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Preventing dedifferentiation of human exocrine enriched pancreatic cells in culture
Poster Abstracts
Preventing dedifferentiation of human exocrine enriched pancreatic cells in culture K R Muir, M J Lima, K Docherty
Abstract Published Online February 27, 2013 Poster 25 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK (KR Muir, M J Lima, K Docherty) Correspondence to: Dr Kenneth Muir, Lab 4.42, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK [email protected]
Background A cell-based therapy offers hope for a cure for type 1 diabetes. Attempts to generate a sizeable population of insulin producing cells from pancreatic cells in vitro have however been largely unsuccessful. This is partly related to beta cells and the exocrine component undergoing rapid dedifferentiation in standard culture conditions. Endocrine and exocrine markers are rapidly lost and the cells take on a mesenchymal phenotype. Because of developmental similarities this exocrine fraction holds potential for transdifferentiation towards a beta-cell lineage. We hypothesise that maintaining an epithelial phenotype will enhance this process. Here we look at the individual and combined effect of transforming growth factor β1 (TGFβ1) and rho-kinase inhibition on dedifferentiation of exocrine enriched pancreatic cells (EEPCs). Methods EEPCs left over from islet transplantation were cultured in RPMI medium with 10% FBS and left for 48 h to attach followed by addition of rho-kinase inhibitor (Y27632), a TGFβ1 inhibitor (SB431542), or both for a further 7 days. Gene expression was measured by real-time quantitative PCR. Findings After treatment with both Y27632 and SB431542, expression of amylase showed a 334% increase compared with untreated control and a 168% increase compared with baseline at 48 h in culture. Insulin showed a 571% increase compared with control but a decrease of 69% compared with baseline. Glucagon expression was 535% higher than control and 4% lower than baseline. Epithelial marker E-cadherin showed a 29% increase compared with control and 12% increase compared with baseline. All these markers were elevated at a statistically significant level compared with untreated controls. Interpretation The data suggest that the addition of Y27632 and SB431542 to EEPCs may slow down this dedifferentiation process. Maintenance of an epithelial morphology may result in cells that are more amenable to transdifferentiation towards beta cells. Funding Wellcome Trust.
80
www.thelancet.com
Preventing dedifferentiation of human exocrine enriched pancreatic cells in culture K R Muir, M J Lima, K Docherty
Abstract Published Online February 27, 2013 Poster 25 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK (KR Muir, M J Lima, K Docherty) Correspondence to: Dr Kenneth Muir, Lab 4.42, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK [email protected]
Background A cell-based therapy offers hope for a cure for type 1 diabetes. Attempts to generate a sizeable population of insulin producing cells from pancreatic cells in vitro have however been largely unsuccessful. This is partly related to beta cells and the exocrine component undergoing rapid dedifferentiation in standard culture conditions. Endocrine and exocrine markers are rapidly lost and the cells take on a mesenchymal phenotype. Because of developmental similarities this exocrine fraction holds potential for transdifferentiation towards a beta-cell lineage. We hypothesise that maintaining an epithelial phenotype will enhance this process. Here we look at the individual and combined effect of transforming growth factor β1 (TGFβ1) and rho-kinase inhibition on dedifferentiation of exocrine enriched pancreatic cells (EEPCs). Methods EEPCs left over from islet transplantation were cultured in RPMI medium with 10% FBS and left for 48 h to attach followed by addition of rho-kinase inhibitor (Y27632), a TGFβ1 inhibitor (SB431542), or both for a further 7 days. Gene expression was measured by real-time quantitative PCR. Findings After treatment with both Y27632 and SB431542, expression of amylase showed a 334% increase compared with untreated control and a 168% increase compared with baseline at 48 h in culture. Insulin showed a 571% increase compared with control but a decrease of 69% compared with baseline. Glucagon expression was 535% higher than control and 4% lower than baseline. Epithelial marker E-cadherin showed a 29% increase compared with control and 12% increase compared with baseline. All these markers were elevated at a statistically significant level compared with untreated controls. Interpretation The data suggest that the addition of Y27632 and SB431542 to EEPCs may slow down this dedifferentiation process. Maintenance of an epithelial morphology may result in cells that are more amenable to transdifferentiation towards beta cells. Funding Wellcome Trust.
80
www.thelancet.com