Stem Cells Tapped to Replenish Organs
Embryonic or adult? The superior source depends on the tissue
By Douglas Steinberg
Editors Note: This is the second of two articles on issues raised by recent stem cell discoveries. The first article appeared in the November 13 issue
"All politics is local" was a famous maxim of Thomas "Tip" O'Neill, the late speaker of the House of Representatives, and the same can be said of medically useful stem cells. Progenitor cells may prove to be more or less pluripotent in the lab, but if they don't succeed on a local level in the body, they won't cure anything. They must be capable of being coaxed into differentiating reliably into the cell types that populate particular organs.
How much can embryonic stem cells (ESCs) and adult stem cells (ASCs) replenish tissues of the brain, pancreas, liver, heart, and blood? So far, researchers have manipulated ESCs to generate a broad span of cell types. ASCs have yielded a narrower range, partly because several subtypes haven't been isolated yet.
The phenomenon of transdifferentiation, however, promises to extend the capabilities of ASCs. And as studies proliferate in the wake of discoveries and the issuance of new guidelines by the National Institutes of Health, the relative advantages and disadvantages of ESCs and ASCs could change considerably within the next few years.
Goal: To replace neurons that have died as a result of degenerative diseases or stroke.
Ronald D.G. McKay and his Laboratory of Molecular Biology at the National Institute of Neurological Disorders and Stroke can efficiently generate dopaminergic and serotonergic functional neurons in vitro from mouse ESCs.2 They can get ASCs, in the form of mesencephalic precursor cells, to induce functional recovery when transplanted into parkinsonian rats.3 But according to McKay, these ASCs stop generating dopaminergic neurons in culture after a week or so.
The yield improves if the cells are grown under low-oxygen conditions, which are characteristic of the fetal environment.4 Still, McKay notes that his lab's experience thus far with several types of ASCs is that "they don't turn into dopaminergic neurons with any kind of efficiency." Referring to a 1999 paper from the Karolinska Institute that reported such a result,5 he wonders whether the final yield is "really a dopaminergic cell or not."
One problem besetting such research is the uncertain identity of ASCs in the mammalian brain. Last year, a Karolinska team led by Jonas FrisÚn announced that the ependymal cells lining the brain's ventricles were neuronal ASCs.6 Five months later, a Rockefeller University group headed by Arturo Alvarez-Buylla countered that subventricular zone (SVZ) astrocytes were the true neuronal ASCs. This group also rejected the ependymal-cell hypothesis after finding that those cells neither formed neurospheres, nor accumulated nucleoside labels, as they would if they divided.7 The New York Times ran a story on the ensuing brouhaha.8
Alvarez-Buylla, who just moved to the neurosurgery department of the University of California, San Francisco, says that the conflict may arise, in part, because SVZ astrocytes "interact very, very closely with the ependymal cells." But he maintains that ependymal cells only serve to create a niche where neurogenesis can occur. His lab is currently examining two signaling systems that seem to prompt SVZ astrocytes into becoming neurogenic.9
Last June, FrisÚn bolstered his theory with a paper showing that neural stem cells had broad differentiation potential.10 The authors couldn't verify that most of their experiments actually involved ependymal cells. But when ependymal-cell-derived neurospheres were injected into the amniotic cavities of chick embryos, the cells showed broad differentiation potential (the data, at footnote 16, weren't published). FrisÚn now says he has additional, unpublished lines of evidence indicating that ependymal cells are neural stem cells.
His theory may need that support. Derek van der Kooy and his colleagues at the University of Toronto weren't able to get ependymal cells to make neurons in vitro.11 A similar negative finding appears in an upcoming paper describing a study led by Eric D. Laywell and Dennis A. Steindler, professors of anatomy and neurobiology at the University of Tennessee in Memphis.12
They and their colleagues, on the other hand, confirmed Alvarez-Buylla's hypothesis by observing that SVZ astrocytes could give rise to neurons, as identified by the expression of ▀-III tubulin and other markers. (Functional studies of the neurons are now under way.) In a significant extension of that hypothesis, they found that astrocytes from cerebral cortex, cerebellum, and spinal cord could also turn into neurons--but only if the astrocytes were derived in the first two postnatal weeks.
"This correlates with what we believe to be the maturation of the astrocyte in the nervous system," notes Steindler. "The end of this critical period in astrocyte multipotency coincides with the end of a period in which the brain's regenerative responses are far more successful than those in the more mature brain."
