Human amniotic fluid‐derived mesenchymal stem cells (AF‐MSCs) may be a valuable source for cell therapy and regenerative medicine. In this study, the potential of DNA methyltransferases (DNMT) inhibitors Decitabine, Zebularine, RG108 alone or combined with Zebularine and p53 inhibitor Pifithrin‐α to induce cardiomyogenic differentiation of AF‐MSCs was investigated. Differentiation into cardiomyocyte‐like cells initiation was indicated with all agents by changes in the cell phenotype, upregulation of the relative expression of the main cardiac genes (NKX2‐5, TNNT2, MYH6, and DES) as well as of cardiac ion channels genes (sodium, calcium, and potassium) as determined by reverse‐transcription quantitative polymerase chain reaction and the increase in Connexin43 levels as detected from Western blot and immunofluorescence data. Cellular energetics and mitochondrial function in induced cells were assessed using Seahorse analyzer and revealed the initiation of AF‐MSCs metabolic transformation into cardiomyocyte‐like cells. All used inducers were nontoxic to AF‐MSCs, arrested cell cycle at the G0/G1 phase, and upregulated p53 and p21 expression. The relative expression of miR‐34a and miR‐145 that are related to cell cycle regulation was also observed. Furthermore, the evaluated levels of chromatin remodeling proteins enhancer of zeste homolog 2, suppressor of zeste 12 protein homolog, DNMT1, histone deacetylase 1 (HDAC1), HDAC2, and heterochromatin protein 1α, as well as the rate of activating histone modifications, exhibited rearrangements of chromatin after the induction of cardiomyogenic differentiation. In conclusion, we demonstrated that all explored DNMT and p53 inhibitors initiated cardiomyogenesis‐related alterations in AF‐MSCs through rather similar mechanisms but to a different extent providing useful insights for the future research and potential applications of AF‐MSCs.
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- β 1) as potential cardiomyogenic differentiation inducers of AF-MSCs. Methods and Results AF-MSCs 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 pluripotency genes ( OCT4 , SOX2 , NANOG , REX1 ). Cardiomyogenic differentiation was induced using different concentrations of angiotensin II and TGF- β 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 RT-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 SUZ12 as well as of modified histones H3 and H4 indicated global chromatin remodeling during the induced differentiation. Conclusions Angiotensin II and TGF- β 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.
Human mesenchymal stem cells isolated from amniotic fluid (AF‐MSCs) demonstrate the potency for self‐renewal and multidifferentiation, and can, therefore, be a potential alternative source of stem cells adapted for therapeutic purposes. The object of this study is to evaluate the efficacy of MSCs from AF when the pregnancy is normal or when the fetus is affected during pregnancy to differentiate into mesodermal lineage tissues and to elucidate epigenetic states responsible for terminal adipogenic and osteogenic differentiation. The morphology of AF‐MSCs from two cell sources and the expression of the cell surface‐specific (CD44, CD90, and CD105) markers and pluripotency (Oct4, Nanog, Sox2, and Rex1) genes were quite similar and underwent mesodermal lineage differentiation because this is shown by the typical cell morphology and of genes’ expression specific for adipogenic (peroxisome proliferator‐activated receptor‐ɣ, adiponectin) and osteoblastic (alkaline phosphatase, osteopontin, and osteocalcin) differentiation. Terminal lineage‐specific differentiation was related to differential expression of miR‐17, miR‐21, miR‐34a, and miR‐146a, decreased levels of acetylated H4 and H3K9, trimethylated H3K4 and H3K9, and the retention of H3K27me3 along with a reduction in the levels of HDAC1, DNMT1, and PRC1/2 proteins (BMI1/SUZ12). No significant distinction could be identified in the levels of expression of all epigenetic or pluripotency markers between undifferentiated MSCs isolated from AF of normal gestation and pregnancy where the fetus was damaged and between those differentiated toward adipocytes or osteoblasts. The expressional changes of those marks and microRNAs that occurred during terminal differentiation to mesodermal tissues indicate subtle epigenetic regulation in AF‐MSCs when the condition of the fetus is healthy normal or diseased. More detailed studies of epigenetic mechanisms may offer a better understanding of AF‐MSCs differentiation in fetus‐diseased conditions and their usage in an autologous therapeutic application and prenatal disease research.
