Fig. 3: Time course analysis of early response of stem cell-derived islets under hypoxia.

a RNA velocity visualized on UMAP of SC-islets cultured in 21% oxygen for 1 day or cultured in 2% oxygen for 1 day, 3 days, 5 days, 2 weeks, and 4 weeks. Cells are colored according to their assigned cell type. b UMAP of cell types shown in (a), colored according to acquisition time point. a, b represent the same UMAP colored as indicated above. c Correlation of expression profiles for major cell types, including SC-β cells, SC-α cells, and SC-enterochromaffin cells (SC-EC) in 2% oxygen, broken down by acquisition time point. d RNA velocity trajectories visualized on UMAP of SC-β cells cultured in 21% oxygen for 1 day or cultured in 2% oxygen for 1 day, 3 days, 5 days, 2 weeks, and 4 weeks. e Representative plots of low-to-high (bottom) and high-to-low (top) insulin trajectories. f UMAP of SC-β cells colored according to acquisition time point. g Representative gene expression visualized on UMAP of SC-β cells in response to hypoxia (INS, left; HIF1α, right). h Expression of selected marker genes as a function of latent time in (d). Dots show expression in single cells, sorted and shaded according to pseudotime order. The lines represent the regression fitted as a function of pseudotime. i Pathway enrichment of differential genes of SC-β cells in response to hypoxia. Gene Set Enrichment Analysis (GSEA) is applied to analyze KEGG pathways. Multiple comparison was adjusted by Benjamini-Hochberg method. j Principal component analysis (PCA) of proteome profiles of SC-β cells cultured in 2% oxygen for 1 day, 3 days, and 5 days compared to those of SC-β cells cultured in 21% oxygen (one-way ANOVA P < 0.01). (n = 3 biological replicates). k KEGG pathway analysis of proteome profiles of SC-β cells cultured in 2% oxygen for 5 days compared to that of SC-β cells cultured in 21% oxygen, downregulated pathways (blue) or upregulated pathways (red). Gene Set Enrichment Analysis (GSEA) is applied to analyze KEGG pathways. Multiple comparison was adjusted by Benjamini-Hochberg method. l Fold changes of protein expression of SC-β cells cultured in 2% oxygen for 1 day (red), 3 days (green), 5 days (orange) to protein expression of SC-β cells cultured in 21% oxygen; dot size represents statistical significance (one-way ANOVA) (n = 3 biological replicates). Proteins were selected as differentially expressed if they met the criteria of an adjusted p-value < 0.01 (one-way ANOVA) and a log fold change (LFC) > 1 at least at one time point (days 1, 3, or 5) under 2% oxygen conditions.