Local activation of cardiac stem cells for post‐myocardial infarction cardiac repair

The prognosis of patients with myocardial infarction (MI) and resultant chronic heart failure remains extremely poor despite continuous advancements in optimal medical therapy and interventional procedures. Animal experiments and clinical trials using adult stem cell therapy following MI have shown a global improvement of myocardial function. The emergence of stem cell transplantation approaches has recently represented promising alternatives to stimulate myocardial regeneration. Regarding their tissue‐specific properties, cardiac stem cells (CSCs) residing within the heart have advantages over other stem cell types to be the best cell source for cell transplantation. However, time‐consuming and costly procedures to expanse cells prior to cell transplantation and the reliability of cell culture and expansion may both be major obstacles in the clinical application of CSC‐based transplantation therapy after MI. The recognition that the adult heart possesses endogenous CSCs that can regenerate cardiomyocytes and vascular cells has raised the unique therapeutic strategy to reconstitute dead myocardium via activating these cells post‐MI. Several strategies, such as growth factors, mircoRNAs and drugs, may be implemented to potentiate endogenous CSCs to repair infarcted heart without cell transplantation. Most molecular and cellular mechanism involved in the process of CSC‐based endogenous regeneration after MI is far from understanding. This article reviews current knowledge opening up the possibilities of cardiac repair through CSCs activation in situ in the setting of MI.     

Bone marrow‐derived cells can acquire cardiac stem cells properties in damaged heart

Experimental data suggest that cell‐based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit⁺ CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild‐type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate ‘cardiospheres’, a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit⁺ CSCs.     

Therapeutic benefits of CD90‐negative cardiac stromal cells in rats with a 30‐day chronic infarct

Cardiac stromal cells (CSCs) can be derived from explant cultures, and a subgroup of these cells is viewed as cardiac mesenchymal stem cells due to their expression of CD90. Here, we sought to determine the therapeutic potential of CD90‐positive and CD90‐negative CSCs in a rat model of chronic myocardial infarction. We obtain CD90‐positive and CD90‐negative fractions of CSCs from rat myocardial tissue explant cultures by magnetically activated cell sorting. In vitro, CD90‐negative CSCs outperform CD90‐positive CSCs in tube formation and cardiomyocyte functional assays. In rats with a 30‐day infarct, injection of CD90‐negative CSCs augments cardiac function in the infarct in a way superior to that from CD90‐positive CSCs and unsorted CSCs. Histological analysis revealed that CD90‐negative CSCs increase vascularization in the infarct. Our results suggest that CD90‐negative CSCs could be a development candidate as a new cell therapy product for chronic myocardial infarction.     

Characterization of contracting cardiomyocyte colonies in the primary culture of neonatal rat myocardial cells: A model of in vitro cardiomyogenesis

The unmet clinical need for myocardial repair after irreversible ischemic injury requires a better understanding of the biological properties of cardiac stem cells (CSCs). Using a primary culture of neonatal rat myocardial cells, we describe the formation and maturation of contracting cardiomyocyte colonies stemming from c-kit⁺, Sca⁺, or Isl1⁺ CSCs, which occurs in parallel to the hypertrophy of the major cardiac myocyte population. The contracting cardiomyocyte colonies (~1–2 colonies per 1 × 10⁵ of myocardial cells) were identified starting from eighth day of culturing. At first, spontaneous weak, asynchronous, and arrhythmic contractions of the colonies at a rate of 2–3 beats/min were registered. Over time, the contractions of the colonies became more synchronous and frequent, with a contraction rate of 58–60 beats/min by the 30th day of culturing. The colonies were characterized by the CSCs subtype-specific pattern of growth and structure. The cells of the colonies were capable of spontaneous cardiomyogenic differentiation, demonstrating expression of both sarcomeric α-actinin and α-sarcomeric actin as well as the maturation of contractile machinery and typical Ca²⁺ responses to caffeine (5 mМ) and K⁺ (120 mМ). Electromechanical coupling, characterized by cardiac muscle-specific Ca²⁺-induced Ca²⁺ release, was evident at 3 weeks of culturing. Thus, the co-cultivation of CSCs with mature cardiac cells resulted in the formation of contracting cardiomyocyte colonies, resembling the characteristics of in vivo cardiomyogenesis. The proposed model can be used for the investigation of fundamental mechanisms underlying cardiomyogenic differentiation of CSCs as well as for drug testing and/or other applications.     

