The Published Record
John Gearhart, Ph.D.
C. Michael Armstrong Professor, Institute for Cell Engineering,
Johns Hopkins University School of Medicine
There have been but two original research articles published
on human embryonic germ cell in the period covered by this
report. It is appropriate that only peer-reviewed articles
be considered in this report, as the field of stem cell
research is rife with undocumented or unsubstantiated claims.
There have been several publications on EG cells derived
in mice and chick and comment will be made on these reports
as they may impact on the eventual use or study of the human
There is a concern, given the imprinting-based developmental
abnormalities observed in humans, and those that have been
produced experimentally in animals, that dysregulated gene
expression in stem cell derived tissues could pose a serious
problem in the use of such tissues for cell-based therapies.
Genomic imprinting is defined as an epigenetic modification
of the DNA (other than sequence) in the germ line that leads
to the preferential expression of a specific allele (monoallelic
expression) of some genes in the somatic cells of the offspring
in a parental dependent manner. Because a maternal gene
allele may be a paternal allele in the next generation,
the imprinting must be reprogrammed in the germ line, that
is, the epigenetic 'marks' must be erased (during germ cell
development) and established in the newly formed embryo.
The timing of the erasure in humans is not known. Imprinting
involves methylation at specific sites in the DNA and most
likely, other changes as well.
There have been several reports from experiments with the
mouse that indicate that imprinting is abnormal in pluripotent
stem cells derived from mouse embryos. These studies include
abnormal or variable imprints in mouse EG cells, abnormal
imprints in ES cells derived from interspecific crosses,
and abnormal gene expression in mice derived from ES cell
The results of the study by Onyango et al. utilizing
EG cell lines derived at John Hopkins, clearly demonstrate
that general dysregulation of imprinted genes will not be
a barrier to their use in transplantation therapies. The
report has determined that the EG cells are not imprinted,
that is, imprinting has been erased in the primordial germ
cells that gave rise to the EG cells, that the erasure is
maintained in the EG cells, but in all informative cases,
they observed the transcription of only a single allele
in differentiated cells derived from the human EG cells.
These results, although on a limited number of lines and
only a few imprinted genes, would indicate that these human
EG cell lines will serve as reliable and safe sources for
the study of EG cell differentiation and, perhaps, cells
for cell-based interventions.
Another area of interest in genetic regulation within EG
cells is that of X inactivation, the mammalian method for
equalization of the dosage of X-linked genes in males and
females. This equalization is accomplished by the down
regulation of the transcriptional output of the X chromosomes
in females, so that only one X is active in diploid somatic
cells of both sexes. Inactivation is initiated in female
blastocysts. Both X chromosomes in female primordial germ
cells are active. Migeon et al. (2001) have demonstrated
that in the very early stages of differentiation of cells
from human EGs, only one X chromosome is active, indicating
normal genetic regulation has occurred. In a report by Nesterova
et al. (2002) on the use of mouse female EG cells
in the study of X chromosome inactivation/reactivation during
primordial germ cell migration and EG cell formation. Both
X chromosomes appear to be active in XX EG cells, and presumably,
one becomes inactive when cells differentiate from the EG
One of the goals of stem cell research is to provide sources
of cells for cell-based therapies. As a step in this direction,
proof of concept or proof of principle studies involve the
use of human cells in animal models of human disease or
injury. Although few, if any, animal models are true models
for the human diseases, they are the closest approximation
that can be made. The first report on the use of cells
derived from stem cells of human embryonic sources was recently
published: Kerr et al. Human Embryonic Germ Cell
Derivatives Facilitate Motor Recovery of Rats with Diffuse
Motor Neuron Injury, J. Neuroscience 23, June 15, 2003.
This is the first demonstration that a human pluripotent
stem cell derived form embryonic or fetal tissue can ameliorate
a disease process in an animal model.
Neural progenitor cells, derived from human EG cells, were
introduced into the cerebrospinal spinal fluid of rats that
had been paralyzed as a result of infection with a neuroadapted
Sindbis virus that specifically targets motor neurons in
the spinal cord. All animals in which human cells were
found had some degree of hindlimb recovery. It was clear
from the histology of the animals that the human cells had
differentiated into appropriate neural cell types within
the ventral horns, including motor neurons, the results
indicated that the major effect of the human cells was to
protect host neurons from death and to facilitate reafferentiation
of motor neuron cell bodies. Growth factors responsible
for this recovery, produced by the human cells, were identified
as brain-derived neurotrophic factor and transforming growth
The significance of this experiment appears to be that
for this disease model, the human cells supply factors that
facilitate motor neuron recovery following viral damage.
