A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors.

Abstract

Efficient differentiation of human induced pluripotent stem cells (hiPSCs) into functional pancreatic cells holds great promise for diabetes research and treatment. However, a robust culture strategy for producing pancreatic progenitors with high homogeneity is lacking. Here, we established a simple differentiation strategy for generating synchronous iPSC-derived pancreatic progenitors via a two-step method of sequential cell synchronization using botulinum hemagglutinin (HA), an E-cadherin function-blocking agent. Of the various methods tested, the first-step synchronization method with HA exposure induces a synchronous switch from E- to N-cadherin and N- to E-cadherin expression by spatially controlling heterogeneous cell distribution, subsequently improving their competency for directed differentiation into definitive endodermal cells from iPSCs. The iPSC-derived definitive endodermal cells can efficiently generate PDX1+ and NKX6.1+ pancreatic progenitor cells in high yields. The PDX1+ and PDX1+ /NKX6.1+ cell densities showed 1.6- and 2.2-fold increases, respectively, compared with those from unsynchronized cultures. The intra-run and inter-run coefficient of variation were below 10%, indicating stable and robust differentiation across different cultures and runs. Our approach is a simple and efficient strategy to produce large quantities of differentiated cells with the highest homogeneity during multistage pancreatic progenitor differentiation, providing a potential tool for guided differentiation of iPSCs to functional insulin-producing cells.