NEWS

Stem Cell Discoveries Stir Debate

Issues take on greater urgency as NIH guidelines go into effect

By Douglas Steinberg

Researchers first isolated embryonic stem cells (ESCs) from mouse blastocysts almost 20 years ago, and a paper announcing the discovery of human ESCs emerged in 1998. Adult-derived stem cells (ASCs) have since become the rage in certain quarters of biology, with unexpected--and sometimes downright weird--findings surfacing regularly in the top journals. Last month, a typical paper reported that neural ASCs can be coaxed into differentiating into skeletal muscle cells.¹

As long-held notions about biological development are challenged,² the therapeutic and ethical implications of stem cell work are also generating controversy. Scientific and "cultural" debates promise to sharpen further once reviewers, under National Institutes of Health guidelines issued in September,³ start to consider applications to fund studies on human ESCs.

Several questions may then come to the fore: Can ASCs do anything ESCs do? Are embryonic germ cells as useful as embryonic stem cells? Is a cloning-type situation possible in which human ESCs would begin to form embryos in a culture dish, much like human eggs do after in vitro fertilization? Given repeated demonstrations of transdifferentiation (a neural ASC, say, developing into a blood cell), is there one basic ASC or many independent varieties?

Some opponents of human ESC research propose that the newfound plasticity of ASCs render these cells as suitable as ESCs for most, if not all, therapeutic uses. This argument has been voiced at recent hearings on stem cells held by the Senate subcommittee on Labor, Health and Human Services, and Education.⁴

One witness before the subcommittee last September was David A. Prentice, a professor of life sciences at Indiana State University in Terre Haute and a founder of Do No Harm (www.stemcellresearch.org), an Alexandria, Va.-based group that opposes the new NIH guidelines. While acknowledging the theological and philosophical underpinnings of his opinions, he contends that scientifically, "we can do everything that we would hope to do with the human embryonic stem cells and do it with a patient's own cells."

Embryonic vs. Adult Stem Cells

Prentice also points out that such ASC transplants won't face immune-system rejection, won't become malignant as long-cultured ESCs may, and will generate desired target cells more reliably than will ESCs, which must undergo lengthier, more complex differentiation.

Margaret A. Goodell, an assistant professor in the Center for Cell & Gene Therapy at Baylor College of Medicine, is well aware of the potential of ASCs, having turned skeletal muscle stem cells into blood cells.⁵ But she also appreciates their limitations, asserting that "in no case do any of those [adult] stem cells really behave like ES cells, where they readily differentiate into a variety of tissue types."

When ESCs are injected into a mouse blastocyst, they are pluripotent, contributing generously and reliably to all tissues and to the germline of the chimeric animal that develops from that blastocyst. But when a group headed by Jonas Frisén at the Karolinska Institute injected neural ASCs into mice blastocysts,⁶ the cells "really didn't behave like ES cells," observes Goodell. "They contributed to a variety of tissues but not all tissues. And every embryo had a different contribution." Injecting hematopoietic ASCs into a blastocyst also fails to show their pluripotency, she adds.

For Goodell, ESCs are indispensable because they can develop into whole organs. "A bone contains bone marrow and stromal cells, and the matrix itself," she says. "You couldn't [generate] that with a bone-marrow stem cell right now because it couldn't make all those tissue types. But an ES cell could." Researchers, however, have to learn first how to direct ESCs down very specific pathways. In a study published last month, an Israeli team used growth factors to systematically turn human ESCs into cells of all three embryonic germ layers. Yet the group couldn't engineer differentiation exclusively into any single cell type.⁷

Findings by Catherine M. Verfaille, director of the Stem Cell Institute at the University of Minnesota Medical School in Minneapolis, suggest the tantalizing possibility of an intermediate between ESCs and ASCs. A few years ago, her lab was trying to purify bone-making mesenchymal stem cells from humans. In a departure from the usual protocol, cells were cultured in serum-free media. The serendipitous yield was adult cells eerily similar to ESCs. The cells divide 70 to 80 times without becoming senescent; express genes and surface markers characteristic of ESCs; and differentiate into many cell types--though not into blood cells.

This work isn't published yet, and animal-model studies are just beginning. Meanwhile, Verfaille speculates that during embryonic development, "nature left behind some cells with much more potential than we ever thought" to serve as backups to organ-specific stem cells after major injury.

