Extended Data Fig. 9: Endocrine differentiation is modulated by ECM composition-mediated integrin signalling.

a–e, Pancreatic progenitors sorted at single-cell density on dishes coated with fibronectin, laminin, vitronectin or collagen, fixed after 24 h and stained for PDX1 (fibronectin, n = 436; laminin, n = 252; vitronectin, n = 133; collagen, n = 227 cells), YAP1 (vitronectin, n = 430; collagen, n = 316 cells), NGN3 (vitronectin, n = 300; collagen, n = 205 cells) or YAP1–NGN3 nuclear intensity (vitronectin, n = 300; collagen, n = 216 cells) were quantified as in Fig. 1a. Small blue boxes in e show the YAP1⁻ cell population quantified. Graphs present plots of individual cell data aggregated from three independent experiments. f, Comparison quantification, as shown in Fig. 4c. Each data point representing YAP1⁺, NGN3⁺ cells and density corresponds to mean ± s.e.m. of three independent experiments. Percentage of NGN3⁺ cell number was determined as in Extended Data Fig. 8a. Two-tailed unpaired t-test; ****P ≤ 0.0001, n.s., non-significant; data are mean ± s.e.m. g, Pancreatic progenitor cells sorted as in Fig. 4c and analysed after 24 h culture on vitronectin or collagen. Cell area and nuclear intensities of YAP1⁺ and NGN3⁺ cells were measured for cells plated on both ECMs. Two-tailed unpaired t-test; ****P ≤ 0.0001, mean ± s.e.m. of cell data aggregated from three independent experiments. h, i, Co-immunofluorescence and quantification from 30-μm sections from E15.5 wild-type pancreata co-stained for NGN3, fibronectin and laminin (h). NGN3⁺ cells were segmented using IMARIS surface module. Identification of cells in contact with either laminin only or fibronectin only or both was computed using percentage intensity overlap. The threshold for the intensity overlap was selected manually by examining individual cells. Pie chart showing the percentage of segmented NGN3⁺ cells in contact with fibronectin or laminin or both. Shown is the mean percentage and confidence interval data of 1,390 NGN3⁺ cells from 4 independent wild-type E15.5 embryos analysed. i, Pie chart showing the percentage of segmented Sox9⁺ cells in contact with fibronectin or laminin or both. Shown is the mean percentage and confidence interval data of 2,637 Sox9⁺ cells from 4 independent wild-type E15.5 embryos analysed. j, Sorted single pancreatic progenitor cells confined (top) or spread (bottom), stained for integrin α5 (ITGa5; red) and NGN3 (grey), with CellMask (green) and DAPI (blue). Scale bar, 10 μm. Representative images from three independent experiments are shown. k, Western blots from sorted hESC-derived cells expressing NGN3–GFP as in Extended Data Fig. 3e. Pancreatic progenitor differentiation performed with the NGN3–GFP reporter line. NGN3^high and NGN3^low cells (endocrine precursors) express significantly lower levels of integrin α5 (ITGa5) and YAP1 protein than NGN3⁻ cells (pancreatic progenitors). Representative blots from two independent experiments shown. l, Integrin α5 (ITGA5) mRNA expression analysis of NGN3⁻, NGN3^low and NGN3^high populations derived from Extended Data Fig. 3e. ITGA5 expression mirrors YAP1 expression during endocrine differentiation (Extended Data Fig. 3e). Expression is presented relative to GAPDH expression. Data are mean ± s.e.m. from two independent differentiation experiments. m, IgG or integrin α5 (ITGa5) antibody staining and flow cytometric analysis of differentiated hESC NGN3–GFP reporter cells. Left, histogram overlay and mean fluorescence intensities (MFI) of IgG-stained (MFI = 100), NGN3–GFP⁺ endocrine precursor (MFI = 310) and NGN3–GFP⁻ pancreatic progenitor cell (MFI = 704) fractions. Right, staining distribution and MFI of each cell population. NGN3⁺ endocrine precursors display lower protein expression levels of integrin α5 compared to NGN3⁻ pancreatic progenitors. Data represent three independent experiments. n, mRNA expression analysis of other α integrins (as in l ) that remain unchanged during differentiation of NGN3⁺ cells from pancreatic progenitors. Expression is shown relative to GAPDH expression; n = 1 experiment.

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