Fig. 4

Transcriptome dissection reveals distinctive roles of ALK5 and CDK8/19 inhibition for endocrine cell induction. a, b iPICs were differentiated with 10 µM ALK5iII or another ALK5 inhibitor, 3 µM SB431542, and/or the CDK8/19 inhibitor, 0.3 µM senexin B to replace ALK5iII. Representative dot plots from FCM analysis (a, left), average proportions of β-cells (a, right), and density of obtained cells (b) are illustrated. Data are representative of three independent experiments and presented as the mean ± SD (n = 4 technical replicates). *P < 0.05 and **P < 0.01 versus data from the ALK5iII-treated cells, Dunnett’s test. c and d Individual (c) and merged (d) distributions of cells based on their gene expression profiles, shown as uniform manifold projections for four types of iPICs treated with ALK5 and CDK8/19 inhibitors as described in a and b. e Shared nearest neighbor clustering divided iPICs into seventeen cell clusters. Superimposed annotation indicated identified cell types based on both DEG-driven enrichment analysis and expression levels of well-validated cellular markers. f The proportion of cells assigned from each sample in each cluster. Total cell numbers were uniformly targeted to 3000 cells for all four samples. For “Common clusters among ALK5iII-, Sen- and SB/Sen-cells groups,” less than 5% of cells were categorized from SB-cells. For “SB-cells dominant clusters” groups, more than 66% of cells were SB-cells. g Internal composition of cells highly expressing INS and NKX6.1 (clusters #1, 3, 6, and 12) within each sample. Pie charts describe the proportions of the cells allocated to each cluster. h Differentially gene expression analysis in iPIC samples compared to data from ALK5iII-treated cells. The number of up- and down-regulated genes (P < 0.05 and FC < 2 or >  − 2) were presented in the scatter plots in red or blue