Transdifferentiation of α-1,3-galactosyltransferase knockout pig bone marrow derived mesenchymal stem cells into pancreatic β-like cells by microenvironment modulation

Objective: To evaluate the pancreatic differentiation potential of alpha-1,3-galactosyltransferase knockout (GalTKO) pig-derived BM-MSCs using epigenetic modifiers with different pancreatic induction media. Methods: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been differentiated into pancreatic ss-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of alpha-1,3-galactosyltransferase knockout (GalTKO) pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - Advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by RT-qPCR, Western blotting, and immunofluorescence. Morphological changes and changes in the CpG island methylation patterns were also evaluated. Results: The expression of the pluripotent marker (OCT4) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of NEUROD1 in the ADMEM-based (5-Aza) media, while the expression of NKX6 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented transdifferentiation ability of GalTKO BM-MSCs into pancreatic ss-like cells in the N2B27-based media than in the ADMEM-based media. Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their transdifferentiation ability into pancreatic ss-like cells.