Fig. 4

Induction from hiPSC-derived pancreatic endoderm to pancreatic acinar progenitor cells. (A) Candidate factors (left) and signaling pathways (right) involved in the differentiation of pancreatic acinar progenitor cells, as identified using pathway analysis by IPA. (B) Comparison of effects of 9 candidate differentiation-inducing factors on the expression of REG4 and PRSS1, as evaluated by qRT-PCR. The treatment started on Stage 4 Day 6 (total day 17) and continued for 8 days, ending on total day 25. The following three concentrations were examined for each factor: 10, 50, and 100 ng/mL EGF; 0.2, 1, and 2 ng/mL IL-1B; 10, 50, and 100 ng/mL HGF; 10, 50, and 100 µM pCPT-cAMP (pC); 2, 10, and 20 nM orskolin (FS); 10, 50, and 100 ng/mL FGF2; 0.02, 0.1, and 0.2 nM bexarotene (B); 5, 25, and 50 ng/mL IGF1; and 1, 5, and 10 µg/mL lipopolysaccharide (LPS). PE, pancreatic endoderm. Red arrows indicate factors that upregulated the expression of REG4 or PRSS1 more than 1.5 times compared to the untreated control (Control). Data from three independent experiments are presented as mean ± SD (n = 3). We normalized the expression values against the house keeping gene GAPDH and then against the untreated control. (C) Effects of treatment with 20 nM forskolin on the expression of acinar lineage markers, REG4, PTF1A, CPA1, PRSS1, SPINK1, and CTRC, as evaluated by qRT-PCR. The treatment started on Stage 4 Day 6 (total day 17) and continued for 8 days, ending on total day 25. Graft indicates one graft sample on day 30 after implantation, which differs from grafts used for scRNA-seq. The graft sample on day 30 after implantation was isolated from the host renal parenchyma and capsule, and cells were dispersed in a collagenase/dispase solution for 15 min at 37 °C, dissociated into single cells by gentle pipetting, and used for RNA isolation without sorting for human cells. Data from three independent experiments are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 by two-tailed Student’s t-test. We normalized the expression values against GAPDH and then against the untreated control (Control). (D) Immunostaining of day 30 grafts from hiPSC-derived pancreatic endoderm cells with or without forskolin treatment for PRSS1 (green) and nuclei (blue; left and upper right panels), and CPA1 (green) and NKX6.1 (red; middle right panels) and quantification of the percentage (%) of PRSS1⁺ or CPA1⁺ cells from all cells within the engraftment region (lower right panel). Data from three independent experiments are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 by two-tailed Student’s t-test. Scale bars: 300 μm in (D; left panels) and 100 μm in (D; right panels).