Fig. 2

Differentiation of hiPSC lines into germ cell-like cells. A Western blot detected the expression of PIWIL2 using an antibody whose antigen was the middle part of PIWIL2 in human testis with normal spermatogenesis, human fibroblasts, and different hiPSC lines. Very faint band appeared in PIWIL2^−/− hiPSCs might because the antibody bound to nonspecific proteins. B PGCLCs were stained with ITGA6 and EpCAM and the percentage of PGCLCs reflecting the PGCLC induction efficiency was determined at 4 days of differentiation. n = 3, n.s., not significant. C SSCLCs were stained with PLZF and the percentage of SSCLCs reflecting the SSCLC induction efficiency was determined at 8, 10, 12, and 14 days of differentiation. n = 3, * versus Control, # versus PIWIL2^+/−, p < 0.05. D Detection of SSC-related gens in the control, PIWIL2^−/−, PIWIL2^+/− and patient groups by RT-qPCR at 12 days of differentiation. n = 3, * versus Control, # versus PIWIL2^+/−, p < 0.05. E Immunofluorescence staining of PLZF (green) and GPR125 (red) in the control, PIWIL2^−/−, PIWIL2^+/− and patient groups at 12 days of differentiation. The nuclei were stained with DAPI (blue). PLZF⁺/GPR125⁺ cells represented SSCLCs; they had relatively small nuclei and grew in clusters. There were less PLZF⁺/GPR125⁺ cells in the patient and PIWIL2^−/− groups, but cells with larger nuclei represent different differentiated cells