Fig 2: (A) Differentiation markers for the endoderm (FOXA2, PDX1), mesoderm (α-actin, brachyury), and ectoderm (β3 Tubulin, Otx2) were detected for the MUCG01, MUCG02, and MUCG03 clinical-grade hESC lines by immunocytochemistry. (B) Standard karyotypes without chromosomal aberration were detected for all hESC lines MUCG01: 46,XX; MUCG02: 46,XX; and MUCG03: 46,XY. (C) Pluripotency markers Oct3/4, Sox2, and Nanog were detected for the MUCG01, MUCG02, and MUCG03 clinical-grade hESC lines by immunocytochemistry.

Fig 3: CHD8 is dispensable for pluripotency maintenance of human ESCs.A Left, schematic diagram showing the gRNA targeting the exon 2 (E2) of CHD8; Right, schematic diagram showing the genotype (frameshift) of the three CHD8 knockout (CHD8-KO) clones generated by Crispr/Cas9 technology. B Top, Western blot analysis showing expression levels of CHD8 in wildtype (WT) and CHD8-KO ESCs (n = 3); Bottom, RT-qPCR of CHD8 expression in WT and CHD8-KO (n = 4). C Top, Bright-field and enlarger images showing the morphology of WT and CHD8-KO clones (KO#1, KO#2, KO#3) (n = 3); Bottom, Representative alkaline phosphatase staining of WT and CHD8-KO ESCs (n = 3). Scale bar = 200 µm. D RT-qPCR of representative pluripotent and lineage genes expression in WT and CHD8-KO ESCs (n = 4). E Western blot analysis showing the protein levels of pluripotent markers in WT and CHD8-KO ESCs (n = 3). F Representative immunofluorescence staining of pluripotent marker NANOG and OCT4 for WT and CHD8-KO ESCs (n = 3). Scale bar = 100 µm. The error bars represented the mean ± SD and the significance level was calculated by Student’s t-test (two-tailed, equal variance) (ns means not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4: GATA3 Is Necessary for Timely Differentiation and Can Drive Differentiation in the Absence of BMP4 Signals(A) Quantification of GATA3 expression during 3 days of BMP4 (50 ng/mL) treatment.(B) Left: representative images of GATA3 and NANOG expression after BMP4 (50 ng/mL) treatment. Scale bar represents 100 µm. Center: GATA3 and NANOG expression in space after BMP4 stimulation, assuming a spherical geometry for hESC colonies. Right: quantification of GATA3 and NANOG levels in single cells after BMP4 treatment. More than 10,000 cells were analyzed for each experimental condition. n = 3 independent experiments.(C) Quantification of GATA3 ChIP-qPCR in the presence (red) or absence (blue) of BMP4 (50 ng/mL) treatment of hESC (for 6 h). BMP4, HES1, CDX2, and SNAI2 are genes shown to be involved in hESC differentiation. ISL1 and FOXC1 are lateral mesoderm (cardiac)-specific genes. A primer set -3 kb from BMP4 was used as a negative control (Neg control). Error bars represent means ± SDs for n = 2 biological replicates (**p < 0.001, ***p < 0.0001, ns is not significant).(D) Quantification of fold change of pluripotency (blue) and lineage-specific (red) genes in response to BMP4 stimulation (left) or tamoxifen-induced GATA3 expression (iGATA3) (right) relative to untreated control cells. The housekeeping gene GUSB was used for normalization. Error bars represent ±SDs from n = 3 biological replicates.(E) Quantification of the fraction of positive cells for pluripotency gene NANOG and lineage-specific markers BRY, SOX17, and CDX2 in response to BMP4 (50 ng/mL) stimulation in GATA3-/- (GATA3 KO) cells (red) or control cells (blue). n > 500 cells were analyzed for each experimental condition.(F) scRNA-seq data of wild type (WT) and GATA3 KO hESC stimulated with BMP4 for 48 h. Combined cell transcriptomes were analyzed with a mean gene per cell value of ~6,500 and a total read depth of ~600 million reads per cell. Cell transcriptomes were projected onto a diffusion map with shared 14 nearest-neighbor clusters highlighted (first 30 dimensions, resolution 0.8).(G) Heatmaps showing normalized counts per cell for GATA3 and mesoderm differentiation genes for WT (left) and GATA3 KO (right) hESCs after 48 h BMP4 stimulation. The left panel highlights a gradient of GATA3 expression.

Fig 5: NANOG overexpression downregulates ICAM1 expression. (a) RNAseq analysis of WT-, GFP-, and GN-DU145 cells. The expression levels of NK cell inhibitory receptors and activation receptors were compared among these cells. (b) ICAM1 expression was detected in WT-, GFPand GN-DU145 cells by western blot. (c) ICAM1 localization was detected in WT- and GN-DU145 cells by immunofluorescence staining