Niche Extracellular Matrix (Ecm) and Hypoxia Promote Symmetric Self-Renewal Proliferation and Migration in Embryonic Germline Stem Cells through Igf-1r-Hif-2a Central Signaling Pathway

Microenvironment niche and hypoxia stress are involving in germline stem cell (GSC) development. During embryogenesis, the competence and specification occur in early primordial germ cell (PGC), and subsequently execute self-renewal and migration toward the genital ridges. However, niche extracellular matrix (ECM) cooperates with hypoxia-derived signals to modulate pluripotency and migration of PGC still remains largely unknown. Gaps in the understanding of niche and endocrine signals in GSC development may be due to the absence of an appropriate in vitro stem cell model. Although regular serum-containing medium should enhance the stem cells proliferation, but it also greatly reduce the cell stemness and interfere the identification of potentially endocrinal factors for stem cell determination. In this regard, we have previously established an in vitro serum-free culture system to generate PGC-like pluripotent stem cells and identified a IGF-1/IGF-1R dependent pathway in maintenance of germ cell pluripotency, and also uncovered a regulatory loop of IGF-1R–HIF-2 signal loop in proliferation and OCT-4 maintenance in Alkaline Phosphatase (AP)+ GSCs under hypoxia. Our preliminary results found that the AP+GSCs showed significant AP activity and expressed stemness-related genes particularly when cultivated on Laminin-coated culture dishes. Moreover, we found that Laminin, CD49f, HIF-2 and OCT-4 expressed in the GSCs of P2 postnatal mouse testis. Treatment of IGF-1 increased expression levels of CD49f, IGF-1R, HIF-2, and OCT-4 in CD49f+AP+GSCs. Knockdown of endogenous IGF-1R effectively suppressed the expressions of CD49f. In addition to ECM niche, hypoxia niche increased AP+GSCs colony formation, colony size, AP activity, and migration ability. Furthermore, we identified SDF-1 protein in conditioned medium under hypoxia. We also confirmed that Sdf-1 and its receptor Cxcr4, as well as Igf-1r, Hif-2, and Oct-4 genes were up-regulated in CD49f+AP+GSCs under hypoxia. Together with these results highlight the potential role of IGF-1R in cell self-renewal proliferation and migration with underlying mechanism of CD49f and CXCR4 during early germ cell development. Thus, to address the point, this proposal aims to identify the embryonic signaling networks of niche and endocrinal IGF-1 in stem cell maintenance and migratory regulation of CD49f+AP+GSCs under hypoxia and ECM surrounding. Three aims will be addressed as listed: Aim 1: To examine the cross-talk between niche ECM and IGF-1R–HIF-2 signals in regulating pluripotent transcription factor OCT-4 expression in CD49f+AP+ germline stem cells (GSCs). (1st and 2nd year) Aim 2: To examine how the niche hypoxia mediates self-renewal proliferation and migration in CD49f+AP+GSCs. (2nd and 3rd year) Aim 3: To examine the cross-talk between the IGF-1R and CXCR4 signaling in regulating cell migration of CD49f+AP+GSCs under hypoxia. (3rd year)