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In vitro generation of insulin-secreting cells from human pancreatic exocrine cells.

ABSTRACT: Transplantation of surrogate β-cells is a promising option for the treatment of insulin-deficient diabetes mellitus in the future. Although pancreatic exocrine cells of rodents have been shown to transdifferentiate into insulin-secreting cells, no studies are reported on human exocrine cells. Here, we report the generation of insulin-secreting cells from exocrine cells of the human pancreas. When cultured in suspension with epidermal growth factor, human pancreatic exocrine cells readily formed spherical cell clusters. Expression of Pdx1 was induced in all 19 cases in which we successfully isolated exocrine cells, and insulin expression was induced in 11 cases. In addition, insulin secretion was evaluated in four cases, and the newly-made cells were found to secrete insulin in response to various stimuli. Although further studies are required to improve both the quality and quantity of such insulin-secreting cells, our data suggest that pancreatic exocrine cells represent a potential source of insulin-secreting cells for treatment of type 1 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00095.x, 2011).

SUBMITTER: Minami K

PROVIDER: S-EPMC4014966 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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In vitro generation of insulin-secreting cells from human pancreatic exocrine cells.

Minami Kohtaro K Doi Ryuichiro R Kawaguchi Yoshiya Y Nukaya Daiki D Hagiwara Yoshiaki Y Noguchi Hirofumi H Matsumoto Shinichi S Seino Susumu S

Journal of diabetes investigation 20110801 4

Transplantation of surrogate β-cells is a promising option for the treatment of insulin-deficient diabetes mellitus in the future. Although pancreatic exocrine cells of rodents have been shown to transdifferentiate into insulin-secreting cells, no studies are reported on human exocrine cells. Here, we report the generation of insulin-secreting cells from exocrine cells of the human pancreas. When cultured in suspension with epidermal growth factor, human pancreatic exocrine cells readily formed ...[more]

PMID: 24843497

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Project description:Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide-inactivating and nucleotide-phosphorylating ecto-enzymes. We suggest that extracellular ATP homeostasis in pancreatic ducts may be important in pancreas physiology and potentially in pancreas pathophysiology.

Project description:BackgroundExpansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Methodology/principal findingRedifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.Conclusions/significanceThese findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.

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In vitro generation of insulin-secreting cells from human pancreatic exocrine cells.

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In vitro generation of insulin-secreting cells from human pancreatic exocrine cells.

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