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Differentiation of Sendai Virus-Reprogrammed iPSC into ? Cells, Compared with Human Pancreatic Islets and Immortalized ? Cell Line.

ABSTRACT: BACKGROUND:New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (i?). However, the gene expression profile and secretory function of i? still need to be validated in comparison with native ? cells. METHODS:Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into i? and compared with donor pancreatic islets and EndoC-?H1, an immortalized human ? cell line. RESULTS:Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). i? were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. i? basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that i? are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of i? to respond to glucose instead was more related to that of EndoC-?H1. DISCUSSION:We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human ? cells.

SUBMITTER: Pellegrini S

PROVIDER: S-EPMC6180725 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Differentiation of Sendai Virus-Reprogrammed iPSC into β Cells, Compared with Human Pancreatic Islets and Immortalized β Cell Line.

Pellegrini Silvia S Manenti Fabio F Chimienti Raniero R Nano Rita R Ottoboni Linda L Ruffini Francesca F Martino Gianvito G Ravassard Philippe P Piemonti Lorenzo L Sordi Valeria V

Cell transplantation 20180925 10

<h4>Background</h4>New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iβ). However, the gene expression profile and secretory function of iβ still need to be validated in comparison with native β cells.<h4>Methods</h4>Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iβ and compared with d ...[more]

PMID: 30251567

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Project description:microRNAs (miRNAs) play an important role in pancreatic development and adult ?-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in ?-and ?-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human ?-and ?-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in ?-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in ?-cells (?-miRNAs) and 134 were expressed more in ?-cells (?-miRNAs). Bioinformatic analysis identified potential targets of ?-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in ?-cells (TF?) is targeted by ?-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line ?TC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TF? that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic ?- and ?-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

Dataset Information

Differentiation of Sendai Virus-Reprogrammed iPSC into ? Cells, Compared with Human Pancreatic Islets and Immortalized ? Cell Line.

Publications

Differentiation of Sendai Virus-Reprogrammed iPSC into β Cells, Compared with Human Pancreatic Islets and Immortalized β Cell Line.

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