Fig. 5

Transcriptomic analysis reveals altered expression of pro-endocrine genes following loss of RREB1 in human beta cells. (a) Schematic of the in vitro differentiation protocol stages: definitive endoderm (DE) cells, primitive gut tube (PGT) cells, posterior foregut (PFG) cells, pancreatic endoderm (PE) cells, endocrine progenitor (EP) cells, endocrine (EN) cells and BLCs. Growth factors and small molecules (listed underneath each stage) were added for the indicated amounts of time. (b) RREB1 expression in RREB1^WT/WT (n=3) and RREB1^KO/KO (n=4) hiPSC cell lines during in vitro differentiation towards BLCs. (c) The first two principal components (PC1, PC2) were calculated using normalised gene counts of RREB1^KO/KO (circles; n=4) and RREB1^WT/WT (squares; n=3) cell lines for all seven stages of in vitro beta cell differentiation. (d) Differential expression of endocrine cell genes in PE cells, EP cells, EN cells and BLCs. (e) Analysis of modules of co-expressed genes using WGCNA: bar plot showing module epigengene (ME) expression of the module enriched for endocrine progenitor and endocrine genes. (f) Venn diagrams of the overlap of DEGs between siRREB1 knockdown EndoC-βH1 cells, RREB1-KO EndoC-βH1 cells and hiPSC-derived RREB1^KO/KO BLCs. Data are presented as means±SEM. **p<0.01 (unpaired t test). Act A, activin A; Alk5i II, ALK5 inhibitor II; CHIR99021, GSK-3 inhibitor; KGF, keratinocyte growth factor; LDN, LDN193189 BMP type 1 receptor inhibitor; N-cys, N-acetyl cysteine; R428, AXL inhibitor; RA, retinoic acid; SANT-1, hedgehog signalling inhibitor; T3, triiodothyronine; TPB, PKC activator; Trolox, vitamin E; Y inh XX, gamma secretase inhibitor; Vit C, vitamin C