An inhibitor of fibroblast growth factor receptor-1 (FGFR1) promotes late-stage terminal differentiation from NGN3+ pancreatic endocrine progenitors.

Yzumi Yamashita-Sugahara,Manami Ohtaka,Mahito Nakanishi,Masahito Matsumoto,Ken Nishimura,Kohnosuke Mitani,Yasushi Okazaki

doi:10.1038/srep35908

Abstract

Human induced pluripotent stem cells (hiPSCs) provide a potential resource for regenerative medicine. To identify the signalling pathway(s) contributing to the development of functional β cells, we established a tracing model consisting of dual knock-in hiPSCs (INS-Venus/NGN3-mCherry) (hIveNry) expressing the fluorescent proteins Venus and mCherry under the control of intrinsic insulin (INS) and neurogenin 3 (NGN3) promoters, respectively. hIveNry iPSCs differentiated into NGN3- and mCherry-positive endocrine progenitors and then into Venus-positive β cells expressing INS, PDX1, NKX6.1, and glucokinase (GCK). Using these cells, we conducted high-throughput screening of chemicals and identified a specific kinase inhibitor of fibroblast growth factor receptor 1 (FGFR1) that acted in a stage-dependent manner to promote the terminal differentiation of pancreatic endocrine cells, including β cells, from the intermediate stage of pancreatic endocrine progenitors while blocking the early development of pancreatic progenitors. This FGFR1 inhibitor augmented the expression of functional β cell markers (SLC30A8 and ABCC8) and improved glucose-stimulated INS secretion. Our findings indicate that the hIveNry model could provide further insights into the mechanisms of hiPS-derived β cell differentiation controlled by FGFR1-mediated regulatory pathways in a temporal-dependent fashion.

Highlights

Many studies have reported the generation of pancreatic endocrine cells in vitro from human embryonic stem cells/Human induced pluripotent stem cells (hiPSCs)
We examined the differentiation efficiency of 246H1 and TIG3/KOSM #7 (TIG) hiPSC lines reprogrammed with OCT4/SOX2/KLF4/Myc via retrovirus and Sendai virus, respectively
We discovered that inhibition of FGFR1 signalling promotes the intermediate stage of endocrine cell differentiation from endocrine progenitors (EPs), and contributes to their terminal differentiation towards mature functional βcells

Summary

Introduction

Many studies have reported the generation of pancreatic endocrine cells in vitro from human embryonic stem cells (hESCs)/hiPSCs. According to the differentiation protocols used in these studies, FGFR1 or FGFR2 agonists are utilized to drive the PDX1 expression that is essential for the early stages of βcell differentiation in vitro These procedures have been developed mostly based on mimicking normal pancreatic development mediated by the transition from PDX1+PPs to endocrine progenitors (EPs) transiently expressing neurogenin 3 (NGN3) as a master regulator of hormonal endocrine cells. The tracing model was designed to facilitate a better understanding of the developmental signals that contribute to the intermediate and terminal differentiation of EPs and lead to the maturation of βcells Using this reporter system, we have discovered that inhibition of FGFR1-mediated signalling enhances the efficiency of endocrine cell intermediate and terminal differentiation, and improves GSIS while increasing the expression of functional βcell markers. We propose that blockade of the FGFR1-mediated signalling cascade contributes to the efficient intermediate and terminal differentiation of βcells and the subsequent early PDX1+PP stage that is essential for the temporal-dependent action of FGFs and the production of functional βcells responsible for the maintenance of glucose homeostasis

Methods

Results

Conclusion