Human Embryonic Germ Cells
Bonnie Cooper, Dacia Foster, Alberto Gonzalez, Christine Huang, Reema Patel
(in alphabetical order, Fall 2005)
Teaching Assistant: Kelly Corcoran

The review was revised by Meriem Bendaoud & Rasha Nayal
(in Alphabetical order, Fall 2006)
Teaching Assistant: Reema Patel

      Stem Cells have recently been heralded by the media as a promising new tool in the fight of degenerative diseases.  However, much knowledge stands to be gained before stem cell therapies can be used as commonplace.  A full understanding of stem cells in their many forms and the progeny they give rise to are paramount to their future use.  Much attention has been paid to human Embryonic Stem Cells (hESCs).  Since the initial discovery of hESCs, other stem cell sources have been discovered one of which is hEGCs, these cells are a possible substitute with shown pluripotent ability.  Human embryonic germ cells can also serve as an additional tool for fighting disease.

Primordial Germ Cells

Embryonic germ cells (EGC) are the derivatives of primordial germs cells (PGC). They are isolated from the gonadal ridge and mesenchyma of fetal tissue (1,2). PGCs are the embryonic precursors of gametes and when allowed to mature they will form the sperm and egg cells (3). PGCs are first identifiable at the base of the allantois in the 7-day mouse embryo as a cluster of approximately eight cells (4,5). From there, the PGCs invade the endoderm, migrate through the hindgut and ultimately settle into the genital ridge (3). Migration within the hindgut requires the Stem Cell Factor (SCF)/c-kit interaction (6-9). Migration out of the hindgut and into the genital ridge occurs during day 9.5 of the developing mouse embryo (10) and it appears to be directed by factors produced by the genital ridge (4) (Figures 1 & 2). One such factor which is important in attracting PGCs to the genital ridge is the peptide growth factor Stromal Derived Factor 1 (SDF-1), which is produced by the stromal cells of the genital ridge and gonads and which interacts with the receptor CXCR4 receptor expressed on PGCs (11-14).  

EGC and ESC Properties
      Embryonic germ cells (EGC) and embryonic stem cells (ESC) have several similar characteristics. Both cell types replicate for an extended period of time, show no chromosomal abnormalities and express a set of markers regarded as characteristic of pluripotent cells. When culture conditions are adjusted to allow differentiation, both EGC and ESC cells can spontaneously differentiate into derivatives of the primary germ layers, endoderm, mesoderm and ectoderm.  In contrast, the ESCs are derived from the inner cell mass of a 5-day old embryo whereas EGCs are derived from the gonadal ridge of the 5-9 week fetus. Additional experiments using hESCs have shown the formation of teratomas (16). This illustration of teratoma formation is used as standard evidence of hESC pluripotency when injected into an NOD/SCID (immunosuppressed) mice (17). While hESC are known to produce teratomas, the engraftment of hEGCs into immunosuppressed mice will not actually generate teratomas (16,17). In fact, Embryonic Stem cells and Embryonic Germ cells from select species have been shown to form teratomas when injected (2).  Conversely hEGCs have yet to display this behavior.  To date, teratomas from analysis of in vivo hEGCs have not yet been observed (11).

Imprinting
      The imprinting status of EGCs is a major issue of concern in their potential for clinical use. Imprinting is the epigenetic change of DNA in which expression of only one of the parental alleles is evident. Epigenetic changes, which are the alteration of proteins surrounding DNA but not the DNA itself, involve DNA methylation and histone modification (18). Imprinting is necessary for normal function and involves about 100 imprinting genes. Erasure of the imprints is necessary between generations so that gametes of both sexes can be formed and expressed.  Also it’s important so that proper expression can occur when two sets of DNA combine (19,20). Studies with mouse EGCs show that tissues or differentiated cells from EGCs frequently do not exhibit imprinting genes properly (16,21). Studies with human EGCs appear altered, and more importantly show normal imprinting. (22). Understanding when erasure occurs is essential to harvesting EGCs without imprinting erasure for therapeutic use. It is believed that erasure can start as early as during movement towards the genital ridge. However most of the demethylation of PGCs occurs within one day of entering the gonadal ridge (21).

Human EGC Derivation
To understand how human EGCs were developed, it is important to recognize preliminary EGC work done in mice. Murine EGCs were derived years before human EGCs were, and it was the culturing conditions and characterization techniques developed for murine cells that were the basis for human EGC derivation (23). Important factors like Stem Cell Factor (SCF), (LIF), and (bFGF) were found to enhance murine Primordial Germ Cell (PGC) replication and survival, making them key for culturing (24).The PGC culture formed colonies of cells resembling murine ESCs. Initial characterization showed that cells were positive for markers such as SSEA-1.  The cells could also be maintained on feeder layers (5,24).
      Human EGCs were first derived in 1998 by a group led by John Gearhart. The gonadal ridges and mesenteries of 5-9 week fetuses, from therapeutically terminated pregnancies, were mechanically and chemically disaggregated, passaged on mouse fibroblast feeder cells and then cultured in growth medium containing Fetal Bovine Serum (FBS), LIF, bFGF, and forskolin. A significant portion of the cells were positive for AP, had markers similar of ESCs (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), and were morphologically similar to murine EGCs. The cells were also shown to have normal and stable karyotype for over 10 passages. When culturing the embryoid bodies (EB) without LIF, bFGF and forkolin, the pluripotency of the cells became apparent in vitro.  The result was a growth of a variety of cells from each germ layer, accordingly meeting important criteria for pluripotency (41). Later, other groups also derived human EGCs using similar methods (3,25-27). The EGCs were further characterized to show Oct-4 expression and telomerase activity, both of which are both important ESC properties (27).

