Directed Differentiation of hPSCs into Pancreatic β-Cells

Differentiation was initiated when hPSCs reached 80% confluence. We used two protocols to generate MPCs as described in Supplementary Table 1. We used our established protocol of Memon et al. [20 (link)–22 ] to generate MPCs (pancreatic progenitors). For further differentiation of MPCs into pancreatic β-cells, updated version (v8) of Pagliuca et al., was used [16 (link)]. mTeSR1 media was replaced with MCDB131 and/or DMEM/F12 (Thermo Fisher Scientific) supplemented with the stage-specific cytokines and growth factors (the detailed protocol and cytokines were described in Fig. 1A, Supplementary Table 1). p53 inhibitors, pifithrin (PFT)-α and PFT-μ (Sigma, USA), were added at two different concentrations (10 μM and 20 μM) for 4 days during stage 4 of differentiation. During stage 5, 20 μM PFT-μ were added daily.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