Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells
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.
KeywordsBeta-like cells BMP4 Human iPSC Insulin–neurogenin 3 Small molecule Sodium cromoglicate
Nicotinamide forskolin dexamethasone and ALK5 inhibitor
Bone morphogenetic protein
Embryonic stem cell
Human embryonic stem cell
Human induced pluripotent stem cell
Induced pluripotent stem cell
Pancreatic and duodenal homeobox 1
Quantitative real-time RT-PCR
The authors are grateful to S. Yamanaka (CiRA, Kyoto University, Kyoto, Japan), M. Uesugi (Institute for Integrated Cell-Material Sciences/Institute for Chemical Research, Kyoto University), T. Araoka (Salk Institute for Biological Studies, La Jolla, CA, USA), F. Shiota (CiRA, Kyoto University), M. Kotaka (CiRA, Kyoto University), K. Yasuda (CiRA, Kyoto University) and T. Kasahara (CiRA, Kyoto University) for technical support and helpful suggestions. Some of these data were presented as an abstract at the 51st Annual Meeting of the EASD (Stockholm, 2015) and the 7th Scientific Meeting of the Asian Association for the Study of Diabetes and the Annual Scientific Meeting of the Hong Kong Society of Endocrinology, Metabolism and Reproduction, in 2015.
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. All other datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.
This research was supported by the Japan Society for the Promotion of Science (JSPS) through its ‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)’; by the Japan Agency for Medical Research and Development (AMED) through its research grant ‘Core Center for iPS Cell Research, Research Center Network for Realization of Regenerative Medicine’; and by the Japan Diabetes Foundation.
Duality of interest
KO is a founder and member without salary of the scientific advisory boards of iPS Portal, Japan. All other authors declare that there is no duality of interest associated with this manuscript.
All authors contributed to the study conception and design, acquisition of data or analysis and interpretation of data. AO, NI and KO supervised all aspects of the study. YK and KO drafted the manuscript. All authors participated in critical revision of the manuscript and approved the final version. KO is the guarantor of this work.
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