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 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. 

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.

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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.