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. 7.
FIG. 7.

The Rho-kinase and TGF-β1 pathway inhibitors enhance reprogramming toward insulin-producing cells. A: Unpassaged exocrine pancreatic cells were plated in tissue culture dishes. The cells were then cultured for 72 h in SFM containing Y27632 (Y), SB431542 (S), 5-Aza-2′deoxycytidine (A), and sodium butyrate (Bu). They were then transduced with adenoviruses expressing the 4TFs and cultured for 7 days in SFM containing 1 nmol/L betacellulin, 10 nmol/L exendin-4, and 10 mmol/L nicotinamide (BEN). Treated and untreated (N/A) cells then were harvested and RNA was extracted for QRT-PCR analysis. Data are represented as mean ± SEM and expressed relative to glyceraldehyde 3-phosphate dehydrogenase. *P < 0.05 or **P < 0.01 relative to N/A samples. B: Release of C-peptide to the medium in transdifferentiated (S+Y+A+Bu+4TFs+BEN) and N/A cells after incubation with 2.5 or 20 mmol/L of d-glucose for 1 h. The dashed line indicates the assay detection limit. Data are representative of triplicate experiments. ***P < 0.001. C: Insulin content of transdifferentiated (S+Y+A+Bu+4TFs+BEN) and N/A cells normalized to the DNA content of each sample. Data are representative of triplicate experiments. *P < 0.05. D: Immunostaining for insulin (INS) (panels ac and gi), C-peptide (C–PEP) (panels df), Pdx1 (panels gi), and glucagon (GLC) (panels df) of transdifferentiated cells in culture. Nuclei were counterstained with DAPI. Data are representative of triplicate experiments. Inlets show a 2× higher magnification image of stained clusters. Scale bar = 50 µm. AML, amylase; E-CAD, E-cadherin; SST, somatostatin.

This Article

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