Objective To reprogramme the induced pluripotent stem (iPS) cells from Human umbilical cord mesenchymal cells (HuMSCs) and induce the iPS cells into germ cells by BMP4.
Methods OCT4, SOX2, Klf4,c-myc, Nanog, Lin28 were transfected into HuMSCs with lentivirus to reprogram HuMSCs into iPS cells. Morphological observation, Alkaline Phosphatase staining, karyotype analysis, RT-PCR, immunofluorescence staining, tumour formation in vivo and embryniod body formation in vitro were performed to examine the pluripotency of the iPS cell lines. Then we induced one of the iPS cells lines into germ cells by BMP4. Gene expression was measured by qRT-PCR at days 0, 3, 7, 10 and 14. Early-stage germ specific protein VASA and meiosis specific protein SYCP3 were assesssed by immunofluorescence staining.
Result We obtained two iPS cell lines completely reprogrammed, HuMSC-iPS1 and HuMSC-iPS2. HuMSC-iPS1 expresses germ cell markers at undifferentiated state. BMP4 can upregulate germ cell markers at different time points highly while the spontaneous differentiation just upregulate DPPA3, DAZL and VASA modestly at day 3. However, all of these genes were downregulated at day 14. VASA and SYCP3 immunofluorescence staining indicates there is a high VASA expression in BMP4 induced group in contrast to low expression in the spontaneous group at day 7. Meanwhile, there is a modest SYCP3 fluorescence in BMP4 induced group in contrast to no immunofluoresence in the spontaneous group.
Conclusion This system can reprogram HuMSCs into iPS cells effectively. The MSC- iPS1 can differentiate into early germ cells spontaneously while the germ cells induced by BMP4 can enter meiosis.