Extended Data Fig. 10: ARID1B knockdown increases expression of identity genes and chromatin features in SC-β cells.
a, Cross reference map of upregulated SC-β cell and primary β-cell genes (left) or transplanted SC-β cell genes (right) with chromatin associated genes to highlight regulators associated with each cell states. Individual image representative of 1 sample. b, Brightfield images of SC-islets transfected with lentivirus carrying ARID1B shRNA. c, qPCR analysis, plotting mean ± s.e.m. (n = 4 biologically independent samples for shRNA sequence 1; n = 3 biologically independent samples for shRNA sequence 2), showing reduced expression of ARID1B using lentiviruses resulting in increased β cell identity gene expressions. (shRNA sequence 1: ARID1B, P = 5.3 × 10−4; INS, P = 0.0014; ISL1, P = 0.022; DLK1, P = 4.3 × 10−5; NKX6-1, P = 0.0015; IAPP, P = 3.0 × 10−6; G6PC2, P = 2.1 × 10−4) Two shRNA sequences were tested for validation of results. (shRNA sequence 2: ARID1B, P = 0.018; INS, P = 0.0035; IAPP, P = 5.9 × 10−6; DLK1, P = 0.045, G6PC2, P = 0.0054) Statistical significance was assessed by unpaired two-sided t-test. d, Confocal fluorescent images showing increased expression of amylin in SC-islets with ARID1B knockdown. Individual image representative of 5 biologically independent samples. e, Flow cytometry analysis, plotting mean ± s.e.m. (n = 4 biologically independent samples), of SC-islets with ARID1B shRNA showing increased fraction of cells with C-peptide expression (P = 2.2 × 10−5) Statistical significance was assessed by unpaired two-sided t-test. f, ELISA quantification, plotting mean ± s.e.m. (n = 4 biologically independent samples) of glucagon (P = 0.017), and somatostatin (ns, P = 0.087) content. Statistical significance was assessed by unpaired two-sided t-test. g, Glucose stimulated insulin secretion assay, plotting mean ± s.e.m. (by ELISA, n = 4 biologically independent samples), comparing insulin secretion at high glucose (20 mM) stimulation from control (GFP shRNA) and ARID1B shRNA SC-islets in presence of various secretagogues. (Glucose (20 mM), P = 0.27; KCL, P = 0.049; Exendin 4, P = 0.035; IBMX, P = 0.94; Tolbutamide, P = 0.43) Statistical significances were assessed using paired two-sided t-test. h, Single-cell multiomic sequencing UMAPs of ARID1B knockdown SC-Islets, showing identified cell types, and cell knockdown condition using both gene expression and ATAC information (21969 cells from 2 independent biological samples; integration of all samples). i, Differential gene expression analysis of SC-β cells comparing control and ARID1B shRNA. Statistical significance was assessed by Wilcoxon rank sum test. j, Chromatin accessibility around the IAPP genomic region of SC-β cells, showing increased peak signals with ARID1B shRNA. EC, enterochromaffin; ns, not significant.