Proliferation
PGCs can be transiently cultured on feeder cell layers expressing the transmembrane-bound isoform of SCF. SCF, together with LIF, promote PGC survival by suppressing apoptosis. Basic fibroblast growth factor (bFGF) is also active in PGC cultures, promoting germ cell proliferation. If PGCs are cultured with SLF, LIF, and bFGF, they can form cell lines termed EGCs. This could be attributed to the fact that hEGCs have been less successful at generating long-term, robust cultures as described earlier. It has been shown that they do not have quite the same set of pluripotency, or “stemness” markers as hESCs, which suggests they may be more mature or slightly more differentiated than hESCs (28). The loss of stemness as EGCs remain in culture may be due to EGCs continuing to follow PGC fate in the embryo or imprinting alteration and erasure in culture (19,20).

Clinical Applications
      Human EGCs spontaneously form EBs in culture as testament to their pluripotent capabilities.  Embryonic Body Cells (EBCs) are clusters of cells formed when the EGCs aggregate and randomly differentiate into precursors of the embryonic germ cell layers, simulating an environment of early embryonic development. Unfortunately, the only definitive way to ascertain a stem cell's pluripotency is from the formation of chimeric offspring (in trials with mice), but due to ethical considerations this is not practical with hEGC lines (1,29).                     The goal of current research is to develop “normal” cells that have the ability to function in vivo (28). EB formation is essential for applications in regenerative medicine, because it is a stepping stone towards the derivation of other cell lineages (2).  In vitro experiments with EGC-derived EBCs have been promising in yielding neuronal (26) and musculoskeletal cells (21).  In neuronal differentiation, EGC-derived EBCs in culture were shown to express typical neuronal progenitor markers such as nestin and N-CAM, while “stem” indicative markers such as Oct-4 and hTERT were undetectable.  When prolonged in culture, the cells began to physically resemble neurons, generating long tubular projections to interconnect with other cell aggregates (26). In another study their gene expression and protein synthesis were shown to follow chondrogenic and mesenchymal differentiation patterns (21).
      Of particular clinical interest are hEGC in vivo experiments, which are limited in number.  Recently, hEGC derived EBCs cultured for neuronal differentiation were implanted into damaged neural tissue in paralyzed rats. Over a period of time, the transplanted animals regained function whereas the controls remained paralyzed.  This could not be directly attributed to the function of the implanted cells, although they are thought to have directed regrowth and prevented neural cell death (30).  More recently, hEGC-derived human neural stem cells (NSC) were shown to repair brain damage in newborn mice. This suggests that the NSCs were able to engraft and repair the lost neurons (22). In addition, experiments have indicated that, by seeding a graft structure with hEGC derived cells, the bladder of a rat was reformed without loss of function or more importantly evidence of graft rejection (31).
      These preliminary studies with human embryonic germ cells have yielded very promising results for clinical application starting from nerve regeneration and beyond. hEGCs are particularly appealing because of one of their primary shortcomings, namely that they do not form teratomas upon injection. This may very well show that they are further along the differentiation pathway than embryonic stem cells (ESC).  This could be a blessing in disguise, making them much more conducive to predictable behavior in vivo, and thereby making hEGCs more appealing for clinical trials.  Hopeful thinking aside, much research yet needs to be done before regenerative medicine with stem cells of any kind moves to a patient's bedside. Much is to be learned and characterized about the differentiation of embryonic germ cells to make them of medicinal value, and in the process, researchers may stand to gain a greater depth of knowledge in the events of early development.
Oocyte Renewal
      The widely held dogma of female reproductive biology is that females of most mammalian species lose the ability to replenish their oocyte population altogether during fetal development. Thus, unlike their male-counterparts, females are born with a finite number of oocytes (16,23), which will be eventually become exhausted and lead to menopause (28). However, contrary to this long-standing belief, it has been recently shown that oogenesis, the production of new oocytes, continues into adult life. It has been demonstrated that germline stem cells (GSC) in female mice are capable of replenishing the oocyte population in mature females (27). Furthermore, and even more surprisingly, it has been suggested that these GSC’s in adult female mammals may actually reside in the bone marrow and peripheral blood (32). However, it is important to note that further research is needed to determine whether or not the putative oocytes generated from GSC’s are competent for fertilization, capable of embryonic development or creation of a viable fetus.
Research Guidelines
Research conducted using EGCs are under the jurisdiction of the federal government and private sectors. The federal policy regarding embryonic stem cells (ESC) is that they must be obtained from a list of stem cells approved by President Bush in 2001. These 22 cell lines are already established. The National Institute for Health (NIH), a federal government medical and research organization, and the Office for Human Research Protections Department of Health and Human Services (OHRP) also mandate the use of these cells (14). Researcher's seeking to use new embryonic stem cell lines or those stem cell lines that are derived from existing cells lines is prohibited from obtaining federal government funding. Regarding embryonic germ cells, researchers have the liberty to use new or derived versions of embryonic germ cells with Institutional Review Board approvals by the NIH's Human Pluripotent Stem Cell Review Group (HPSCRG) and still receive federal funding (33).
      Alternatives to cell lines not approved by the federal government include obtaining funding from private sectors and using animal models. Currently there are no policies banning the use of new and derived embryonic stem cell for research funded by private companies (14). However, this alternative alters the right to use embryonic germ cells for researchers because they are permitted to use any germ cell with federal funding (33). Animal models of germ cells are the second alternative. Researchers are free to use animal embryonic germ stem cells under federal and private funding. However, these models are limited because extrapolations must be made to human models and the use of animal models contains their own regulations and could therefore alter data and results (33).

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