A-366 induces cell cycle arrest and increases adipogenic differentiation potential of rat mesenchymal stem cells

Hedyeh Khanban,1,* Hedyeh khanban,2

1. Department of Animal Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University
2. Department of Biology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran

Abstract

Introduction

Epigenetic mechanisms such as histone methylation are considered as one of the most important mediators that control msc behaviors such as proliferation, senescence and differentiation. g9a, a histone methyltransferase, have recently generated intense attention as potential target for controlling many diseases such as cancer. the aim of the present study was to evaluate the effect of in vivo administration of a-366, a g9a inhibitor, on proliferative and differentiation capacity of bone marrow-derived mesenchymal stem cells (bm-mscs).

Methods

Rat bm-msc was treated with a-366 in vitro and then their proliferation and differentiation potential were analyzed using flow cytometry, population doubling time, and colony forming assay, oil red and alizarin red staining and real-time pcr. next, g9a was inhibited in vivo using intraperitoneally administration of a-366, and then bm-msc proliferation and differentiation behaviors were evaluated.

Results

We found that in vitro treatment of bm-mscs with a-366 didn’t affect their proliferation behavior as determined by calculating population doubling time and analysis of cell cycle. however, a high dose of a-366 increased pdt and induced cell cycle arrest. interestingly, in vitro treatment of bm-mscs with a-366 enhanced adipogenesis and reduced osteogenesis as determined by oil red and alizarin red staining, respectively. on the other hand, colony formation assay of bm-msc at primary culture showed that in vivo administration of a-366 reduced the colony formation capacity of bm-derived cells. moreover, pdt of bm-msc isolated from a-366 treated rats was higher than control, especially in earlier passages. more importantly, bm-msc isolated from a-366 treated rats showed higher adipogenic capacity than control at earlier passage as determined by expression of adipocyte specific genes and oil red staining. whereas, osteogenic propensity of bm-msc isolated from a-366 treated rats was lower than control, especially at lower passage.

Conclusion

Our results suggest that the epigenetic modifier such as a-366, which seems to be a therapeutic approach for controlling diseases such as cancer, might also influence the proliferation and differentiation capacity of mscs both in vitro and in vivo. moreover, epigenetic modifying chemicals seem to be a good strategy to manipulate msc expansion rate, differentiation propensity, as well as to efficiently use them in cell-based therapy and tissue engineering.

Keywords

Bm-mscs, g9a, adipogenesis, osteogenesis, a-366