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Embryonic Germ Cells

(A Lay Summary)

An extensive effort is being put forth by the scientific community in search of human pluripotent cells for use in regenerative medicine and cell replacement techniques. Much attention has been paid worldwide to human Embryonic Stem Cells (hEGCs), however, other viable options exist. Human Embryonic Germ Cells (hEGCs), like hESCs, are capable of forming cells from all three primary germ layers (endo, ecto, and mesoderm). In addition, they have been shown to proliferate in culture and display a set of cell surface markers characteristic of pluripotency. Pluripotency refers to a cell's ability to give rise to different cell lineages

hEGCs are derived from cells isolated from the gonadal ridge of 5-to-9-week-old fetal tissue known as Primordial Germ Cells (PGCs). PGCs are diploid embryonic precursors of cells that will mature as the source for haploid gametes found in the testes or ovaries of adults. PGCs originate in the yolk sack and migrate to the developing genital region via the allantois, through the hind gut, and past the mesentery, until they reach the developing gonadal ridge. The PGC journey is guided by the permissive environment of the developing embryo, and proliferation is fostered by the factors Leukemic Inhibitory Factor (LIF) and Stem Cell Factor (SCF; also known as Steel factor or mastocyte growth factor). These factors are small, quickly degradable molecules known as cytokines. LIF and SCF help cells maintain pluripotency while proliferating, making them key cytokines in development. Once reaching the ridges, the cells will engraft. However, in some instances the cells follow inappropriate migration patterns. Cells usually undergo apoptosis, which is programmed cell death, but in rare cases misguided cells will form teratomas (tumors).

As the PGCs migrate towards the gonadal ridge, they undergo a process known as erasure. Organisms that are the product of sexual reproduction have a method for selecting which copy of select genes they will express, whether from the father's genetic contribution, or from the mothers. Called imprinting, it silences one copy while letting the other be expressed without changing the DNA. Erasure is the deleting of the imprinting surrounding the genetic content of the PGCs and is necessary so that their haploid progeny will be able to form their own imprinting. Erasure is in the interest of the offspring, but is a complication for using PGCs as pluripotent cells, as it could lead to abnormal gene expression. As a result, much research is being made to determine when erasure occurs and how to select PGCs with their imprinting intact.

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

hEGCs are derived from these PGCs as follows. The gonadal ridge tissue is isolated from a human fetus (from therapeutically terminated pregnancies) to be mechanically and chemically broken-up. The cells are then maintained in a cocktail of enzymes and growth media. SCF, LIF as well as basic Fibroblast Growth Factor (bFGF) are included in the growth media to promote cell proliferation and prevent cell death (apoptosis). In culture, hEGCs have been characterized to be morphologically similar to hESCs and to share many of the same surface markers. Surface markers are membrane- bound protein receptors involved in cellular communication, and can be indicative of cell type and function.

The phrase "don't judge a book by its cover" holds true for any type of stem cell, hEGCs included. In other words, just because they carry the same surface markers as hESCs, there is no guarantee that they will behave as pluripotent stem cells. A common test of pluripotency is to inject the cells of interest into an immunocompromised mouse with the observance of teratomas being the desired outcome. Although hESCs and EGCs from other species have been shown to form this type of tumor, human EGCs have yet to display this behavior, although it is unclear whether this is due to an intrinsic property of hEGCs or a consequence of working with a murine model. Nevertheless, hEGCs spontaneously form aggregates of cells with precursors of the embryonic germ layers known as Embryoid Bodies (EB). The ability to create EBs is one indicator of pluripotent ability. However, the only definitive way to ascertain pluripotency is by forming chimeric offspring; but due to ethical considerations this is not possible with hEGC lines.

Embryonic body formation is of importance to regenerative medicine as it is used as a stepping stone towards the derivation of different cell types. The aim of current research is to take these hEGC derived EB cells (EBCs) and guide their differentiation in culture into "normal" cells that will function in vivo. Current in vitro studies have been successful at creating neuronal and musculoskeletal cells. In vivo, scientists have succeeded using rodent models to use hEGCs to regenerate motor neuron function, repairing brain damage. Unfortunately, it is difficult to say whether the implanted cells were functioning as neurons to regain function, of if they were creating a microenvironment to foster regrowth of existing nerve tissue.

Figure 2: Embryonic Germ Cell Derivation

Figure 3: Experimental Models

These preliminary studies have yielded promising results for clinical applications. Armed with their capability to derive other cells types, hEGCs are also appealing because they have not be shown to form teratomas upon injection. Ironically this was one of their shortcomings and may be a blessing in disguise, making them much more conducive to predictable behavior in vivo, and therefore favorable for clinical applications (a current hurdle with hESCs).

However, much is left to be learned about these ESC alternatives before they can be used in a clinical setting. Much is left to be learned and characterized about the differentiation of hEGCs to make them of medicinal value. Fortunately for hEGC researchers there are comparatively liberal restrictions in place. In 2001, President Bush limited hESC research to a list of 68 stem cell lines. Of which, only ~22 are of use to research. Although legislation may change, the current restrictions have severely limited hESC research with federal funds in the United States. On the other hand, research regarding EGCs is at liberty to use new or derived EGC lines with federal funds. In summary, research with EGCs may be far from providing a patient's bedside cure, but it has to potential to do just that. In the process we stand to gain insight to events early in our development.

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.