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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.19.0796    [Accepted] Published online February 25, 2020.
Transdifferentiation of α-1,3-galactosyltransferase knockout pig bone marrow derived mesenchymal stem cells into pancreatic β-like cells by microenvironment modulation
Imran Ullah1,2  , Ran Lee1  , Keon Bong Oh1  , Seongsoo Hwang1  , Youngim Kim1  , Tai-Young Hur1  , Sun A Ock1,* 
1Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 565-851, Korea
2Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Correspondence:  Sun A Ock, Tel: +82-10-2907-0274, Fax: +82-63-238-7000, Email: ocksamoon@gmail.com
Received: 14 October 2019   • Revised: 21 November 2019   • Accepted: 15 January 2020
Abstract
Objective
To evaluate the pancreatic differentiation potential of α-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 α-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.
Keywords: N2B27; GalTKO BM-MSCs; Epigenetic Modifications; Cytidine; Pancreatic ß-like Cells
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