Figure 1.

Differentiation of Human PSC INS Reporter Cells

(A) A diagram showing control (left panel) and experimental (right panel) INS reporter cells.

(B) Immunohistochemistry (IHC) staining with an anti-human-C-peptide antibody in β-like cells differentiated from INS:tdT, INS:CD19, and parental PSCs (right three panels) and undifferentiated PSCs (left panel). Scale bar, 50 μm.

(C) Representative flow cytometry results of PSCs (left panels) and β-like cells (right panels) differentiated from INS:tdT (top panel) and INS:CD19 (bottom panel), showing the GFP signal on the vertical axis and the tdTomato fluorescence signal (top panel) or anti-CD19 signal on the horizontal axis (bottom panel).

(D) Quantifications of the proportion of INS reporter-expressing cells at the end of differentiation (n = 3).

(E) Quantification of the luciferase signal in undifferentiated ESCs and β-like cells differentiated from the INS reporter cells (n = 4).

(F) In vitro glucose-stimulated insulin secretion (GSIS) assays of PSC-differentiated β-like cells. The stimulation index shows the ratio of the amounts of secreted human insulin released when β-like cells were treated with 20 mM glucose compared with 2.5 mM glucose (n = 4).

(G) Confocal micrographs with an anti-tdTomato antibody (red) and an anti-C-peptide antibody (green) of β-like cells differentiated from INS:tdT PSCs. Scale bar, 10 μm.

(H) Confocal micrographs with an anti-CD19 antibody (red) and an anti-C-peptide antibody (green) of INS:tdT β-like cells (top panel), INS:CD19 PSCs (center panel), and differentiated β-like cells (bottom panel). Scale bar, 10 μm.

(I) Quantitative RT-PCR analysis of the β-cell marker genes on RNA samples extracted from sorted INS marker-expressing cells differentiated from INS:tdT and INS:CD19 PSCs (n = 4).

∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, non-paired t tests.

See also Figures S1–S3.