Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells

Yasushi Kondo, Taro Toyoda, Ryo Ito, Michinori Funato, Yoshiya Hosokawa, Satoshi Matsui, Tomomi Sudo, Masahiro Nakamura, Chihiro Okada, Xiaotong Zhuang, Akira Watanabe, Akira Ohta, Nobuya Inagaki, Kenji Osafune

Pancreatic beta-like cells generated from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) offer an appealing donor tissue source. However, differentiation protocols that mainly use growth factors are costly. Therefore, in this study, we aimed to establish efficient differentiation protocols to change hiPSCs/hESCs to insulin (INS)(+) cells using novel small-molecule inducers. We screened small molecules that increased the induction rate of INS(+) cells from hESC-derived pancreatic and duodenal homeobox 1 (PDX1)(+) pancreatic progenitor cells. The differentiation protocol to generate INS(+) cells from hiPSCs/hESCs was optimised using hit compounds, and INS(+) cells induced with the compounds were characterised for their in vitro and in vivo functions. The inducing activity of the hit compounds was also examined using mouse embryonic pancreatic tissues in an explant culture system. Finally, RNA sequencing analyses were performed on the INS(+) cells to elucidate the mechanisms of action by which the hit compounds induced pancreatic endocrine differentiation. One hit compound, sodium cromoglicate (SCG), was identified out of approximately 1250 small molecules screened. When SCG was combined with a previously described protocol, the induction rate of INS(+) cells increased from a mean ± SD of 5.9 ± 1.5% (n = 3) to 16.5 ± 2.1% (n = 3). SCG induced neurogenin 3-positive cells at a mean ± SD of 32.6 ± 4.6% (n = 3) compared with 14.2 ± 3.6% (n = 3) for control treatment without SCG, resulting in an increased generation of endocrine cells including insulin-producing cells. Similar induction by SCG was confirmed using mouse embryonic pancreatic explants. We also confirmed that the mechanisms of action by which SCG induced pancreatic endocrine differentiation included the inhibition of bone morphogenetic protein 4 signalling. SCG improves the generation of pancreatic endocrine cells from multiple hiPSC/hESC lines and mouse embryonic pancreatic explants by facilitating the differentiation of endocrine precursors. This discovery will contribute to elucidating the mechanisms of pancreatic endocrine development and facilitate cost-effective generation of INS(+) cells from hiPSCs/hESCs. The RNA sequencing data generated during the current study are available in the Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo ) with series accession number GSE89973.