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Mesenchymal stem cells (MSCs) from healthy donors improve cardiac function in experimental acute myocardial infarction (AMI) models. However, little is known about the therapeutic capacity of human MSCs (hMSCs) from patients with ischemic heart disease (IHD). Therefore, the behavior of hMSCs from IHD patients in an immunecompromised mouse AMI model was studied. Enhanced green fluorescent protein-labeled hMSCs from IHD patients (hMSC group: 2 x [10.sup.5] cells in 20 [micro]l, n = 12) or vehicle only (medium group: n = 14) were injected into infarcted myocardium of NOD/scid mice. Sham-operated mice were used as the control (n = 10). Cardiac anatomy and function were serially assessed using 9.4-T magnetic resonance imaging (MRI); 2 wk after cell transplantation, immunohistological analysis was performed. At day 2, delayed-enhancement MRI showed no difference in myocardial infarction (MI) size between the hMSC and medium groups (33 [+ or -] 2% vs. 36 [+ or -] 2%: P = not significant). A comparable increase in left ventricular (LV) volume and decrease in ejection fraction (EF) was observed in both MI groups. However, at day 14, EF was higher in the hMSC than in the medium group (24 [+ or -] 3% vs. 16 [+ or -] 2%; P < 0.05). This was accompanied by increased vascularity and reduced thinning of the infarct scar. Engrafted hMSCs (4.1 [+ or -] 0.3% of injected cells) expressed von Willebrand factor (16.9 [+ or -] 2.7%) but no stringent cardiac or smooth muscle markers. hMSCs from patients with IHD engraft in infarcted mouse myocardium and preserve LV function 2 wk after AMI, potentially through an enhancement of scar vascularity and a reduction of wall thinning. magnetic resonance imaging; cell therapy; animal model

Myocardial scar formation impairs heart function by inducing cardiac remodeling, decreasing myocardial compliance, and compromising normal electrical conduction. Conversion of myocardial scar fibroblasts (MSFs) into (functional) cardiomyocytes may be an effective alternative treatment to limit loss of cardiac performance after myocardial injury. In this study, we investigated whether the phenotype of MSFs can be modified by gene transfer into cells with properties of cardiomyocytes. To this end, fibroblasts from postmyocardial infarction scars of human left ventricles were isolated and characterized by cell biological, immunological, and molecular biological assays. Cultured human MSFs express GATA4 and connexin 43 and display adipogenic differentiation potential. Infection of human MSFs with a lentivirus vector encoding the potent cardiogenic transcription factor myocardin renders them positive for a wide variety of cardiomyocytespecific proteins, including sarcomeric components, transcription factors, and ion channels, and induces the expression of several smooth muscle marker genes. Forced myocardin expression also endowed human MSFs with the ability to transmit an action potential and to repair an artificially created conduction block in cardiomyocyte cultures. These finding indicate that in vivo myocardin gene transfer may potentially limit cardiomyocyte loss, myocardial fibrosis, and disturbances in electrical conduction caused by myocardial infarction. [ABSTRACT FROM AUTHOR]

