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. 2021 Nov 10;10(11):3099. doi: 10.3390/cells10113099

Figure 6.

Figure 6

Dynamics of pluripotent and germ cells markers during hPGCLCs -TS induction. (A) Schematic protocol used to generate hPGCLCs. (B) hiPSCs-TS (p20), hEpiLC-TS (D.2), and hPGCLCs-TS (D.6) quantification of the relative expression of OCT4, SOX2, NANOG, DPPA3, TFAP2C, SOX17, PRDM14, NANOS3, VASA, and DAZL genes associated with germ cells development. Individual reactions of qRT-PCR were normalized to the Β-ACTIN gene. The data was compiled from at least four biological replicates and three technical replicates. p values were calculated by Two-way ANOVA, followed by Tukey’s multiple comparisons test as appropriate. (letters a-b-c show difference between groups). Error bars represent SEM. (C–F) PGCLCs-TS of Pt.1; PGCLCs-TS of Pt.2; PGCLCs-TS of Pt.3; PGCLCs-TS of Pt.4 showed. Phase contrast image showed hiPSCs-TS maintained in mTeSR-1 medium on Geltrex; hiPSCs-TS(p20) stimulated via hEpiLC; PGCLCs -TS (d4) cultured in Agreewell plates. Scale bars: 200 μm; hPGCLCs-TS (D.6) isolated for further characterization. Scale bars: 400 μm. Immunofluorescence analysis showed pluripotency and germ cell markers DDPA3, VASA, OCT4, DAZL, SOX17, and AP-2γ. Nuclei were stained with Hoechst (blue). Scale bars: 20 μm. Immunofluorescence analyzed showing the epigenetic profile of histones H3K27me3, H3K9me2, and H4K20; and DNMT3B and KDM6A protein markers. Nuclei were stained with Hoechst (blue). Scale bars: 20 μm. The data was compiled from at least two technical replicates for all staining.