However, the cells did migrate to the site of injury in
the spinal cord, which differentiates them from other cellular
grafts, and then delivered the factors. Also, there was
considerable differentiation of the human cells into various
neural cells types in the cord.
In a review article on deriving glucose-responsive insulin-producing
cells from stem cells (Kaczorowski et al. 2002),
mention is made on the isolation of such cells from mouse
and human ES cells and human EG cells, but no new data was
presented, only a reference to a paper published in 2001.
EG cell studies in other species
Several studies on EG cells from other species have been
published. EG cells from the chick have been demonstrated
to yield germ line chimeras when transferred to early embryos
(Park et al. 2003). EG cell lines of the mouse were
found to colonize not only the epiblast but also the primary
endoderm of the gastrulating embryo following aggregation
with 8-cell embryos (Durcova-Hills et al. 2003).
This observation was permitted as a result of using a transgenic
construct effect as a lineage marker for cells of the early
embryo. Horii et al. (2002) report the serum-free
culture of mouse EG cells, a system which inhibits the 'spontaneous'
differentiation due to the presence of various growth factors
in the serum. The cells are, however, are cultured on a
An area of great interest in EG cell derivation is the
basis of the underlying mechanism for the conversion, derivation
or transformation (terms reflecting our ignorance of the
process) of primordial germ cells to EG cells. Kimura et
al. (2003) report that the loss of a tumor suppressor
gene in mice, PTEN (phosphatase and tensin homology deleted
form chromosome ten, aka MMAC1 and TEP1), leads to a high
incidence of testicular teratomas and enhances the production
of EG cells from PGCs of the mutant mice. While it is highly
unlikely that the loss of this gene in the culture of normal
PGCs leads to the derivation of EG cells, it may implicate
downstream signaling pathways that are involved in cell
cycle progression, cell survival and cell migration as important
players in the process.
Durcova-Hills, G., Wianny, F., Merriman,
J., Zernicka-Goetz, M., and McLaren, A. 2003. Developmental
fate of embryonic stem cells (EGCs), in vivo and
in vitro. Differentiation 71, 135-141.
Horii, T., Nagao, Y., Tokunaga, T.,
and Imai, H. 20003. Serum-free culture of murine primordial
germ cells and embryonic germ cells. Theriogen. 59, 1257-1264.
Kaczorowski, D.J., Patterson, E.S.,
Jastromb, W.E., Shamblott, M.J. 2002. Review Article. Glucose-responsive
insulin-producing cells from stem cells. Diabetes Metab.
Res. Rev. 18, 442-450.
Kerr, D.A., Lladó, J., Shamblott, M.J.,
Maragakis, N.J., Irani, D.N., Crawford, T.O, Krishnan, C.,
Dike, S., Gearhart, J.D., and Rothstein, J.D. 2003. Human
embryonic germ cell derivatives facilitate motor recovery
of rats with diffuse motor neuron injury. J. Neurosci. 23,
Kimura, T., Suzuki, A., Fujita, Y.,
Yomogida, K., Lomeli, H., Asada, N., Ikeuchi, M., Nagy,
A., Mak, T.W., and Nakano, T. 2003. Development and Disease.
Conditional loss of PTEN leads to testicular teratoma and
enhances embryonic germ cell production. Development 130,
Migeon, B.R., Chowdry, A.K., Dunston,
J.A., and McIntosh, I. 2001. Identification of TSIX,
encoding an RNA antisense to human XIST, reveals
differences from its murine counterpart: Implications for
X inactivation. Am. J. Human Genet. 69, 951-960.
Onyango, P., Jiang, S., Uejima, H.,
Shamblott, M.J., Gearhart, J.D., Cui, H., and Feinberg,
A.P. 2002. Monoallelic expression and methylation of imprinted
genes in human and mouse embryonic germ cell lineages. Proc.
Natl. Acad. Sci. USA 99, 10599-10604.
Park, T.S., Hong, Y.H., Kwon, S.C.,
Lim, J.M., and Han, J.Y. 2003. Birth of germline chimeras
by transfer of chicken embryonic germ (EG) cells into recipient
embryos. Molec. Reprod, Develop. 65, 389-395.
Sapienza, C. 2002. Commentary. Imprinted gene expression,
transplantation medicine, and the "other" human embryonic
stem cell. Proc. Natl. Acad. Sci. USA 99, 10243-10245.