The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Abstract

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

Histone modifications interact with DNA methylation at the GATA4 promoter during differentiation of mesenchymal stem cells into cardiomyocyte‐like cells

“…Research has shown that 5‐aza inhibits DNA methylation; possible mechanisms for this process may be that 5‐aza induces a loss in the ability of DNA to accept methylation via its integration into the DNA or that 5‐aza reduces DNMT activity by reducing DNMT expression . In this study, we observed that 5‐aza not only reduced CpG methylation levels at the GATA4 promoter but also increased H3K9 acetylation levels and decreased H3K9 methylation levels in this region.…”

Pharmacologic concentrations of linezolid modify oxidative phosphorylation function and adipocyte secretome

“…It was previously reported that inhibiting nDNA methylation in 3T3-L1 preadipocytes, or hMSCs, by 5azadC significantly inhibited adipogenesis [38], [39], [40]. Thus, demethylating and upregulating WNT10A gene expression, which codes for a key factor to determine the fate of hMSCs, provoke a decreased PPARγ expression and reduced adipocyte differentiation [39], [40]. LIN, inhibiting mitochondrial translation, is able to affect nDNA methylation and reduce PPARγ mRNA levels.…”

Polysome Profiling Shows the Identity of Human Adipose-Derived Stromal/Stem Cells in Detail and Clearly Distinguishes Them from Dermal Fibroblasts

“…The proliferation assay was performed according to [26]. In brief, fibroblasts and ADSCs at 70% confluence were incubated with 100 mM bromodeoxyuridine (BrdU; Invitrogen) for 24 h. The cells were detached with trypsin and fixed by incubation with 100% ethanol for 30 min on ice.…”