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 cells.
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 nuclear transfer.
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 cells.
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 factor – a.
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 feeder layer.
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.
References
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, 5131-5140.
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, 1691-1700.
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.
