Document Type : Original Article
Authors
1
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran;Department of Cell Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical
2
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran;Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3
Department of Cell Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4
4Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran
5
5Department of Medical Biotechnology, School of Advanced Medical Technology, Tehran University of Medical Sciences, Tehran, Iran
6
6Department of Stem Cell Biology, Stem Cell Technology Research Center, Tehran, Iran
7
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract
Objective
Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer (SCNT). We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells (BADSCs) would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and histone deacetyltransferses (HDAC1, HDAC2, HDAC3) in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression (OCT4) and acetylation of H3K9 (H3K9ac) in BADSCs cultures and different passages in vitro.
Materials and Methods
In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction (q-PCR), and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 (P3), 5 (P5) and 7 (P7).
Results
The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7.
Conclusion
Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages.
Keywords
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