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 a–c and g–i), C-peptide (C–PEP) (panels d–f), Pdx1 (panels g–i), and glucagon (GLC) (panels d–f) 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,