Myocardial and coronary development are both critically dependent on epicardial cells. During cardiomorphogenesis, a subset of epicardial cells undergoes an epithelial-to-mesenchymal transition (EMT) and invades the myocardium to differentiate into various cell types, including coronary smooth muscle cells and perivascular and cardiac interstitial fibroblasts. Our current knowledge of epicardial EMT and the ensuing epicardium-derived cells (EPDCs) comes primarily from studies of chick and mouse embryonic development. Due to the absence of an in vitro culture system, very little is known about human EPDCs. Here, we report for the first time the establishment of cultures of primary epicardial cells from human adults and describe their immunophenotype, transcriptome, transducibility, and differentiation potential in vitro. Changes in morphology and β-catenin staining pattern indicated that human epicardial cells spontaneously undergo EMT early during ex vivo culture. The surface antigen profile of the cells after EMT closely resembles that of subepithelial fibroblasts; however, only EPDCs express the cardiac marker genes GATA4 and cardiac troponin T. After infection with an adenovirus vector encoding the transcription factor myocardin or after treatment with transforming growth factor-β1 or bone morphogenetic protein-2, EPDCs obtain characteristics of smooth muscle cells. Moreover, EPDCs can undergo osteogenesis but fail to form adipocytes or endothelial cells in vitro. Cultured epicardial cells from human adults recapitulate at least part of the differentiation potential of their embryonic counterparts and represent an excellent model system to explore the biological properties and therapeutic potential of these cells. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The purpose of the study was to investigate the development of electrical transmission across human adult bone marrow-derived mesenchymal stem cells (hMSCs) during long-term co-incubation with cardiomyocytes (CMCs). Methods: Neonatal rat CMCs were cultured in multi-electrode array dishes. A conduction block was induced by creating a central acellular channel, yielding two asynchronously beating CMC fields. Enhanced green fluorescent protein (eGFP)-labeled hMSCs from ischemic heart disease patients (n =8), eGFP-labeled hMSCs having RNA interference-mediated connexin43 (Cx43) knockdown (n =6), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (Dil)-labeled CMCs (n =6), or no cells (n =9) were seeded in the channel. Assessment of conduction velocity (CV), Cx expression and localization, gap junctional coupling, and intracellular electrical recordings were performed for up to 14 days. Results: Resynchronization of the two CMC fields occurred within 24 h after seeding of hMSCs. CV across hMSCs increased from 1.4±0.4 cm/s at day 7 to 3.5±0.1 cm/s (p <0.05) at day 14. CV across seeded CMCs was 16.8±0.2 cm/s throughout this period. No resynchronization occurred in the absence of seeded cells. Knockdown of Cx43 in hMSCs abolished conduction across the channel completely. Time-dependent increase of CV across hMSCs was associated with increased Cx43 mRNA and protein expression resulting in increased gap junctional coupling. Intracellular recordings in coupled hMSCs showed increased conducted action potential (AP) amplitude, lower resting membrane potential, and decreased duration of conducted AP (p <0.05, day 14 versus day 1). Conclusions: CV across hMSCs increases progressively after 7 days of co-incubation with CMCs, most likely via improved electrotonic interaction. This is associated with increased Cx43 expression, increased functional gap junctional coupling, and enhanced intercellular electrical coupling between hMSCs and CMCs. [Copyright &y& Elsevier]

Abstract: Objective: Myocardin is a recently discovered transcriptional regulator of cardiac and smooth muscle development. Its ability to transactivate smooth muscle-specific genes has been firmly established in animal cells but its effect on heart muscle genes has been investigated less extensively and the consequences of ectopic myocardin expression in human cells are unknown. Methods: In this study, primary human mesenchymal stem cells and foreskin fibroblasts were transduced with human adenovirus vectors expressing the longest splice variant of the human myocardin gene (hAd5/F50.CMV.myocL) or with control vectors. One week later, the expression of muscle-restricted genes in these cells was analyzed by reverse transcription-polymerase chain reactions and immunofluorescence microscopy. Results: Forced expression of myocardin induced transcription of cardiac and smooth muscle genes in both cell types but did not lead to activation of skeletal muscle-specific genes. Double labeling experiments using monoclonal antibodies directed against striated (i.e. sarcomeric α-actin and sarcomeric α-actinin) and cardiac (i.e. natriuretic peptide precursor A) muscle-specific proteins together with a polyclonal antiserum specific for smooth muscle myosin heavy chain revealed that hAd5/F50.CMV.myocL-transduced cells co-express heart and smooth muscle-specific genes. Conclusions: These data indicate that the myocardin protein is a strong inducer of both smooth and cardiac muscle genes, but that additional factors are necessary to fully commit cells to either cardiac or smooth muscle cell fates. [Copyright &y& Elsevier]