Fig 1: Transcriptomic changes in pancreatic endocrine progenitors (EPs) derived from FOXA2+/− iPSCs.A Volcano plot plotting the log2 fold change (FC) and the adjusted p-value for all the transcripts. B Pathway enrichment analysis on upregulated and downregulated differentially expressed genes (DEGs) in FOXA2+/− iPSC-derived EPs using DAVID function annotation tool. The enriched pathways were plotted against −log10 (p-value). C Heatmaps of pathway-associated DEGs in FOXA2+/− iPSC-derived EPs compared to those derived from Ctr-iPSCs. The downregulated genes were associated with pancreatic development, maturity-onset diabetes of the young (MODY), and NOTCH signaling pathways, while the upregulated genes were associated with the WNT signaling pathway and nervous system development. The relative value for each gene is depicted by color intensity, with red indicating upregulated and blue indicating downregulated genes. D RNA-Seq results were validated using qPCR for the upregulated genes associated with WNT signaling and nervous system development. Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: Effect of FOXA2 haploinsufficiency on definitive endoderm (DE) formation.A Western blotting of FOXA2 expression in the definitive endoderm (DE) derived from Ctr-iPSCs and FOXA2+/− iPSCs. B qPCR quantification of the expression of DE markers, FOXA2 and SOX17 in DE derived from Ctr-iPSCs and FOXA2+/− iPSCs (n = 3). C Representative immunofluorescent images showing the expression of SOX17 and OCT4 or FOXA2 and SOX2 in DE derived from Ctr-iPSCs and FOXA2+/− iPSCs. D Representative flow cytometry histograms and its quantification showing the expression of FOXA2, SOX17, and OCT4 in DE derived from Ctr-iPSCs and FOXA2+/− iPSCs. Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: Effect of FOXA2 haploinsufficiency on iPSC-derived beta cells.Representative immunofluorescent images (A) and flow cytometry histograms (B) showing the expression of INSULIN (INS), NKX6.1, C-PEPTIDE (C-PEP), and GLUCAGON (GCG) in stage 7 derived from Ctr-iPSCs and FOXA2+/− iPSCs. C Flow cytometry quantification for the data shown in B. D qPCR quantification for the expression of the pancreatic markers, FOXA2, INS, GCG, ABCC8, KCNJ11, NKX6.1, NKX2.2, and PDX1 in beta cells derived from Ctr-iPSCs and FOXA2+/− iPSCs. GSIS assay: fold change of C-PEPTIDE secretion in iPSC-derived beta cells challenged with low (LG; 2.8 mM) and high glucose (HG; 20 mM) (E) or stimulated with or without 30 mM KCl in the presence of LG (F). Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: FOXA2 overexpression in PP2 reverse the phenotype of FOXA2 deficiency.A Representative western blotting showing the overexpression of FOXA2 in the PP2 derived from FOXA2+/− iPSCs, and FOXA2−/− iPSCs, 48 h after transfection with FOXA2 plasmid or empty vector (EV). B Real-time PCR analysis for the expression of PDX1, NKX6.1, SOX9, GATA6, PAX4, PTF1A, AMYLASE, TCF7L2, INSR, NOTCH, HES1, MNX1, ISL1, PROX1, PTPRN1, ADRA2A, and CDX2 in PP2 derived from FOXA2+/− iPSCs and FOXA2−/− iPSCs 48 h after transfection. C Real-time PCR analysis for the expression of BMP4, NOG, MSX1, MSX2, DKK1, DLX2, DLX5, EMX2, ERG2, OTX1, OTX2, and RFX6 in PP2 derived from FOXA2+/− iPSCs and FOXA2−/− iPSCs 48 h after transfection. D Real-time PCR analysis for the expression of INS, GCG, SST, PDX1, NKX6.1, ABCC8, KCNJ11, HHEX, and NEUROD1 in stage 7 derived from FOXA2+/− iPSCs and FOXA2−/− iPSCs transfected with FOXA2 plasmid or EV at stage 4. E Representative immunofluorescence images showing the expression of INS and GCG in stage 7 derived from FOXA2+/− iPSCs and FOXA2−/− iPSCs transfected with FOXA2 plasmid or EV at stage 4. F C-PEPTIDE (C-PEP) secretion (% of content) after stimulation with or without 30 mM KCl in the presence of low glucose (LG) in FOXA2+/− iPSC-derived beta cells transfected with FOXA2 plasmid or EV at stage 4. The data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: Effect of FOXA2 haploinsufficiency on pancreatic progenitor (PP2) differentiation.Western blotting (A) and qPCR analysis (B) for FOXA2 protein and mRNA expression levels in iPSC-derived PP2. C Representative immunofluorescent images showing the expression of PDX1 and NKX6.1 in the PP2 derived from Ctr-iPSCs and FOXA2+/- iPSCs. D Flow cytometry histograms showing the expression of PDX1 and NKX6.1 in the PP2 derived from Ctr-iPSCs and FOXA2+/- iPSCs. E Quantification of the expression of PDX1 and NKX6.1 shown in D. F qPCR quantification for the expression of key PP2 markers, PDX1, NKX6.1, SOX9, HNF1A, GATA6, PAX4, ONECUT1 (HNF6), PTF1A, and AMYLASE in the generated PP2. Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from DNASU for FOXA2 (Homo sapiens) in pLenti6.2/V5-DEST (V5-tagged lentiviral mammalian expression vector)