Comparison of the Induction Effect of 5-Azacytidine and Trichostatin A on the Cardiac Differentiation of Human Adipose-Derived Stem Cells Encapsulated in Fibrin Scaffolds

Background and Aim: Human adipose-derived stem cells (hADSCs)are capable of differentiating into many cells including cardiac cells.Several chemical agents and different types of scaffolds are used for celldifferentiation, but the best is yet to be determined. The main purpose ofthis study focused on the possibility of better differentiation of hADSCstowards to the cardiac-like cells in fabricated fibrin scaffold (3D) incomparison with culture plates (2D) in the presence of 5- Azacytidine(5-Aza) and trichostatin A (TSA).Methods: After approving the characteristics of hADSCs by flowcytometryand differentiation of hADSCs into adipocytes, osteocytes, andchondrocytes, cells were cultured in fibrin scaffold (3D) that fabricatedusing human plasma fibrinogen and culture plate (2D) and treatedwith 10 μM of 5-Aza and TSA, then followed weekly up to 4 weeks.The morphology of the scaffold was characterized by Scanning ElectronMicroscopy (SEM). In 2D culture, the differentiated cells were examinedfor cell growth and morphological changes every day by using aninverted phase contrast microscope. Immunochemistry assays and qRTPCRwere used to evaluate the expression of special cardiac genes suchas NKX2.5, Cx43, and cTnI in the treated hADSCs with 5-Aza and TSA inthe 3D and 2D groups. In addition, induced cells in each of the groupswere examined by Transmission Electron Microscopy (TEM).Results: The SEM images of scaffolds showed that a uniform structure ofthe scaffold with a mean pore size of 113.14 ± 26 μm. The fibroblastlikemorphology was the dominant form in the negative control group.Gradual morphological changes of the induced hADSCs using TSA in the2D group were multinuclear, elongated, ball-like myotube-like structuresand fork like and star-like during the 4th week. Immunochemistry assayand qRT-PCR showed the significantly higher expression of specialcardiac genes such as NKX2.5, Cx43 and cTnI in the treated hADSCswith TSA in the 3D and 2D groups in comparison with 5-Aza groups(P < 0.05). Also, treated hADSCs with TSA, within 4 weeks, showedthe hADSCs could differentiate to cardiomyocyte-like cells. The TEMimages showed that hADSCs in the treatment groups using TSA all hadmyofilaments in the cytoplasm when compared with the 5-Aza groups.Conclusion: Taken together, these results indicated that the TSA allowsa higher and sooner differentiation of hADSCs into cardiomyocyte-likecells treated alone or with fibrin scaffold and it can lead to generatingof cardiac-like cells in a shorter period of time in comparison to 5-Aza.In addition to the combination of TSA and fibrin scaffold effectivelypromotes the cardiomyogenic differentiation of hADSCs in vitro theirapplication may represent a therapeutic strategy for the treatment ofischemic heart disease.

Keywords:

Human ADSCs , Cardiomyocyte-like cells , Fibrin scaffold , TSA , 5-Aza