notably Neuronyx Inc. of Malvern, Pa., ReNeuron of London, and
StemCells Inc. of Sunnyvale, Calif, are working primarily with neuronal cells, developing the potential to perhaps
repair damage from a stroke or ameliorate the effects of Parkinson's or Alzheimer's diseases.
A Growing Need
More companies seem to be interested in using stem cells for therapeutic purposes than in offering products and
services for researchers using stem cells in their own labs. But this is likely to change with the new government
guidelines. Culturing stem cells has all the usual tissue culture requirements for temperature- and
atmosphere-controlled incubation, nonpyrogenic flasks, biosafety cabinets, and cryogenic storage, but with the
added complexity of maintaining layers of feeder cells and stringently controlling the composition of the media and
the addition of growth factors and cytokines. Working with stem cells also often requires access to a good suite of
microscopy tools, micromanipulators, microinjectors, pipette pullers, and other specialized equipment.
Many of the current suppliers of stem cells and stem cell-specific reagents also are involved in knockout mouse
technology and have therefore recently been profiled in LabConsumer.8 For companies focused more on stem
cells, the Poietics™ division of BioWhittaker carries a wide selection of stem cells, including erythroid and CD34+
progenitors, CD4+ T cells, dendritic cell precursors, and mesenchymal stem cells (licensed from Osiris
Therapeutics). The company also has irradiated stromal cells as an alternative to mouse embryonic fibroblast feeder
cells. Medicore Inc. supplies engineered feeder cells and mouse embryonic fibroblasts carrying resistance to
neomycin or hygromycin and also offers bulk cell culture services.
Buying cells from a commercial source involves a certain amount of trust. Many questions can arise during work
with purchased stem cells, and it can take a long time before problems manifest themselves. The presence, or
absence, of specific cell-surface antigens defines many stem cells in part, so it is important that they be tested for
the appropriate surface features. As cells are repeatedly passaged, viability can decrease and chromosomal
aberrations can accumulate, making the number of passages an important parameter. The proportion of viable cells
in a batch is another variable. Ensuring that stocks have been screened for pathogens is also important. Bacteria,
yeast, and other fungi are relatively easy to detect; mycoplasmas are more difficult, and harder still are viral
contaminants such as hepatitis B and C and HIV, yet these can destroy an experiment after several weeks--or even
years.
If research needs go beyond what is commercially available, stem cells can be isolated from scratch. There are
many different ways to do this, often dependent on the type of cells desired. Anything from microdissection of
individual cells to gradient centrifugation of enzymatically digested tissue might be necessary. As mentioned above,
many stem cells are characterized by specific cell-surface components, suiting them to purification through Miltenyi
Biotec's MACS technology.9 Miltenyi offers MACS beads for purifying human hematopoietic stem and progenitor
cells; T, B, DC, ACP, and NK cells; B monocytes; granulocytes and myeloids; erythroid and proliferating cells;
tumor cells; megakaryocytes and platelets; and endothelial cells. Similar products are available for mice and rats.
Embryonic stem cells must be grown on a layer of feeder cells, commonly mouse fibroblasts. To remain pluripotent,
they must meet two conditions: The cells must constantly receive an extracellular signal from the cytokine leukemia
inhibitory factor (LIF) and must continually express the Oct4 protein. In the absence of these molecules, embryonic
stem cells form small aggregates that spontaneously differentiate into a variety of cell types, including blood, nerve,
epithelia, and even beating heart cells.10-12 Details about which combination of cytokines forces these cells down
an avenue of ever-decreasing choices to a final cell type are largely unknown. With more than 2,000 cytokines and
growth factors purified or cloned and many more identified or postulated, the array of available combinations is
enormous. Cell-to-cell contacts also may be crucial for stem cell proliferation and differentiation.
Obviously there is still an enormous amount to learn before researchers can grow new kidneys or knees, but with
regulatory roadblocks removed, the pace most likely will quicken. In any event, when LabConsumer revisits this
topic in the future, the small commercial stem cell industry will likely have proliferated and differentiated into an
intricate array of products, services, and support. S
Bob Sinclair (bobsinclair@tech-write-edit.com) is a freelance writer in Salt Lake City.
References
1. F.E. Bloom, "Breakthroughs 1999," Science, 286:2267, Dec. 17, 1999.
2. J.A. Thomson et al., "Embryonic stem cell lines derived from human blastocysts," Science,
282:1145-7, 1998.
3. M.J. Shamblott et al., "Derivation of pluripotent stem cells from cultured human primordial germ cells,"
Proceedings of the National Academy of Sciences, 95:13726-31, 1998.
4. K.A. Jackson et al., "Hematopoietic potential of stem cells isolated from murine skeletal muscle,"
Proceedings of the National Academy of Sciences, 96:14482-6, Dec. 7, 1999.
5. I. Wilmut et al., "Viable offspring derived from fetal and adult mammalian cells," Nature, 385:810-3,
1997.
6. Department of Health and Human Services, National Institutes of Health, "National Institutes of Health
Guidelines for Research Using Human Pluripotent Stem Cells," 65[166]:51975-81, Aug. 25, 2000.
7. G. Vogel, "NIH sets guidelines for funding embryonic stem cell research," Science, 286:2050-1, Dec.
10, 1999.
8. C.M. Smith, "Technical knockout," The Scientist, 14[15]:32-4, July 24, 2000.
9. A.L. Francis, "Selection perfection," The Scientist, 14[4]:21, Feb. 21, 2000.
10. J. Nichols et al., "Formation of pluripotent stem cells in the mammalian embryo depends on the POU
transcription factor Oct4," Cell, 95:379-91, 1998.
11. H. Niwa et al., "Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3,"
Genes & Development, 12:1048-60, 1998.
12. A. Bradley, "Embryonic stem cells: proliferation and differentiation," Current Opinion in Cell Biology,
2:1013-7, 1990.
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