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Combined biophysical and soluble factor modulation induces cardiomyocyte differentiation from human muscle derived stem cells.

ABSTRACT: Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function of injured failing myocardium. Human multipotent muscle derived stem cells (MDSC) can be a potential abundant, autologous cell source for cardiac repair. However, robust conditions for cardiomyocyte (CM) differentiation are not well established for this cell type. We have developed a new method for CM differentiation from human MDSC that combines 3-dimensional artificial muscle tissue (AMT) culture with temporally controlled biophysical cell aggregation and delivery of 4 soluble factors (microRNA-206 inhibitor, IWR-1, Lithium Chloride, and BMP-4) (4F-AG-AMT). The 4F-AG-AMT displayed cardiac-like response to ?-adrenergic stimulation and contractile properties. 4F-AG-AMT expressed major cardiac (NKX2-5, GATA4, TBX5, MEF2C) transcription factors and structural proteins. They also express cardiac gap-junction protein, connexin-43, similar to CMs and synchronized spontaneous calcium transients. These results highlight the importance of temporal control of biophysical and soluble factors for CM differentiation from MDSCs.

SUBMITTER: Tchao J

PROVIDER: S-EPMC4196107 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Combined biophysical and soluble factor modulation induces cardiomyocyte differentiation from human muscle derived stem cells.

Tchao Jason J Han Lu L Lin Bo B Yang Lei L Tobita Kimimasa K

Scientific reports 20141014

Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function of injured failing myocardium. Human multipotent muscle derived stem cells (MDSC) can be a potential abundant, autologous cell source for cardiac repair. However, robust conditions for cardiomyocyte (CM) differentiation are not well established for this cell type. We have developed a new method for CM differentiation from human MDSC that combines 3-dimensional artificial muscle tissue (AMT) culture with tempo ...[more]

PMID: 25310989

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Project description:Direct cell reprogramming represents a promising new myocardial regeneration strategy involving in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocytes. Adult human cells are relatively resistant to reprogramming, however, likely because of epigenetic restraints on reprogramming gene activation. We hypothesized that modulation of the epigenetic regulator gene p63 could improve the efficiency of human cell cardio-differentiation. qRT-PCR analysis demonstrated significantly increased expression of a panel of cardiomyocyte marker genes in neonatal rat and adult rat and human cardiac fibroblasts treated with p63 shRNA (shp63) and the cardio-differentiation factors Hand2/Myocardin (H/M) versus treatment with Gata4, Mef2c and Tbx5 (GMT) with or without shp63 (p < 0.001). FACS analysis demonstrated that shp63+ H/M treatment of human cardiac fibroblasts significantly increased the percentage of cells expressing the cardiomyocyte marker cTnT compared to GMT treatment with or without shp63 (14.8% ± 1.4% versus 4.3% ± 1.1% and 3.1% ± 0.98%, respectively; p < 0.001). We further demonstrated that overexpression of the p63-transactivation inhibitory domain (TID) interferes with the physical interaction of p63 with the epigenetic regulator HDAC1 and that human cardiac fibroblasts treated with p63-TID+ H/M demonstrate increased cardiomyocyte marker gene expression compared to cells treated with shp63+ H/M (p < 0.05). Whereas human cardiac fibroblasts treated with GMT alone failed to contract in co-culture experiments, human cardiac fibroblasts treated with shp63+ HM or p63-TID+ H/M demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes. These findings demonstrate that p63 silencing provides enhanced rat and human cardiac fibroblast transdifferentiation into induced cardiomyocytes compared to a standard reprogramming strategy. p63-TID overexpression may be a useful reprogramming strategy for overcoming epigenetic barriers to human fibroblast cardio-differentiation.

Project description:BackgroundCellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported.Methods and resultsSkeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed.Conclusions and significanceSkeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

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Combined biophysical and soluble factor modulation induces cardiomyocyte differentiation from human muscle derived stem cells.

Publications

Combined biophysical and soluble factor modulation induces cardiomyocyte differentiation from human muscle derived stem cells.

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