Fig 2: Enhanced the expression of the stem cell markers. A & B. Cells were cultured under normoxia (N) and in hypoxia (H) for 72 h and stained for Sox2 and Oct4 by immunocytochemistry. The Images were taken with DMi8 Leica microscope and prepared and quantified using ImageJ. C. Western blotting was done by the SDS-PAGE method and densitometry done using ImageJ. The relative expression was normalized to tubulin. Analysis was done using student t test using graph pad prism. Each value represents the mean ± SD of three independent experiments, * indicates p < 0.05. Scale bar = 50 μm. Representative Western blots are shown as cropped images. Full-length blots are presented in Additional File 2 (Fig. S2)

Fig 3: Inhibition of ALK4/5/7 inhibits male fetal germ cell differentiation.A) qRTPCR analysis of Nodal and Lefty1, and the male germ cell differentiation markers Dppa4 and Dnmt3l in E12.5 testes cultured for 72 hours with DMSO or SB431542. All of these genes are specifically expressed by the germ cells. Relative expression (± SEM, n = 3) was calculated after normalization to Mapk1 and Canx [64]. B–C) Immunofluorescence analysis of E12.5 testes cultured for 72 hours in the presence of DMSO or SB431542. B) DPPA4 is in red, KI67 is in Pacific Blue and MVH is in green. In the SB431542 treated images the arrows indicate germ cells that express low levels of DPPA4 and remain proliferative (KI67 positive), while the arrow-heads indicate germ cells that express high levels of DPPA4 and have exited the cell cycle (KI67 negative); C) MILI (red, left panel); FGFR3 (red right panel); In C–D MVH staining (green) marks the germ cells while DAPI (blue) marks nuclear DNA. Scale bars; 20 µm. D) qRTPCR analysis of Nodal and the pluripotency markers Nanog and Sox2 in E12.5 testes cultured for 72 hours with DMSO or SB431542. Relative expression (± SEM, n = 3) was calculated after normalization to Mapk1 and Canx. E) Immunofluorescence analysis of E12.5 testes cultured for 72 hours in the presence of DMSO or SB431542. SOX2 (red, left panel); OCT4 (red right panel). MVH staining (green) marks the germ cells. DAPI (blue) marks nuclear DNA. Scale bars; 20 µm.

Fig 4: PD-MK conditioned mESCs retain pluripotency.(A) Growth curve of mESC conditioned to Std, 2i, and PD-MK conditions. Error bars indicate SD (n = 3). (B) Percentage of apoptotic cells of mESC conditioned to Std, 2i, and PD-MK conditions. Error bars indicate SD (n = 3). (C) Cell cycle distribution of mESCs conditioned to Std, 2i, and PD-MK conditions. (D) Immunofluorescence of pluripotency markers (NANOG, OCT4, and SSEA1) of mESCs conditioned to Std, 2i, and PD-MK conditions. The images show maximum intensity projections of stacks. Scale bar, 50 µm. (E) Chimeric mice with black coat color generated from C57BL/6NCr ES cells conditioned to the PD-MK condition and then to the Std condition before injection into albino host embryos. Photo credit: T. Okamura (National Center for Global Health and Medicine).

Fig 5: In silico subpopulation analysis reveals mESCs exit pluripotency toward TE-like lineage in 2iJ+B-A condition; related to Fig 5 In silico subpopulation analysis of possible signaling input combinations with 2iL and 2i-L. The threshold values for predicted expression levels of lineage specifiers in each SCC are set as follows: Oct4 = 0.3, EpiTFs = 0.2, and Gata6 = 0.5 and Cdx2 = 0.7.Frequency of Cdx2+ population including TE-like subpopulation (Cdx2+/Oct4-) after 5 days in culture measured by immunostaining. Data represent the means and the error bars represent s.d. of six replicates consisting of two biological replicates with three technical replicates in each. The representative density plot of immunostaining of Cdx2 on day 5 is shown in the left panel. The significance was assessed by Wilcoxon exact rank test (*P < 0.05, **P < 0.0005) for the frequencies of total Cdx2+ and Cdx2+Oct4- comparing 2iL+B-A and 2iJ+B-A.Histograms for expression of TE-enriched cell surface markers, CD40 and CDCP1. Shown are mESCs treated in 2iJ (1st column), 2i-L (2nd column), or 2iL (3rd column), in unsupplemented medium (1st row) or BMP4 and ALKi supplemented medium (+B-A; 2nd row). Control mESCs (kept in control LS) and TSCs are shown at the bottom. In vivo lineage contribution frequency and chimera efficiency of H2B-Tomato ESCs (Morgani et al, 2013) treated with either 2iL or 2iJ in the presence of serum. Source data are available online for this figure.