Differences between ASCs and ESCs would presumably be apparent in the expression patterns of large numbers of genes. But characterization of such patterns is just beginning, and companies are said to be doing much of this expensive work in secret. At a mouse molecular genetics meeting last summer at Cold Spring Harbor Laboratory, Derek J. Symula, a postdoc in Edward M. Rubin's lab at Lawrence Berkeley National Laboratory, reported on a DNA chip analysis that showed ESCs expressing far more genes than hepatocytes, epithelial cells, or teratoma cells.

Shouldn't cells turn on more genes as they differentiate? John D. Gearhart, a professor of gynecology and obstetrics at Johns Hopkins University School of Medicine, has also performed limited gene-expression analysis of primordial cells and recalls being "stifled by the amount of information coming back." He suggests that cell-culture heterogeneity may be a factor in such results. "Suppose you're looking at millions of cells, but they're not all absolute stem cells from the standpoint that they are at ground zero," he says. "Some are differentiating in a slight way, and this causes up-regulations and down-regulations of appropriate genes."

Germ vs. Stem Cells

In 1998, two teams of investigators isolated human ESCs and embryonic germ cells (EGCs), respectively.^8,9 ESCs come from the inner cell mass of the blastocyst. EGCs appear later in embryogenesis. Derived from primordial germ cells, EGCs are the ancestors of sperm and egg cells. The growth requirements, surface antigens, and morphologies of ESCs and EGCs differ.

Embryonic stem cells and germ cells, nevertheless, are both pluripotent. "One of the tasks that we've had from the mouse side is to show that mouse EG cells can do everything that mouse ES cells can do," says Gearhart, who headed the team that discovered human EGCs.⁹ "This we have done." Papers are now in preparation.

Some ethicists argue that EGC research is less objectionable because the cells are obtained at a later stage of development from already aborted material. ESC work, in contrast, requires the destruction of an embryo. Yet a cloud hovers over the future of EGCs, at least as therapeutic tools.

Several years ago, M. Azim Surani, a professor in the Welcome/CRC Institute of Cancer and Developmental Biology at the University of Cambridge, found that, although chimeric mice grown from ESC-injected embryos were normal, chimeras resulting from EGC injections displayed fetal overgrowth and skeletal abnormalities.¹⁰ He surmised that imprinting--the selective inactivation, often linked to methylation, of the maternal or paternal copy of a gene--was to blame. Imprinting is "erased" in primordial germ cells.¹¹ When EGCs develop soon afterward, imprinting might not yet be fully reestablished.

Colin L. Stewart, head of the Laboratory of Cancer and Developmental Biology at the National Cancer Institute, had different results when he injected EGCs into mouse embryos.¹² "We got normal chimeras [from] which we could breed," he recalls. Even subtle abnormalities weren't apparent, much less the gross deficits Stewart has seen in cell lines and chimeras derived from androgenetic and parthenogenetic ESCs. These ESCs indicate what might occur if imprinting were erased in EGCs. Containing either a wholly sperm-derived or wholly egg-derived genome, respectively, they express a double dose of any paternal or maternal gene subject to imprinting, since neither allele comes from the silenced parent. (By the same token, the cells--unlike EGCs--don't express other imprinted genes at all.)

Researchers haven't reconciled the discrepant EGC findings. Peter J. Donovan, an associate professor of microbiology and immunology at Thomas Jefferson University, notes that the imprinting status of genes in cultured embryonic cells "can be very different, depending on the culture conditions." For his part, Surani says: "I would be very interested for people who claim that they have different results to publish their work. I haven't seen anything published. I have seen people make kind of odd, off-the-cuff remarks about, 'Oh, our EG cells are fine.'"

Cloning by Cell Culture?

Mouse ESCs are pluripotent, not totipotent, because they develop unaided into almost all cell types of the embryo except those of the trophoblast, the section that becomes the placenta, umbilical cord, and amnion. (Mouse ESCs, however, are readily nudged into the trophoblast lineage by repressing the gene encoding the transcription factor Oct-3/4.¹³) Human and primate ESCs, in contrast, seem to be totipotent. Cultures of these cells release chorionic gonadotropin (CG) into the medium^8,14; CG is produced only by trophoblasts and by certain tumors. According to Do No Harm's Prentice, this finding signifies that "potentially, you could generate embryos in culture."

