PMC

(A) Immunofluorescence staining with SCP3 is shown. Scale bars, 10 μm. (B) FISH with probe against autosomal chromosome 16 and chromosome 22 in the undifferentiated hESCs group and two induced groups. Scale bars, 10 μm.

(A) Immunofluorescence straining of β-catenin and OCT4 in 3 d CHIR99021 plus IWR-1 co-culture group and undifferentiated group. Scale bars, 50 μm. (B) Immunofluorescence straining of DAZL in 12 d CHIR99021, IWR-1 and RA co-culture group and undifferentiated group. Scale bars, 50 μm.

(A) Immunofluorescence analysis showing the expression of OCT4, DAZL, and β-catenin after the treatment of 3 d CHIR99021. Scale bars, 25 μm. (B) Statistical analysis of the percentage of β-catenin in the nuclei. All data are presented as means ± SEM. *p < 0.05, **p < 0.01,***p < 0.001.

(A) The protocol used for the differentiation of hESCs into PGCs. (B) The hESCs were treated by CHIR99021 and/or RA as indicated in (A). The DAZL positive cells were detected by using immunofluorescence staining nuclei were counterstained with DAPI. Scale bars, 25 μm. (C) Summary data showing the mean number of the DAZL positive cells in the different treatments. All data are presented as means ± SEM. *p < 0.05, **p < 0.01.

(A) mRNA levels of DDX4, Blimp-1, Nanos and TFAP2C was determined by RT-qPCR. The two hESCs lines were treated by 12 d CHIR99021 plus RA co-culture and 3 d CHIR99021 plus 9 d RA. The undifferentiated hESCs were used as control. (B) The two hESCs lines were treated by 12 d CHIR99021 plus RA co-culture or 3 d CHIR99021 plus 9 d RA. The undifferentiated hESCs were used as control. Then, Acrosin, DDX4, β-catenin and β-actin were detected by Immunoblots. All data are presented as means ± SEM in three independent experiments vs undifferentiated hESCs. (C–E) Flow cytometry analysis of c-Kit⁺, CXCR4⁺ and DAZL⁺ cells. The undifferentiated hESCs were used as a control shown on the left. The percentage shown in the histogram is the rate of positive cells. The middle icon represents 3 d CHIR99021 plus 9 d RA, the right icon stands for 12 d CHIR99021 and RA co-culture treatment. All data are presented as means ± SEM in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.