Fig. 3: SC-islets, used as a human model, recapitulate β-cell identity loss resulting from zinc accumulation.

a Schematic flowchart of the SC-islet differentiation process. b, c Representative immunostaining images (b) and corresponding quantification (c) showing the percentages of INS-GFP⁺ cells (green) and GCG⁺ cells (red) among the total number of DAPI⁺ cells (blue), as well as the proportion of bi-hormonal INS-GFP⁺GCG⁺ cells among the total INS-GFP⁺ cells, in SC-islets cultured with or without high glucose for 10 days. n = 9. Mannitol was used as an osmotic control. White arrows indicate bi-hormonal INS-GFP⁺GCG⁺ cells. Scale bar (low magnification), 50 μm; scale bar (high magnification), 5 μm. d qPCR analysis of relative SLC30A8 (ZnT8) mRNA expression levels at various time points (CT, n = 7; 4 days, n = 5; 8 days, n = 7; 12 days, n = 7; and 16 days, n = 7) during high-glucose treatment (33 mM). e, f Representative zinquin staining (e) and corresponding measurements of mean fluorescence intensity (f) at different time points during high-glucose treatment: CT (n = 8), 4 days (n = 10), 8 days (n = 11), 12 days (n = 11), and 16 days (n = 10). Scale bar, 50 μm. g, h Representative immunostaining images (g) and corresponding quantification (h) showing the percentages of INS-GFP⁺ cells (green) and GCG⁺ cells (red) among the total number of DAPI⁺ cells (blue), as well as the proportion of bi-hormonal INS-GFP⁺GCG⁺ cells among the total INS-GFP⁺ cells in SC-islets cultured in the absence (n = 8) or presence (n = 9) of excessive zinc under high glucose for 10 days. White arrows indicate bi-hormonal INS-GFP⁺GCG⁺ cells. Scale bar, 50 μm. i UMAP analysis showing the distribution of cell types in SC-islets. j Proportions of cell types in groups of SC-islets treated with or without excessive zinc under high glucose. k, l Violin plots showing the distribution of INS (k) and NKX6.1 (l) expression levels in SC-islets treated with or without excessive zinc under high glucose. m Single-cell trajectory of SC-β cells treated with or without excessive zinc under high glucose; cells were ordered by pseudotime along the path from INS^highGCG^low SC-β cells to INS^lowGCG^high SC-β cells. n, o Changes in INS and GCG expression levels along pseudotime. One-way ANOVA with Dunnett’s multiple-comparisons test was used to analyze data in d, f. Unpaired two-tailed t-test was used to analyze data in c, h. ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. Individual data points are shown for all bar graphs.