Figure 4.

Decreased Levels of GATA6 at the DE Stage Influence Downstream Pancreatic Differentiation

(A) Expression of DE (SOX17, GATA6, GATA4, and FOXA2), pancreatic (HNF4A, HLXB9, and PDX1), and endocrine (INSULIN) marker genes in H9^∗ and H9-derived GATA6^4ins/+ and GATA6^Δ4/Δ4 mutant cells at the four key stages of the 24-day pancreatic differentiation protocol (Figure S1A).

(B) Percentage of PDX1-positive cells in H9-derived GATA6^4ins/+ and GATA6^GFP/+ lines on day 12 shown relative to H9^∗ (100%) as measured by FACS.

(C) Percentage of C-PEPTIDE-positive cells in H9^∗ and H9-derived GATA6^4ins/+ and GATA6^GFP/+ mutant lines at the EP stage (day 24) as measured by FACS.

(D) Immunofluorescence analyses for the key PE markers SOMATOSTATIN (SST), C-PEPTIDE (C-PEP), and GLUCAGON (GCG) in H9^∗ and H9-derived GATA6^4ins/+ cells at the EP stage (day 24). DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride. Scale bars, 100 μm.

(E) Heatmap illustrating differential gene expression of key pancreatic progenitor markers via RNA-seq between H9^∗cells and H9-derived GATA6^4ins/+ mutant cells at the PE stage. n = 3 biological replicates for each cell line.

(F) Expression of DE (GATA6 and GATA4) and pancreatic (HNF4A, PDX1, and NKX6-1) marker genes in H9^∗ and H9-derived GATA6^4ins/+ and GATA6^Δ4/Δ4 mutant cells at key stages of the differentiation protocol using the STEMdiff pancreatic progenitor kit.

(G) Percentage of PDX1-positive cells in GATA6^4ins/+ and GATA6^GFP/+ lines on day 12 shown relative to H9^∗ (100%) as measured by FACS in cells differentiated using the STEMdiff pancreatic progenitor kit.

(A–C, F, and G) Error bars represent the SE of three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.