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. 2015 Winter;16(4):466-75.
doi: 10.22074/cellj.2015.492. Epub 2015 Jan 13.

DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs)

Beheshteh Abouhamzeh  1 , Mohammad Salehi  2 , Ahmad Hosseini  3 , Ali Reza Masteri-Farahani  4 , Fatemeh Fadai  4 , Mohammad Hasan Heidari  4 , Mohsen Nourozian  4 , Masoud Soleimani  5 , Mohsen Khorashadizadeh  6 , Majid Mossahebi-Mohammadi  7 , Ardalan Mansouri  3
Affiliations

DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs)

Beheshteh Abouhamzeh et al. Cell J. 2015 Winter.

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: DNA Methyltransferases; Epigenetics; Histone Deacetyltransferses; Somatic Cell Nuclear Transfer.

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Figures

Fig 1
Fig 1
Microscopic images of BADSCs (A) differentiated into adipocytes stained by Oil Red (B) differentiated into osteocytes stained by Alizarin Red, and undifferentiated (C). Bar=50 μ. BADSCs; Bovine adipose tissue-derived stem cells.
Fig 2
Fig 2
Histograms showing average relative transcription levels of HDAC1 (A), HDAC2 (B), HDAC3 (C), DNMT1 (D), DNMT3a (E) and DNMT3b (F) in BADSCs at P5 and P7 compared to P3. Gene transcription levels of the P3 cells were used as the calibrator. P; Passage number, HDAC; Histone deacetylases, DNMT; DNA methyltransferases and BADSCs; Bovine adipose derived stem cells.
Fig 3
Fig 3
Histogram indicating distribution of acetylation H3K9 using flow cytometry in BADSCs at P3 (A), P5 (B), P7 (C) and (D) positive control (HeLa cell). P; Passage number, H3K9; Histone H3 at Lysine 9 and BADSCs; Bovine adipose derived stem cells.
Fig 4
Fig 4
Histogram indicating distribution of Oct4 using flow cytometry in BADSCs at P3 (A), P5 (B), P7 (C) and (D) positive control (mouse embryonic stem cell). P; Passage number and BADSCs; Bovine adipose derived stem cells.
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