p53 Inhibition in Pancreatic Progenitors Enhances the Differentiation of Human Pluripotent Stem Cells into Pancreatic β-Cells

The multipotent pancreatic progenitor cells (MPCs) co-expressing the transcription factors, PDX1 and NKX6.1, are the source of functional pancreatic beta-cells. The aim of this study was to examine the effect of p53 inhibition in MPCs on the generation of PDX1(+)/NKX6.1(+) MPCs and pancreatic beta-cell generation. Human embryonic stem cells (hESCs) were differentiated into MPCs and beta-cells. hESC-MPCs (stage 4) were treated with different concentrations of p53 inhibitors, and their effect was evaluated using different approaches. NKX6.1 was overexpressed during MPCs specification. Inhibition of p53 using pifithrin-mu (PFT-mu) at the MPC stage resulted in a significant increase in the number of PDX1(+)/NKX6.1(+) cells and a reduction in the number of CHGA(+)/NKX6.1(-) cells. Further differentiation of MPCs treated with PFT-mu into pancreatic beta-cells showed that PFT-mu treatment did not significantly change the number of C-Peptide+ cells; however, the number of C-PEP+ cells co-expressing glucagon (polyhormonal) was significantly reduced in the PFT-mu treated cells. Interestingly, overexpression of NKX6.1 in hESC-MPCs enhanced the expression of key MPC genes and dramatically suppressed p53 expression. Our findings demonstrated that the p53 inhibition during stage 4 of differentiation enhanced MPC generation, prevented premature endocrine induction and favored the differentiation into monohormonal beta-cells. These findings suggest that adding a p53 inhibitor to the differentiation media can significantly enhance the generation of monohormonal beta-cells.