The Scientist 14[23]:20, Nov. 27, 2000

RESEARCH

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.

Brain

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 Frisén 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, Frisén 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). Frisén 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 ß-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."

Pancreas

Goal: To replace insulin-producing islet ß 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 ß 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.

Liver

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.

Heart

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."

Transdifferentiation

Researchers will have to understand transdifferentiation better before they can deploy adult stem cells (ASCs) as broadly and effectively as possible. Transdifferentiation is the phenomenon whereby a muscle ASC, say, can give rise to a blood cell.

Margaret A. Goodell, who studies stem cells at Baylor College of Medicine's Center for Cell & Gene Therapy, foresees that once biologists begin to "rationalize" the recent spate of observations of this phenomenon, "it won't turn out to be just this wild free-for-all where anything can differentiate into anything." Rules discovered over the last 20 years, she adds, "must have some meaning because otherwise you wouldn't get the development of a very highly organized animal."

Richard C. Mulligan, a professor of genetics at Harvard Medical School, has proposed alternative hypotheses that could help explain transdifferentiation. One theory is that ASCs in various organs all originate from ASCs in bone marrow; these ASCs then adopt organ-specific traits after being seeded in local environments. The other theory is that ASCs arise independently in various organs but share phenotypic and functional characteristics. Thus, ASCs from one organ can generate mature cells of another organ because the ASCs of both organs have a common origin and/or exhibit certain common features.

In a 1999 Nature paper, a team headed by Mulligan and his Harvard colleague, Louis M. Kunkel, reported that injecting muscle-derived ASCs into irradiated mice led to reconstitution of the recipients' hematopoietic compartment.¹ ASCs with this capability were designated muscle SP ("side population") cells. Like hematopoietic SP cells, muscle SP cells resisted staining by a Hoechst dye. The two SP cell types weren't identical, however.

In unpublished work since then, "we've marched from organ to organ and tissue to tissue, looking for these SP cells in the heart, the liver, the kidney, the CNS [central nervous system]," recalls Mulligan. "From each of these tissues, it appears you can isolate a putative SP population that bears many of the surface characteristics of both the bone-marrow and muscle SP population." He contends that the sheer quantity of these cells means they aren't blood contaminants. Mulligan's lab is now looking for the origin of SP cells, the notion being that they might be recent offshoots of a common bone-marrow ASC. The lab is also trying to define what's common about their surface phenotypes. He hopes his work will guide decisions on "the practical utility of bone-marrow SP versus organ SP cells for transplantation purposes."

Another of Mulligan's new findings is that bone-marrow ASCs give rise to endothelial cells only if the recipient is injured, for example, by an induced heart attack or by receiving an organ transplant. His 1999 Nature paper reported that bone-marrow ASCs generated muscle in a murine model of Duchenne's muscular dystrophy, arguably a form of injury. As a result, Mulligan is betting that injury is also a prerequisite for hematopoietic-to-muscle transdifferentiation.

--Douglas Steinberg

1. E. Gussoni et al., "Dystrophin expression in the mdx mouse restored by stem cell transplantation," Nature, 401:390-4, 1999.

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.

Blood

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.

References

1. D. Steinberg, "Stem cell discoveries stir debate," The Scientist, 14[22]: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.

Precursor Cells to the Rescue?

Two reports out this month suggest that less-than-fully differentiated cells--whether embryonic or adult--could help humans recover from a host of nervous system ailments, ranging from motor neuron diseases to brain cancer.

In a study announced at the Society for Neuroscience meeting in New Orleans Nov. 4-9, researchers from Johns Hopkins University School of Medicine and Harvard Medical School infected rodents with Sindbis virus, which causes limb paralysis by attacking the motor neurons that thread from the spinal cord to the muscles. The team then injected embryonic germ cells (EGCs), primordial cells that are pluripotent like stem cells, into the cerebrospinal fluid (CSF) at the base of the animals' spines. The EGCs were pretreated with growth factors to nudge them onto the path of neural differentiation, thereby preventing them from developing into tumors.

Within several weeks, the EGCs had migrated to the ventral horn of the spinal cord, which contains the cell bodies of motor neurons. And by eight weeks after injection, 11 out of 18 animals had regained the ability to place the soles of one or both of their hind feet on the ground. Yet, only about 6 percent of the migrating EGCs seemed to have differentiated into neurons, as indicated by expression of cell-surface markers.

Earlier animal studies showed that applying stem cells to the site of a traumatic spinal-cord injury leads to some functional recovery. The newly reported experiments were the first involving a diffuse disease that affects the whole spinal cord and the first in which primordial cells were delivered via CSF, according to lead researcher Douglas A. Kerr, an assistant professor of neurology at Hopkins. In ongoing work, "We're trying to characterize why the animals recovered," he says. "And we're also trying to trick the cells prior to implantation into really thinking that they are to be motor neurons. Presumably then we might even see a better functional recovery."

Before Kerr and his colleagues turned to EGCs, their study used human neural stem cells (NSCs) derived from a fetus' telencephalon by Evan Y. Snyder, an assistant professor of neurology at Harvard Medical School.¹ Kerr recalls that these NSCs restored some function to a handful of rodents, but that they showed no effect in later experiments on a larger group of animals.

Snyder and a Harvard team, meanwhile, used the same NSCs in a newly published study on brain cancer.² After the researchers implanted glioblastoma cells into rodents, the animals developed intracranial tumors. NSCs, which were implanted several days later, infiltrated and surrounded the tumors, and chased down malignant cells that were migrating into normal tissue. Tumor targeting occurred even when the NSCs were introduced far from a tumor, such as into the vein of an animal's tail.

When NSCs expressed cytosine deaminase, an enzyme that converts a non-toxic pro-drug into a chemotherapeutic agent, one mouse's tumor shrank about 80 percent. The researchers found that the NSCs neither differentiated nor turned tumorigenic in the recipient rodents.

One message of the study, says Snyder, is that "if there's pathology, not only can there be very dramatic, extensive [NSC] migration, but it happens along nonstereotypical, unpredicted pathways." Why do NSCs track brain tumor cells? He suggests some possibilities: Some oncologists view brain-tumor cells as NSCs "gone bad," and these two similar cell types might respond to the same cues. In addition--or alternatively--tumors or the brain cells that they're killing might secrete factors that attract stem cells.

Snyder and his colleagues have been holding talks with the Food and Drug Administration about using NSCs as adjunctive therapy to treat brain tumors, which are now almost always incurable. He notes that NSCs could be equipped with transgenes that fight cancer by promoting differentiation or blocking angiogenesis.

--Douglas Steinberg

References

1. J.D. Flax et al., "Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes," Nature Biotechnology, 16:1033-9, 1998.

2. K.S. Aboody et al., "Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas," Proceedings of the National Academy of Sciences, 97:12846-51, Nov. 7, 2000.

The Scientist 14[23]:20, Nov. 27, 2000