Angiotensin Ii And Tgf-Beta 1 Induce Alterations In Human Amniotic Fluid-Derived Mesenchymal Stem Cells Leading To Cardiomyogenic Differentiation Initiation

Background and Objectives: Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be a valuable source for cardiovascular tissue engineering and cell therapy. The aim of this study is to verify angiotensin II and transforming growth factor-beta 1 (TGF-beta 1) as potential cardiomyogenic differentiation inducers of AF-MSCs.Methods and Results: AF-NISCs were obtained from amniocentesis samples from second-trimester pregnant women, isolated and characterized by the expression of cell surface markers (CD44, CD90, CD105 positive; CD34 negative) and pluripolency genes (OCT4, SOX2, NANOG, REX1). Cardiomyogenic differentiation was induced using different concentrations of angiotensin H and TGF-beta 1. Successful initiation of differentiation was confirmed by alterations in cell morphology, upregulation of cardiac genes-markers NKX2-5, TBX5, GATA4, MYH6, TNNT2, DES and main cardiac ion channels genes (sodium, calcium, potassium) as determined by Kr-qPCR. Western blot and immunofluorescence analysis revealed the increased expression of Connexin43, the main component of gap junctions, and Nkx2.5, the early cardiac transcription factor. Induced AF-MSCs switched their phenotype towards more energetic and started utilizing oxidative phosphorylation more than glycolysis for energy production as assessed using Agilent Seahorse XF analyzer. The immune analysis of chromatin-modifying enzymes DNMT1, HDAC1/2 and Polycomb repressive complex 1 and 2 (PRC1/2) proteins BMI1, EZH2 and SUZI2 as well as of modified histones H3 and H4 indicated global chromatin remodeling during the induced differentiation.Conclusions: Angiotensin II and TGF-beta 1 are efficient cardiomyogenic inducers of human AF-MSCs; they initiate alterations at the gene and protein expression, metabolic and epigenetic levels in stem cells leading towards cardiomyocyte-like phenotype formation.