Suppression of Epithelial-to-Mesenchymal Transitioning Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue Toward Functional Insulin-Producing β-Like Cells

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FIG. 6.
FIG. 6.

The Rho-kinase and TGF-β1 pathway inhibitors suppress dedifferentiation of cultured pancreatic exocrine cells. A: Unpassaged exocrine pancreatic cells were plated in tissue culture dishes. After 48 h to allow attachment, cells were untreated (N/A) or treated with 2 μmol/L ρ-kinase inhibitor Y27632 (Y) and 10 μmol/L TGF-β1 inhibitor SB431542 (S) individually or in combination, and the cells were incubated for another 5 days. Treated and N/A cells as well as baseline samples after 48 h in culture (day 0) were then harvested and RNA was extracted for QRT-PCR analysis for expression of pancreatic, epithelial, and mesenchymal markers. Data are expressed relative to glyceraldehyde 3-phosphate dehydrogenase and presented as mean ± SEM (n = 3). A one-way ANOVA was performed with Dunnet post hoc test comparing treatment groups with N/A. A t test was used to compare day 2 with Y + S. For all analyses, *P < 0.05, **P < 0.01. B: Immunocytochemistry for the pancreatic markers amylase (AML) and Pdx1, the epithelial marker E-cadherin (ECAD), and the mesenchymal marker vimentin (VIM) in cultured exocrine pancreatic cells after 10 days in the presence of Y27632 and SB431542 (Y+S). N/A cells also were analyzed for the same markers. Nuclei were counterstained with DAPI. Scale bar = 20 μm. EPCAM, epithelial cell adhesion molecule; GLC, glucagon; INS, insulin; SST, somatostatin.

This Article

  1. Diabetes vol. 62 no. 8 2821-2833