Fig. 3. PAX4 knockout human induced pluripotent stem cell lines have defects in endocrine cell formation during in vitro differentiation.

a CRISPR-Cas9 genome editing was used to generate three independent hiPSC PAX4 homozygous knockout cell lines (PAX4^KO/KO) and three unedited wildtype control cell lines (PAX4^WT/WT). b Sanger sequencing for exon 2 and exon 5 of compound heterozygous PAX4^KO/KO and homozygous deletion PAX4^KO/KO hiPSC lines. c Schematic outline of seven-stage protocol A and six-stage protocol B from human induced pluripotent stem cells (hiPSC), definitive endoderm (DE), primitive gut tube (PGT), posterior foregut (PF) or pancreatic progenitor 1 (PP1), pancreatic endoderm (PE) or pancreatic progenitor 2 (PP2), endocrine progenitor (EP), endocrine (Endo), towards islet-like cells (SC-islets). RNA-seq samples were collected at the end of stages highlighted in blue. d Expression of PAX4 in transcripts per million (TPM) in PAX4^WT/WT and PAX4^KO/KO cells at DE, PE, EP and SC-islets derived from Protocol A. e Volcano plot of differentially expressed genes in PAX4^KO/KO versus PAX4^WT/WT SC-islets derived from Protocol A. The top ten differentially expressed genes are highlighted. f Expression of PAX4 in TPM in PAX4^WT/WT and PAX4^KO/KO cells at hiPSCs, EPs and SC-islets derived from Protocol B. g Volcano plot of differentially expressed genes in PAX4^KO/KO versus PAX4^WT/WT SC-islets derived from Protocol B. The top ten differentially expressed genes are highlighted. h Gene Ontology (GO) analysis of differentially expressed genes in PAX4^KO/KO compared to PAX4^WT/WT SC-islets from Protocol B. Bolded are the terms that include ARX. i Heatmap of relative gene expression for pancreatic endocrine genes in SC-islets derived from Protocol B.