Goal: To replace insulin-producing islet ▀ cells destroyed in some types of diabetes.
Stem cell research involving the pancreas seemed to score two home runs this year. In February, Bernat Soria and colleagues at the Universidad Miguel Hernandez in San Juan, Spain, reported that they had obtained insulin-secreting cells from mouse ESCs by using antibiotic selection under the control of the insulin gene's regulatory regions.13 Soria says he is now trying to replicate his results using human ESCs. (A poster at a recent diabetes meeting, meanwhile, is said to have announced that human ESCs differentiate spontaneously into insulin-positive cells.)
A month after the Soria paper came out, a team of researchers led by Ammon B. Peck, a professor of pathology, immunology, and laboratory medicine at the University of Florida College of Medicine in Gainesville, reported a second major advance. They claimed to have reversed diabetes in non-obese diabetic (NOD) mice by transplanting islets generated in vitro from pancreatic ASCs, which had not been previously isolated.14 NOD mice are the best current model for autoimmune diabetes.
Nora D. Sarvetnick, a professor of immunology at Scripps Research Institute, is puzzled by Peck's results. "Unless you immunosuppress the mouse"--which wasn't done--"the mouse is just going to reject the ▀ cells," she contends. Peck responds that cells grown in culture, such as his ASC-derived islets, sometimes exhibit lower antigenicity for unknown reasons.
Susan Bonner-Weir, an associate professor of medicine at Harvard Medical School, objects that the amount of insulin in Peck's ASC-derived islet cells was "orders of magnitude" too low. "What they were putting in [the NOD mice] would have been a very minuscule amount," she says, though she concedes that more insulin might have been made if the islet cells differentiated further inside the mice. Bonner-Weir's own work involves expanding human pancreatic duct cells in vitro, then turning them into insulin-producing islet cells.15 She calls the duct cells, which are differentiated, "functional stem cells" because they undergo scores of doublings in culture and help to regrow pancreas after a portion is removed.
Goal: To develop a plentiful source of hepatocytes for regenerating damaged livers and treating some metabolic diseases.
Another functional stem cell is the hepatocyte. "For liver repopulation purposes and transplantation, the best cell type is the differentiated hepatocyte," says Markus Grompe, a professor of molecular and medical genetics and pediatrics at Oregon Health Sciences University. He adds that in transplants, hepatocytes are "far superior" to liver stem cells, whose existence has been established only in the past 12 months or so.
The major source of hepatocytes for therapeutic purposes, however, is human cadavers. More accessible and plentiful are the liver stem cells residing in the bone marrow, discovered by Neil D. Theise, an associate professor of pathology at New York University School of Medicine, and colleagues. Their proof: The Y chromosome pops up in some hepatocytes after male marrow transplants into females.
Are these new liver cells functional? In a small-scale study of human transplants,16 "We show such extensive engraftment that it's hard to avoid the conclusion that this is a part of physiological regeneration," asserts Theise. The next test is to use bone-marrow transplants to correct defective liver function in animal models of some human metabolic diseases. Grompe and a team of researchers published a paper this month reporting such a finding in a mouse model of tyrosinemia.17
The roles played by ASCs in the liver are still far from clear. Intrahepatic oval cells have recently--and grudgingly--won full acceptance as stem cells, particularly after injury. (Theise proposes that oval cells ultimately derive from the bone marrow.) Apparently no one has yet generated liver cells from ESCs. The growth factors "are just absolutely not known," notes Grompe.
Goal: To replace cardiomyocytes that have died during heart attacks.
Several years ago, the lab of Loren J. Field, a professor of medicine and pediatrics at Indiana University School of Medicine in Indianapolis, derived relatively pure cardiomyocyte cultures from transfected mouse ESCs.18 The cardiomyocytes weren't identical to their adult counterparts. But according to Field, experimental data suggest that under appropriate humoral and neuronal stimulation, a cardiomyocyte derived from ESCs "will adapt the characteristics typical for the adult cell."
The number of heart muscle cells in a mouse is several orders of magnitude lower than the number in a human. Now that his lab has refined its methods, Field is optimistic that "with bio-processing and growth factors, we can produce sufficient cells for therapeutic applications." To address the low efficiency at which the cardiomyocytes seed into recipient hearts, he is testing such strategies as blocking apoptosis, making the cells more resistant to ischemia, and boosting their capacity to divide.
