Fig 2: The clearance of ROS can rescue the impaired DE differentiation due to mitochondrial dysfunction.A Flow cytometric analysis to determine the changes of intracellular ROS level during DE differentiation treated with (DYNA) or without (CTRL) dynasore. The mean fluorescence intensity (MFI) of flow cytometry as shown on the right (n = 3). B Flow cytometry analysis of DE marker with the treatment of dynasore, and dynasore plus 3 mM NAC, respectively (n = 3). C Flow cytometry analysis of markers of DE cells when treated with ATN (n = 3). D The mRNA expression of FOXA2 during DE differentiation treated with 10 µM ATN. Data are normalized to the mRNA level of untreated (control) DE cells (n = 3). E Percentage of CXCR4-positive cells (flow cytometry) in the endoderm differentiation treated with 3 µM dynasore, 1 mM NAC or 0.5 mM ATP (n = 3). F Schematic illustration of mitochondrial regulation to DE differentiation. All data are shown as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 3: Mitochondria are more mature during human DE differentiation.A Scheme of DE differentiation. FOXA2+ (green), SOX17+, and CXCR4+ cells represented differentiated DE cells, and OCT4+, SOX2+ (red), and NANOG+ represented pluripotent cells (nucleus, DAPI, blue). Scale bar = 400 µm. B RNA expression analysis of differentiation markers and pluripotency markers during DE differentiation. Data are normalized to the mRNA level of PSCs (n = 3). C Representative images of MitoTracker staining observed through confocal in DE differentiation of iPSCs. Scale bar = 5 µm. D Quantitative statistics of mitochondrial average length in (C) using the MINA tool. E Representative mitochondrial images of ESCs, D2, DE were observed through a transmission electron microscope. Scale bar = 2 µm. F The ratio of Max/Min axes and diameter length of mitochondria (E) were statistically analyzed from about 20 independent images. G PCA analysis of all genes (top) and mitochondria-related genes (bottom) in pancreatic differentiation (GSE114099, red) and our DE differentiation (blue). All data are shown as mean ± SD. ns, not significant. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 4: Interference with mitochondrial homeostasis impairs DE differentiation.A The western blot of TFAM and ß-actin protein along with the removal time of doxycycline. B Schematic illustration of the differentiation protocol for inducible TFAM knockout PSCs. C Flow cytometric analysis to determine the change in differentiation efficiency between induced knockout and wild-type iPSCs. SOX17+ and CXCR4+ cells represented differentiated DE cells. D Immunofluorescence analysis of DE cells marked with SOX17 (green) (nucleus, DAPI, blue). Scale bar = 200 µm. E mRNA expression of endoderm- or pluripotency-marker SOX17, OCT4 in DE cells treated with or without 100 ng/ml doxycycline (n = 3). F Flow cytometric analysis to measure the percentage of cells expressing both differentiation markers (SOX17 and CXCR4) treated with XCT790 (XCT) in DE differentiation (n = 3). G mRNA expression of endoderm transcription factors FOXA2, SOX17, and CXCR4 in DE cells treated with XCT (n = 3). H mRNA expression of pluripotent markers OCT4 and SOX2 in DE cells treated with XCT (n = 12). I Flow cytometric analysis to measure the percentage of cells expressing both DE markers (SOX17 and CXCR4) treated with dynasore (DYNA) (n = 3). J mRNA expression of endoderm transcription factors FOXA2, SOX17, and CXCR4 in DE cells treated with dynasore (n = 3). K mRNA expression of pluripotent markers OCT4 and SOX2 in DE cells treated with dynasore (n = 6). The data of RT-qPCR are normalized to the mRNA level of untreated (control) DE cells. All data are shown as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 5: CHD8 knockout does not affect definitive endoderm differentiation.A Left, RT-qPCR analysis of endoderm genes FOXA2, SOX17 and GATA6; Right, RT-qPCR analysis of CHD8 and pluripotent genes OCT4 and SOX2 at day 3 of differentiation (n = 5). Wildtype ESCs at day 0 served as control. B Immunofluorescence of endoderm marker FOXA2 and SOX17 in WT and CHD8KO groups at day 3 of differentiation. The percentage of FOXA2- or SOX17-positive cells was calculated (n = 4). Scale bar = 200 µm. C Flow cytometric analysis showing the percentage of SOX17-positive cells in differentiated WT and CHD8-KO cells (n = 5). D, E AnnexinV/PI assay (D) and Cell cycle analysis using PI staining (E) in differentiated WT and CHD8-KO definitive endoderm cells (n = 5). The populations of different cell cycle phases were calculated in FlowJo (v10.4.0). 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).