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

Abstract

The multipotent pancreatic progenitor cells (MPCs) co-expressing the transcription factors, PDX1 and NKX6.1, are the source of functional pancreatic β-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 β-cell generation. Human embryonic stem cells (hESCs) were differentiated into MPCs and β-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-μ (PFT-μ) 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-μ into pancreatic β-cells showed that PFT-μ 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-μ 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 β-cells. These findings suggest that adding a p53 inhibitor to the differentiation media can significantly enhance the generation of monohormonal β-cells.

Keywords: Differentiation protocol; Insulin; Islets; Monohormonal β-cells; NKX6.1; hESCs.

Fig. 1

Differentiation of hESCs into pancreatic progenitors and pancreatic β-cells. (A) Schematic diagram showing the differentiation protocols used to generate multipotent pancreatic progenitor cells (MPCs) and pancreatic β-cells. (B) Immunofluorescence analysis for the co-expression of PDX1 and NKX6.1 and glucagon (GCG) and insulin (INS) in hESC-derived MPCs and hESC-derived pancreatic β-cells. Scale bars = 50 μm

Fig. 2

Inhibition of p53 enhances generation of hPSC-pancreatic progenitors. (A-B) Flow cytometry analysis and quantification showing the co-expression of PDX1 and NKX6.1 after treating the cells during stage 4 with DMSO, pifithrin-α, and pifithrin-μ. (C) Flow cytometry quantification of the co-expression of PDX1 and NKX6.1 after treating the cells during stage 4 with DMSO, and pifithrin-μ (10 μM and 20 μM). Flow cytometry analysis (D) and quantification (E) of PDX1 and NKX6.1 expression in DMSO or pifithrin-μ - treated pancreatic progenitors (n = 3). Flow cytometry analysis (F) and quantification (G) of CHGA and NKX6.1 expression in DMSO or Pifithrin-μ - treated cells (n = 3). Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

p53 activation is important for endocrine lineage commitment. (A) The cells were treated with DMSO and pifithrin-μ during stage 5 of differentiation. (B) Flow cytometry of CHGA and NKX6.1 expression in DMSO or pifithrin 𝜇 -treated. (C) Quantification of the proportion of CHGA+/NKX6.1+ expressing cells (n = 3). Data are represented as mean ± SD; *p < 0.05, **p < 0.01

Fig. 4

p53 inhibition enhances pancreatic progenitor differentiation into monohormonal β-cells. (A) Experimental design for B and C. Flow cytometry of C-peptide and Glucagon expression in DMSO or pifithrin 𝜇 -treated (B). Quantification of proportion of C- Peptide (C-PEP)/Glucagon (GCG) co-positive β-cells (C)

Fig. 5

NKX6.1 overexpression in hPSC-derived pancreatic progenitors. (A) RT-PCR analysis showing the expression of NKX6.1 in hESC-derived pancreatic progenitors, 48 hours after transfection with NKX6.1 and LacZ control. (B) RT-qPCR analysis of NKX6.1 expression in hESC-derived pancreatic progenitors, 48 hours after transfection with NKX6.1 and LacZ control. (C) Western blot analysis showing the expression of NKX6.1, 48 hours after transfection with NKX6.1 and LacZ control. (D) Flow cytometry analysis of key pancreatic markers, NKX6.1, PDX1, SOX9, and CHGA, 48 hours after transduction of hESC-derived pancreatic progenitors with lentiviruses carrying lacZ control or NKX6.1. (E) Heatmap of Nanostring gene expression analysis comparing lacZ-transduced pancreatic progenitors compared to NKX6.1-transduced progenitors