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Human Embryonic Germ Cells

(A Scientific Review)

Introduction

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 cells can be used in routine therapies. A full understanding of stem cells in their many forms and the progeny they give rise to is paramount to their future use. Much attention has been paid to human Embryonic Stem Cells (hESCs). However, human Embryonic Germs Cells (hEGCs) are a substitute with shown pluripotent ability. If characterized further, hEGCs could 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, 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, shown in Figure 1 (3). Migration within the hindgut requires stem cell factor (SCF, also known as steel factor) and its interaction with its receptor, c-kit. (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 (11). One such factor which is important in attracting PGCs to the genital ridge is stromal derived factor 1 (SDF-1), which is produced by the stromal cells of the genital ridge and gonads. SDF-1 interacts with the CXCR4 receptor expressed on PGCs (9, 12, 13).

Figure 1: Migratory Path of Primordial Germ Cells to the Gonadal Ridge

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 (1, 14) when the culture conditions are favorable, both EGC and ESC cells can spontaneously differentiate into derivatives of endoderm, mesoderm and ectoderm primary germ layers.

On the other hand, EGCs and ESCs differ in origin, tumorogenicity, and robustness in culture. ESCs are derived from the inner cell mass of a 5-day old embryo whereas EGCs are derived from the gonadal ridge of a 5-9 week fetus. The injection of ESCs into immunosuppressed mice reproducibly leads to teratoma formation (15), which is used as standard evidence of ESC pluripotency (16). While ES and EG cells from select species have been shown to form teratomas (2), human EGCs have yet to display this behavior (12, 15, 16). Additionally, EGCs have been less successful at generating long-term robust cultures than ESCs and have not been characterized with regard to signature molecules as compared to hESCs. EGCs are relatively more differentiated than ESCs (17). This is not surprising since studies have shown derivation of EGCs from ESCs (18). The loss of `stemness' as EGCs remain in culture could be due to EGCs continuing to follow the differentiation pathway to PGC fate, or imprinting alterations in culture (19, 20).

Imprinting

A major issue in the potential clinical use of EGCs is their imprinting status. Abnormal imprinting impedes normal cell behavior and would not be desirable for therapies. Imprinting is the epigenetic change of DNA to only express one of the parental alleles. Epigenetic changes, which are the alteration of proteins surrounding DNA but not the DNA itself, involve DNA methylation and histone modification (21). Imprinting is necessary for normal function and to date, 100 genes have been assigned to imprinting properties. Erasure of the imprints is needed between generations 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 properly exhibit imprinting genes (15, 22), whereas studies with human EGCs show altered, but more normal imprinting. (23). Understanding when erasure occurs is important in harvesting EGCs with normal imprinting for therapeutic use. 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 (22).

Figure 2: Embryonic Germ Cell Derivation

Human EGC Derivation (Figure 2)

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, and it was the culturing conditions and characterization techniques developed for murine cells that were the basis for human EGC derivation (24). Important factors such as SCF, leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) were found to enhance murine PGC replication and survival, making them key for culturing (25). SCF and LIF suppress apoptosis, and bFGF promotes germ cell proliferation. The PGC culture formed colonies of cells resembling murine ESCs. Initial characterization showed that cells were positive for alkaline phosphatase (AP) activity, positive for markers such as SSEA-1, and could be maintained on feeder layers (5, 25).

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 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 to 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. The pluripotency of the cells was shown in vitro by culturing the embryoid body cells (EBCs) without LIF, bFGF, and forskolin. EBCs are clusters of cells formed when the EGCs aggregate and randomly differentiate into precursors of the embryonic germ cell layers. Similar EBC behavior has been observed with ESCs. A variety of cells from all three germ layers were formed, thus meeting an important criterion for pluripotency (1). Later, other groups also derived human EGCs using similar methods (14, 26, 27). The EGCs were further characterized to show Oct-4 expression and telomerase activity, which are both important ESC properties (14). Unfortunately, the only definitive way to ascertain a stem cell's pluripotency is the formation of chimeric offspring (in trials with mice), but due to ethical considerations this is not practical with hEGC lines (1) (Figure 3).

Figure 3: Experimental Models

Clinical Applications

The goal for current research is to develop epigenetically normal cells that will be functional in vivo (17). 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 (27) and musculoskeletal cells (22). 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 (27). In another study their gene expression and protein synthesis were shown to follow chondrogenic and mesynchymal differentiation patterns (22).

Of particular clinical interest are in vivo experiments with EGC, 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. Although, this could not be directly attributed to the function of the implanted cells, they are thought to have directed regrowth and prevented neural cell death (28). More recently, hEGC-derived 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 (23). In addition, experiments have indicated that seeding a graft structure with hEGC-derived cells, the bladder of a rat was reformed without loss of function or evidence of graft rejection (29).

The discussed preliminary studies with hEGCs have yielded promising results for clinical application starting from nerve regeneration and beyond. Human EGCs are particularly appealing because of lack of teratomas, as seen by ESCs. This property makes the behavior of EGCs relatively predictable for clinical applications. Despite the current research, much basic research studies are necessary before reaching the bedside.

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 (30, 31), which will be eventually become exhausted and lead to menopause (32). 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 (33). 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 (34). 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 in 2001.These 22 cell lines were 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 for federal-funded research. Researchers are not restricted on the use of EGCs, although their use requires approval by their Institutional Review Board.

Non-federal approved ESCs can still be used, except with funds from private sectors. This restriction does not apply to EGCs, which could use federal funds. Animal models of germ cells are another alternative for non-human studies. However, animal models are limited because extrapolations must be made to human models. The use of animal models is also under strict guidelines.

Acknowledgements

This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey:

Bonnie Cooper, Dacia Foster, Alberto Gonzales, Christine Huang, Reema Patel (in alphabetical order).

Teaching Assistant: Kelly Corcoran

The review was edited by two stem cell biologists.