STARD10 regulates human pancreatic β cell differentiation and triglyceride metabolism

Figures

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  Figure 1. Loss of STARD10 in hESCs impairs the formation of INS+ β-like cells (A) Schematic illustration of the 35-day β cell differentiation protocol used in this study. DE, definitive endoderm; PGT, primitive gut tube; PP1, early pancreatic progenitor; PP2, late pancreatic progenitor; EN, endocrine progenitor. (B and C) Relative mRNA expression at the four differentiation stages (D0, D13, D20, and D35) measured by qPCR (n = 3–5). (B) PDX1, ∗; NKX6.1, #; INS, †; STARD10, ‡. Statistical significance for each gene was calculated by comparing against D0. (C) STARD10, ∗; STARD11, #. Statistical significance for each gene was calculated by comparing against D0. No significant difference for STARD2 and STARD7 at all timepoints. (D–H) Western blot analysis at D35 of β cell differentiation (one representative experiment out of three). Relative mRNA expression at (E–F) D20 and (G–H) D35 of β cell differentiation measured by qPCR (n = 3–4). (I and J) Flow cytometry analyses at D35 of β cell differentiation (n = 3). STARD10 KO represents pooled data from #2 and #4. Error bars denote SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Statistical analyses were performed using unpaired t test…

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  Figure 2. Deficiency of STARD10 leads to downregulation of cell cycle and cell proliferation genes in β-like cells (A) Top 10 downregulated biological processes in STARD10 KO (average of #2 and #4) β-like cells as compared to Parental β-like cells. (B) Heatmap analysis of the 148 mitotic cell cycle genes that were significantly downregulated. Mitotic cell cycle genes that were selected for qPCR validation were highlighted (black box). (C–H) Relative mRNA levels of cell cycle genes measured by qPCR in D35 β-like cells (n = 3). (I and J) Flow cytometry analyses at D35 of β cell differentiation (n = 3). STARD10 KO represents pooled data from #2 and #4. Error bars denote SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Statistical analyses were performed using unpaired t test for two groups and one-way ANOVA, followed by Tukey’s multiple comparison test for three groups. See also Figure S3.

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  Figure 3. Deletion of STARD10 leads to increased triglyceride accumulation and impaired cellular bioenergetics in β-like cells (A) Lipidomics analyses of total TGs on D35 of β cell differentiation (n = 3). (B) Oil red O staining of β-like cells (one representative experiment). Scale bars, 50 μm. (C) Quantification of oil red O staining in Parental and STARD10 KO (#2 and #4 combined) β-like cells (n = 3). (D) LipidSpot610 staining of D35 cryosections (one representative experiment). Scale bars, 100 μm. (E) Mean fluorescence intensity quantification of LipidSpot610 staining between Parental and STARD10 KO (#2 and #4 combined) cells (n = 3). (F) Extracellular acidification rate (ECAR) measurement of D35 β-like cells via Seahorse XF Glycolysis Stress Test Kit (n = 5–6). (G) Oxygen consumption rate (OCR) measurement of D35 β-like cells using the Seahorse XF Cell Mito Stress Test Kit (n = 5–6). (H) OCR measurement of D35 β-like cells treated with palmitate using the Seahorse XF Palmitate Oxidation Stress Test Kit (n = 4–5). Error bars denote SEM. ∗p < 0.05; ∗∗p < 0.01. Statistical analyses were performed using unpaired t test for two groups and one-way ANOVA, followed by Tukey’s multiple…

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  Figure 4. STARD10 regulates INS and Ki67 expression and lipid accumulation via ETFB (A) Relative gene expression analysis in D35 β-like cells (n = 4). (B) Western blot analysis of D35 β-like cells (one representative experiment out of three). (C) mRNA time course analyses throughout β cell differentiation (n = 3). (D) Immunofluorescence analysis of ETFB and INS in D35 cells transduced with empty vector (EV) or ETFB-overexpressing vector (one representative experiment). Scale bars, 100 μm. (E–F) Mean fluorescence intensity quantification of ETFB and INS staining (n = 3). (G) Immunofluorescence analysis of Ki67 in D35 cells transduced with empty vector (EV) or ETFB-overexpressing vector (one representative experiment). Scale bars, 100 μm. (H) Mean fluorescence intensity quantification of Ki67 staining (n = 3). (I) LipidSpot610 staining of D35 cells transduced with empty vector (EV) or ETFB-overexpressing vector (one representative experiment). Scale bars, 100 μm. (J) Mean fluorescence intensity quantification of LipidSpot610 staining (n = 3). Error bars denote SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Statistical analyses were performed using unpaired t test for two groups…