الفهرس 
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Abstract
Cardiomyocytes are terminally differentiated cells that lack the ability to proliferate. Multilineage-differentiating stress enduring (Muse) cells have been previously identified as endogenous non-tumorigenic, self-renewable, and pluripotent-like stem cells. They are collected as stage specific embryonic antigen-3 (SSEA-3)+ mesenchymal stem cells from various easily accessible sources including the bone marrow, adipose tissue, and dermal fibroblasts. We describe in this study the cytokine induction steps for differentiation of human bone marrow derived-Muse stem cells into cardiomyocyte phenotype. The effect of suspension culture and 5’-Azacytidine on pluripotency, global DNA methylation, and Nkx2.5 -cardiac marker- promoter DNA methylation was assessed using Q-PCR, Methylated DNA-specific ELISA, and Bisulfite Sequencing; respectively. Three cytokine induction groups were set in the study: 1) Adherent: Muse cells were induced on adherent all the time; first induction with early cardiac differentiation factors including Wnt3a, BMP2/4, and TGFβ1, then induction with late cardiac differentiation cytokines including cardiotrophin-1. 2) Sus+Ad: this group was initially treated with 5’-azacytidine in suspension then induced as in Adherent group. 3) Sus+Ad+DN: same like Sus+Ad but DKK-1 and Noggin induction step was inserted between the two steps of induction. Cardiac differentiation was evaluated using mRNA and protein expression of several cardiac markers. We used RT-PCR analysis for Nkx2.5, Tbx20, MLC1v, MLC1a, and Q-PCR of α-actinin, MLC2v, and MLC2a. In addition, phase contrast observation of striation-like structures, laser confocal immunocytochemical staining for α-actinin and troponin-I, and western blotting analysis of α-actinin and desmin was done. Cardiorophin-1 main downstream pathway responsible for cardiac induction was studied by using α-actinin expression level in presence of PI3K and MEK1,2 pathway inhibitors. Treatment with 5’-azacytidine in suspension successfully up-regulated pluripotency, decreased global DNA methylation, and accelerated the de-methylation of Nkx2.5 promoter. Induced Muse in all groups expressed Nkx2.5, Tbx20, MLC1v, and MLC1a in RT-PCR experiments. α-actinin mRNA expression in Sus+Ad+DN group was significantly higher than other groups.The Sus+Ad and Sus+Ad+DN induction converted Muse cells into cardiomyocyte-like cells that expressed α-actinin, and troponin-I with striation-like pattern. The striation-like pattern was observed also in phase contrast microscopy.Desmin protein expression level in the Sus+Ad+DN group was significantly higher than other groups. MLC2a mRNA expression in the Sus+Ad group suggested differentiation into atrial subtype, while MLC2v mRNA expression in Sus+Ad+DN suggested differentiation into ventricular subtype. In addition,The PI3K pathway seemed to be the main pathway underlying cardiotrophin-1 cardiac induction effect. Muse cells could be induced into atrial or ventricular cardiomyocyte phenotype via a simple three or four-steps cytokine induction which might be beneficial for clinical applications in chronic cardiac conditions.