That's unlikely, responds Janet Rossant, a professor of molecular and medical genetics at the University of Toronto. One of the investigators who demonstrated that mouse ESCs can't form trophoblasts,¹⁵ Rossant has applied growth factors to generate what she calls mouse trophoblast stem cells (TSCs). These differentiate into trophoblast-derived structures.¹⁶ When ESCs and TSCs are combined into one culture, the result is a cell aggregate, not an organized embryo.

"Making an organized embryo is more than just having cell lines," she explains. "You need some of the structures of the intact embryo itself to help put cells in the right places and subject them to the right organization signals. And it doesn't look as though we can readily, even in a mouse, reconstitute that in vitro."

But what about the potential of single primate ESCs to form trophoblasts first and maybe embryos later? (Experiments using human ESCs to create chimeras or clones are, of course, not permissible.) The best proof that primate ESCs could form trophoblasts would be chimeric animals whose placental cells bear an ESC marker, says Gerard Schatten, a senior scientist at the Oregon Regional Primate Research Center of Oregon Health Sciences University. But he is almost certain that no one has tried yet to produce primate chimeras.

Schatten cites some of the technical roadblocks: Blastocysts don't tend to implant when transferred to monkeys. Mixing small ESCs with large embryo cells is a geometrical challenge and could result in extrusion of one of the cell types. A monkey is rarely pregnant with more than one offspring, so that a researcher can't insert many embryos into its womb and trust that a handful will survive.

Schatten hopes, nevertheless, that techniques developed recently in his lab¹⁷ will aid in efforts to create a chimeric monkey fetus. Predicting "months, maybe years, of frustration" before that point is reached, he stresses that his goal is not to propagate embryos but to create cells for therapeutic purposes. "That's where the nonhuman primate is really an extraordinary bridge," he says. "It's sort of a Rosetta stone for trying to understand how much we can extrapolate from mice and how much we can't." For critics of human ESC work, however, such primate studies may seem to be uncomfortably close to human cloning.

Douglas Steinberg is a freelance writer in New York.

References

1. R. Galli et al., "Skeletal myogenic potential of human and mouse neural stem cells," Nature Neuroscience, 3:986-91, October 2000.

2. R. Lewis, "A paradigm shift in stem cell research?" The Scientist, 14[5]:1, March 6, 2000.

3. K. Devine, "NIH lifts stem cell funding ban, issues guidelines," The Scientist, 14[18]:8, Sept. 18, 2000.

4. The hearings were partly linked to a bill, S.2015, introduced last January by Senators Arlen Spector (R.-Pa.) and Tom Harkin (D.-Iowa). S.2015 would lift the ban on NIH paying for the extraction of stem cells from embryos. The bill was shelved in September, but Spector plans to reintroduce it in the next Congress.

5. K.A. Jackson et al., "Hematopoietic potential of stem cells isolated from murine skeletal muscle," Proceedings of the National Academy of Sciences (PNAS), 96:14482-6, 1999.

6. D.L. Clarke et al., "Generalized potential of adult neural stem cells," Science, 288:1660-3, June 2, 2000.

7. M. Schuldiner et al., "Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells," PNAS, 97:11307-12, Oct. 10, 2000.

8. J.A. Thomson et al., "Embryonic stem cell lines derived from human blastocysts," Science, 282:1145-7, 1998.

9. M.J. Shamblott et al., "Derivation of pluripotent stem cells from cultured human primordial germ cells," PNAS, 95:13726-31, 1998.

10. T. Tada et al., "Epigenotype switching of imprintable loci in embryonic germ cells," Development Genes and Evolution, 207:551-61, 1998.

11. P.E. Szabo, J.R. Mann, "Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting," Genes and Development, 9:1857-68, 1995.

12. C.L. Stewart et al., "Stem cells from primordial germ cells can reenter the germ line," Developmental Biology, 161:626-8, 1994.

13. H. Niwa et al., "Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells," Nature Genetics, 24:372-6, April 2000.

14. U.S. Patent No. 5,843,780 titled "Primate embryonic stem cells"; accessible at www.uspto.gov.

15. A. Nagy et al., "Derivation of completely cell culture-derived mice from early-passage embryonic stem cells," PNAS, 90:8424-8, 1993.

16. S. Tanaka et al., "Promotion of trophoblast stem cell proliferation by FGF4," Science, 282:2072-5, 1998.

17. A.W. Chan et al., "Clonal propagation of primate offspring by embryo splitting," Science, 287:317-9, Jan. 14, 2000.

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