Abstract
Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.
Highlights
Pancreatic beta cells are of great interest for biomedical research and regenerative medicine
Human foreskin fibroblasts were transduced with non-integrating episomal reprogramming factors, OCT4, SOX2, KLF4 and a short hairpin RNA against p5331, recovered in fibroblast medium for 4 days, and cultured in initiation medium containing epidermal growth factor (EGF), basic fibroblast growth factor and CHIR99021 to support cell proliferation (Fig. 1a)
These data demonstrate that human fibroblasts can be converted into converted definitive endodermal progenitor (cDE) cells using an episomal reprogramming system by employing the Cell-Activation and Signalling-Directed (CASD) transdifferentiation approach
Summary
Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Stepwise differentiation protocols have been devised to guide the differentiation of human embryonic stem cells (hESCs)[1], and more recently induced pluripotent stem cells (iPSCs)[2], into definitive endoderm, primitive gut tube endoderm, posterior foregut endoderm and pancreatic endoderm (PE) These hESC-derived PE cells can mature into functional beta-like cells in vivo after prolonged periods following transplantation into immunodeficient mice[3,4,5]. We previously devised a Cell-Activation and Signalling-Directed (CASD) lineage conversion strategy, which employs transient overexpression of iPSC-transcription factors in conjunction with lineage-specific soluble signals to reprogramme somatic cells into diverse lineage-specific cell types without first establishing a pluripotent state[20,21] Using this method, we and other groups have demonstrated the successful reprogramming of fibroblasts and other cells into cardiac cells[22,23], neural progenitors[24,25,26], angioblast-like progenitors[27], endothelial cells[28] and hepatocytes[29]. These findings suggest an approach for the potential production of patient-specific insulin-producing cells to study relevant and unresolved questions in beta-cell biology
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