Figure 3

Differentiation of hLMDFs into insulin-producing cells (stage 3). (A) hLMDFs were cultured in DMEM/F12 medium with ITS mix for 7 days (stage 2: upper panel) and then switched to DMEM (low glucose) supplemented with 10% FBS, ITS mix, and nicotinamide for 7 days (stage 3: lower panel). The cells continued to differentiate and eventually formed larger clusters after completion of stage 3. These clusters were collections of differentiated cells that were intensely stained by DTZ. (B) RT-PCR analysis of endodermal and pancreatic cell-related genes in induced hLMDFs. These cells expressed endodermal-related genes in stage 2, and hLMDF-derived IPCs (stage 3) expressed pancreatic cell-related genes. Capan-2 was used as a control. (C) Western blot analysis of endodermal and pancreatic cell-related genes in induced hLMDFs. (D) hLMDF-derived IPCs stained crimson red by DTZ are visible, with some cells assembled in groups. (E) Immunocytochemical analysis of Pdx1, insulin, and c-peptide in stage 3. Nuclei were stained blue with DAPI. Expression of both insulin and c-peptide was detected in most stage 3 IPCs. All experiments were repeated at least three times. (F) hLMDF-derived IPCs (stage 3) exhibit glucose-stimulated insulin secretion. hLMDF-derived IPCs were sequentially treated with low (3 mM) and high (16.7 mM) concentrations of glucose and the supernatants were collected and analyzed for insulin content by ELISA. ^*P < 0.05 compared to 3 mM glucose. (G) After induction for 7 days in stage 3, hLMDF-derived IPCs secreted insulin in response to KCl. ^*P < 0.05 compared to vehicle treatment.