Modeling Human Fetal Germ Cell Development In Vitro.

Abstract

Primordial Germ Cells (PGCs) express many of the classical markers of pluripotency, such as Oct-4 and Alkaline Phosphatase; suggesting a close relationship exists between germ cells and pluripotent stem cells. Ex vivo, mammalian PGCs undergo a limited number of divisions before arresting and senescing. Under specific culture conditions some PGCs overcome this block and adopt a phenotype of pluripotency and unlimited self-renewal, becoming Embryonic Germ (EG) cells. Mouse EG cells have been shown to re-acquire a germ cell phenotype when co-cultured with gonadal somatic cells, indicating that they retain the ability to respond to the microenvironment of the germ cell niche. EG cells may therefore present an opportunity to study the mechanisms of early germ cell development in vitro. The derivation of human EG cells from PGCs has been reported previously, however the potential of human EG cells to differentiate back into PGCs in vitro has not been investigated. Here we report our efforts to establish and characterize long-term cultures of human PGCs and EG cells. We have successfully maintained human PGCs from several first trimester human fetal gonads in culture for least three weeks on feeder layers of mitotically-inactivated STO feeders. Throughout the period of culture, the germ cells maintained robust expression of alkaline phosphatase. Studies are continuing to characterize the expression of other stem and germ cell markers in our cultures using RT-PCR and immunocytochemistry, and to determine the effects of various growth factors on the survival and proliferation on short term cultures of human PGCs in vitro. By combining human EG cells with gonadal somatic cells we ultimately aim to develop a culture system that allows us to recapitulate human germ cell development in vitro, and use this system to analyze the effect of growth factors, signaling molecules and disruption of genes of candidate fertility genes on human fetal gametogenesis.