Geron Corp., based in Menlo Park, Calif., and a few academic labs have already shown that cultured human ESCs can give rise to cardiomyocytes. Meanwhile, the presence of ASCs in the heart itself still hasn't been proven. "If they exist, they aren't doing their job," Field says, noting the heart's limited capacity to heal after injury. Other researchers have reported finding ASCs for cardiomyocytes in other parts of the body such as the bone marrow, but no such claim has yet won wide acceptance.
Goal: To develop a limitless source of blood cells for transfusions.
Over the past 30 years, a small army of researchers has investigated the culture conditions under which hematopoietic ASCs preferentially give rise to myeloid or lymphoid lineages. (Relatively pure cultures of red blood cells have been the most elusive to produce.) Gordon Keller, a professor at Mount Sinai School of Medicine's Institute for Gene Therapy and Molecular Medicine, has succeeded at differentiating mouse ESCs into a variety of blood cell types, though he admits that generating lymphocytes is still a problem. His lab has developed the requisite protocols by trial and error over the past decade.19
When removed from conditions that keep them in an undifferentiated state, ESCs form clusters of differentiating cells called embryoid bodies. "At that point, we take the cells from the embryoid body and put them into cultures containing cytokines that stimulate the growth and maturation of blood-cell progenitors," Keller recounts. "Alternatively, we can first isolate the blood-cell progenitors from the embryoid bodies by using antibodies to specific cell-surface markers and then put them into culture."
Keller is now searching within embryoid bodies for the hematopoietic stem cell equivalent to the hematopoietic ASC that other labs have isolated in bone marrow. This putative stem cell in the embryoid body has been harder to find, he says, because it "appears to be more immature than the one in adult bone marrow." His approach is to transplant candidate stem cells into mice with drug-damaged hematopoietic systems and then to observe whether blood-cell re-population occurs. When might his methods boost human blood supplies for transfusions? "Some years away" is all that Keller will predict.
Douglas Steinberg is a freelance writer in New York.
1. D. Steinberg, "Stem cell discoveries stir debate," The Scientist, 14:1,14-5, Nov. 13, 2000.
2. S.-H. Lee et al., "Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells," Nature Biotechnology, 18:675-9, June 2000.
3. L. Studer et al., "Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats," Nature Neuroscience, 1:290-5, 1998.
4. L. Studer et al., "Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen," Journal of Neuroscience, 20:7377-83, Oct. 1, 2000.
5. J. Wagner et al., "Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes," Nature Biotechnology, 17:653-9, 1999.
6. C.B. Johansson et al., "Identification of a neural stem cell in the adult mammalian central nervous system," Cell, 96:25-34, 1999.
7. F. Doetsch et al., "Subventricular zone astrocytes are neural stem cells in the adult mammalian brain," Cell, 97:703-16, 1999.
8. N. Wade, "Brain stem cell is discovered, twice," New York Times, p. F3, June 15, 1999.
9. One paper is in press. For the other, see J.C. Conover et al., "Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone," Nature Neuroscience, 3:1091-7, November 2000.
10. D.L. Clarke et al., "Generalized potential of adult neural stem cells," Science, 288:1660-3, June 2, 2000.
11. B.J. Chiasson et al., "Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics," Journal of Neuroscience, 19:4462-71, 1999.
12. E.D. Laywell et al, "Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain," Proceedings of the National Academy of Sciences (PNAS), in press.
13. B. Soria et al., "Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice," Diabetes, 49:157-62, February 2000.
14. V.K. Ramiya et al., "Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells," Nature Medicine, 6:278-82, March 2000.
15. S. Bonner-Weir et al., "In vitro cultivation of human islets from expanded ductal tissue," PNAS, 97:7999-8004, July 5, 2000.
16. N.D. Theise et al., "Liver from bone marrow in humans," Hepatology, 32:11-6, July 2000.
17. E. Lagasse et al., "Purified hematopoietic stem cells can differentiate into hepatocytes in vivo," Nature Medicine, 6:1229-34, November 2000.
18. M.G. Klug et al., "Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts," Journal of Clinical Investigation, 98:216-24, 1996.
19. See, e.g., M. Kennedy et al., "A common precursor for primitive erythropoiesis and definitive haematopoiesis," Nature, 386:488-93, 1997.
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