Characterization of the Self-Renewal/Reprogramming Factors of Human/Mouse Germ Line Stem Cells in Serum-Free Culture System and Its Induction Effect on Human Embryonic Stem Cells to Sperm (Extension Proposal) (II)

Derivation pluripotent stem cells from the adult human testis have important implications for reproductive and regenerative medicine. Successful generation of pluripotent germline stem cells (GSCs) from testis of neonatal/adult mice and adult human have been demonstrated using a high serum-containing culture system. However, the complex serum component in medium not only dramatically reduces the value of translational medicine, but also limits the finding of regulatory factors in germ cell pluripotency. Thus, a serum-free culture system is the critical point for successful clinical use. For this purpose, we have recently established a serum-free stem-niche co-culture system to generate pluripotent stem cells from neonatal mouse testis. By using such an ex-vivo-like cell platform, we uncovered an important role of IGF-1/IGF-1R signaling in regulation of mouse germ cell pluripotency (2009 FASEB J 23: 2076-2087). Furthermore, we found niche hypoxia will enhance the IGF-1R signaling to up-regulates the cell stemness (Oct-4, Sox2, Nanog, and Klf-4), proliferation, and migration of pluripotent GSCs (preliminary results). These observations strongly highlight a hypoxia-IGF-1R signaling circuit in early germ cell development. Therefore, in this proposal, we plan to utilize the serum-free culture system combined with hypoxic condition to derive pluripotent human GSCs in vitro. The finding in this proposal would create an important clinical value in infertility translational medicine; and, the scientific inputs of basic research and biotechnology. Two specific aims will be addressed here: Specific aim 1: To generate potential pluripotent human AP+GSCs in vitro using a serum-free culture system under hypoxia condition Specific aim 2: To identify the essential endocrinal signaling in regulation of human pluripotent AP+GSCs formation under hypoxia condition