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. 2020 Feb 21;16(8):1450–1462. doi: 10.7150/ijbs.44414

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Optimization of epidermal cell fate determination at early stage. See also Figure S2. A. The time course of primitive ectoderm induction. Cells were treated with SB431542 single treatment for 0, 1, 2 or 3 days in differentiation medium, followed by combination treatment with SB431542, BMP4 and CHIR99021 until day 6, and collected on day 6 for TP63 gene expression analysis by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. B. The impact of prolonged exposure to TGFβ inhibitor. H1 cells were treated with TGFβ inhibitor SB431542 (SB) for 2, 4 or 7 days in the presence of BMP4 (Day 1 to 7) and CHIR99021 (CHIR, Day 1 to 5) in differentiation medium, and gene expression was analyzed on day 7 by RT-qPCR. The results were normalized to control (no SB4315432 treatment); Data are presented as the mean±SD of three independent experiments. *, p < 0.05. C. The effect of BMP4 timing on TP63 induction. In the presence of SB431542 (SB, Day 0-6) and CHIR99021 (CHIR, Day 1-5), BMP4 was added for specific time periods, and gene expression was analyzed on day 7 by RT-qPCR. The results were normalized to control (no BMP4 treatment); Data are presented as the mean±SD of three independent experiments. ns, not significant (one-way ANOVA with post hoc multiple comparisons). D. The effect of WNT activation timing on epidermal differentiation. WNT activator CHIR99021 (CHIR) was added in the presence of SB431542 (SB, Day 0-6) and BMP4 (Day 1-7) for specified periods of time, and gene expression was analyzed by RT-qPCR on day 7. The results were normalized to control (no CHIR treatment); Data are presented as the mean±SD of three independent experiments. *, p < 0.05. E. Impact of NOTCH inhibition during epidermal differentiation. Epidermal differentiation was carried out with SB431542 (SB, Day 0-6), CHIR99021 (CHIR, Day 1-6) and BMP4 (Day 1-6). NOTCH inhibitor DAPT was added between Day 4-6, and the gene expression was analyzed by qPCR on Day 6. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. F. The impact of WNT activation along with NOTCH inhibition on epidermal cell fate determination. Cells in epidermal differentiation were treated with DAPT with or without CHIR99021 (CHIR) between day 4 to 6, and the samples were collected on Day 6 for RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05.