Shigeharu G Yabe1, Satsuki Fukuda1, Fujie Takeda1, Kiyoko Nashiro1, Masayuki Shimoda2, Hitoshi Okochi1. 1. Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan. 2. Pancreatic Islet Transplantation Project, Department of Diabetes Research Center, National Center for Global Health and Medicine, Tokyo, Japan.
Abstract
BACKGROUND: Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells. METHODS: A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. RESULTS: Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo. CONCLUSION: Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.
BACKGROUND: Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells. METHODS: A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiencymice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. RESULTS: Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabeticmice had lower blood glucose levels, and islet-like structures were detected in vivo. CONCLUSION: Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.
Authors: Kevin Verhoeff; Nerea Cuesta-Gomez; Ila Jasra; Braulio Marfil-Garza; Nidheesh Dadheech; A M James Shapiro Journal: Stem Cell Rev Rep Date: 2022-05-31 Impact factor: 6.692