Glycogen synthase kinase 3 (GSK3) inhibitor 6- bromoindirubin-3'-oxime (BIO) promotes the proliferation of mouse male germline stem cells (mGSCs) under serum- and feeder-free conditions

The 6-bromoindirubin-3'-oxime (BIO), as one of glycogen synthase kinase 3 (GSK3) inhibitors, is a key regulator of many signaling pathways with the capacity to maintain the pluripotency of human and mouse embryonic stem cells (ESCs). Whether BIO can maintain the pluripotency of mouse male germline stem cells (mGSCs) remains unclear at present. In this study, with BIO and retinol (RE) both in Knock-out serum replacement (KSR) medium, we got a relatively optimal feeder- and serum-free system for mGSCs in vitro. After continuous culturing, the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC-induced embryoid bodies (EBs) were examined as well. Results show that in the presence of LIF or retinol, BIO significantly increased the number of mGSCs and alkaline phosphatase (AP) positive colonies, and the mitosis index through the BrdU assay. BIO also increased the expression of pluripotent markers including Oct4, Nanog, PCNA and c-Myc analysed by real-time polymerase chain reaction (PCR). The mGSCs cultured in RE medium containing BIO can form embryoid bodies (EBs), which consist of three embryonic germ layers analysed by immunofluorescence. When these cells were transplanted into infertile mice, they could well repair the capacity of making male germ cells. Our results demonstrate the combined treatment of BIO and RE could significantly promote the proliferation of mouse mGSC colonies and maintain the undifferentiated status.