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The Scientist - Volume 14, Number 22, November 13, 2000
http://www.the-scientist.com/yr2000/nov/index_001113.html

The Scientist 14[22]:1, Nov. 13, 2000
NEWS
Stem Cell Discoveries Stir Debate
Issues take on greater urgency as NIH guidelines go into effect
By Douglas Steinberg

http://www.the-scientist.com/yr2000/nov/steinberg_p1_001113.html


Editor's Note: This is the first of two articles on questions
raised by recent stem cell discoveries. The second article,
focusing on various organs and the nervous system, will appear in
the Nov. 27 issue of The Scientist.

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?

Pluripotent murine embryonic germ cells identified by red staining.
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."

A phase contrast image of the pluripotent murine embryonic germ
cell.
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."

A culture dish containing hundreds of colonies of murine embryonic
germ cells. Each red-stained spot represents an individual colony
comprised of hundreds or thousands of stem cells.

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.

Photo: Paul Vincent Kuntz

Margaret A. Goodell
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.'"

Reprinted by permission from Nature (359:550-1(1992)©2000
Macmillan Magazines Ltd.

Antigenic and morphologic characterization of PGC-derived EG cells.
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 medium8,14; 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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