원문정보
초록
영어
Diabetes mellitus, a hyperglycemic condition, in which the patients either fail to secrete insulin because of ß-cells destruction (type I) or shows insulin resistance (type II), affecting more than 300 million people around the globe. Generation of effective ß-cells for the treatment of diabetes is a key area in modern translational medicines. Here we tried to generate porcine induced pancreatic ß-cells (piPan ß-cells) from Gal- TKO+MCP porcine bone marrow mesenchymal stem cell (pBM-MSCs) by overexpressing set of transcription factors included EGFP along with step-wise induction of small molecules. pBM-MSCs were cultured in media with 5-azacytidine for 24 hours and then proceeded with transfection using episomal transfection system. Transfected cells were replaced with basal media containing growth factors cocktail, Activin A and 2 uM of A83-01 followed by retinoic acid treatment. Finally, cells were replaced by maintenance media until maturation. After 3 days of transfection, EGFP expression was observed showing successful transfection which disappeared completely after 21 days. pBM-MSCs attained definitive endoderm morphology after Activin A treatment and formed exact pancreatic clusters after 4 weeks and 8 weeks in maintenance media, respectively. After 4 and 8 weeks, piPan ß-cells were analyzed for the expression of pancreatic markers by immunofluorescence (Insulin, PAX6, Somatostatin) and RT-qPCR (GCG, INS, NKX6.1, PDX1 and NEUROD1), which showed significantly higher expression as compared with pBM-MSCs. Morphological changes and expression of pancreatic markers revealed successfully differentiation of PiPan ß-cells; however, more in depth studies for its functional characteristic and in vivo maintenance are still in progress.