Amniotic fluid‐derived mesenchymal stem cells (AF‐MSCs) are autologous to the fetus and represent a potential alternative source for the regenerative medicine and treatment of perinatal disorders. To date, AF‐MSCs differentiation capacity to non‐mesodermal lineages and epigenetic regulation are still poorly characterized. The present study investigated the differentiation potential of AF‐MSCs toward neural‐like cells in comparison to the mesodermal myogenic lineage and assessed epigenetic factors involved in tissue‐specific differentiation. Myogenic and neural differentiation assays were performed by the incubation with specific induction media. Typical MSCs markers were determined by flow cytometry, the expression of lineage‐specific genes, microRNAs and chromatin modifying proteins were examined by RT‐qPCR and Western blot, respectively. AF‐MSCs of normal and fetus‐affected gestations had similar stem cells characteristics and two‐lineage potential, as characterized by cell morphology and the expression of myogenic and neural markers. Two‐lineage differentiation process was associated with the down‐regulation of miR‐17 and miR‐21, the up‐regulation of miR‐34a, miR‐146a and DNMT3a/DNMT3b along with the gradual decrease in the levels of DNMT1, HDAC1, active marks of chromatin (H4hyperAc, H3K9ac, H3K4me3) and the repressive H3K9me3 mark. Differentiation was accompanied by the down‐regulation of PRC1/2 proteins (BMI1/SUZ12, EZH2) and the retention of the repressive H3K27me3 mark. We report that both AF‐MSCs of normal and fetus‐affected gestations possess differentiation capacity toward myogenic and neural lineages through rather similar epigenetic mechanisms that may provide potential applications for further investigation of the molecular basis of prenatal diseases and for the future autologous therapy.
Human amniotic fluid mesenchymal stem cells (AF-MSCs) are a valuable, easily obtainable alternative source of SCs for regenerative medicine. Usually, amounts of cells required for the translational purposes are large thus the goal of this study was to assess the potency of AF-MSCs to proliferate and differentiate during long-term cultivation in vitro. AF-MSCs were isolated from amniotic fluid of healthy women in the second trimester of pregnancy and cultivated in vitro. AF-MSCs were cultivated up to 42 passages and they still maintained pluripotency genes, such as OCT4, SOX2, and NANOG, expression at a similar level as in the initial passages as determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).Fluorescence-activated cell sorting analysis demonstrated that the cell surface markers CD34 (negative), CD44, and CD105 (positive) expression was also stable, only the expression of SCs marker CD90 decreased during the cultivation. The morphology of AF-MSCs changed over passage, acridine orange/ethidium bromide staining revealed that more cells entered into apoptosis and the first signs of aging were detected only at late passages (later than p33) using SA-β-gal assay. Concomitantly, the differentiation potential towards cardiomyogenic lineage, induced with DNA methyltransferases inhibitors decitabine, zebularine, and RG108, was impaired when comparing AF-MSCs at p31/33 with p6. The expression of cardiomyocytes genes MYH6, TNNT2, DES together with ion channels genes of the heart (sodium, calcium, and potassium) decreased in p31/33 induced AF-MSCs. AF-MSCs have a great proliferative capacity and maintain most of the characteristics up to 33 passages; however, the cardiomyogenic differentiation capacity decreases to a certain extent during the long-term cultivation. These results provide useful insights for the potential use of AF-MSCs for biobanking and broad applications requiring high yield of cells or repeated infusions. Hence, it is vital to take into account the passage number of AF-MSCs, cultivated in culture, when utilizing them in vivo or in clinical experiments. K E Y W O R D S amniotic fluid, cardiac, cell differentiation, pregnancy, stem cells Abbreviations: AF-MSCs, amniotic fluid mesenchymal stem cells; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; MSCs, mesenchymal stem cells; PE, phycoerythrin.
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