Bmi‐1 high‐expressing cells enrich cardiac stem/progenitor cells and respond to heart injury

Bmi‐1 gene is well recognized as an oncogene, but has been recently demonstrated to play a role in the self‐renewal of tissue‐specific stem cells. By using Bmi‐1^GFP/+ mice, we investigated the role of Bmi‐1 in cardiac stem/progenitor cells and myocardial repair. RT‐PCR and flow cytometry analysis indicated that the expression of Bmi‐1 was significantly higher in cardiac side population than the main population from CD45^?Ter119^?CD31^? heart cells. More Sca‐1⁺ cardiac stem/progenitor cells were found in Bmi‐1 GFP^hi subpopulation, and these Bmi‐1 GFP^hi heart cells showed the potential of differentiation into SMM⁺ smooth muscle‐like cells and TnT⁺ cardiomyocyte‐like cells in vitro. The silencing of Bmi‐1 significantly inhibited the proliferation and differentiation of heart cells. Otherwise, myocardial infarction induced a significantly increase (2.7‐folds) of Bmi‐1 GFP^hi population, mainly within the infarction and border zones. These preliminary data suggest that Bmi‐1^hi heart cells are enriched in cardiac stem/progenitor cells and may play a role in myocardial repair.     

Human cardiac stem cells isolated from atrial appendages stably express c-kit

The in vivo studies of myocardial infarct using c-kit⁺/Lin⁻ cardiac stem cells (CSCs) are still in the early stage with margin or no beneficial effects for cardiac function. One of the potential reasons may be related to the absence of fully understanding the properties of these cells both in vitro and in vivo. In the present study, we aimed to systematically examine how CSCs adapted to in vitro cell processes and whether there is any cell contamination after long-term culture. Human CSCs were enzymatically isolated from the atrial appendages of patients. The fixed tissue sections, freshly isolated or cultured CSCs were then used for identification of c-kit⁺/Lin⁻ cells, detection of cell contamination, or differentiation of cardiac lineages. By specific antibody staining, we demonstrated that tissue sections from atrial appendages contained less than 0.036% c-kit⁺/Lin⁻ cells. For the first time, we noted that without magnetic activated cell sorting (MACS), the percentages of c-kit⁺/Lin⁻ cells gradually increased up to ∼40% during continuously culture between passage 2 to 8, but could not exceed >80% unless c-kit MACS was carried out. The resulting c-kit⁺/Lin⁻ cells were negative for CD34, CD45, CD133, and Lin markers, but positive for KDR and CD31 in few patients after c-kit MACS. Lin depletion seemed unnecessary for enrichment of c-kit⁺/Lin⁻ cell population. Following induced differentiation, c-kit⁺/Lin⁻ CSCs demonstrated strong differentiation towards cardiomyocytes but less towards smooth and endothelial cells. We concluded that by using an enzymatic dissociation method, a large number, or higher percentage, of relative pure human CSCs with stable expression of c-kit⁺ could be obtained from atrial appendage specimens within ∼4 weeks following c-kit MACS without Lin depletion. This simple but cost-effective approach can be used to obtain enough numbers of stably-expressed c-kit⁺/Lin⁻ cells for clinical trials in repairing myocardial infarction.     

Reaching out: junctions between cardiac telocytes and cardiac stem cells in culture

Telocytes (TCs) were previously shown by our group to form a tandem with stem/progenitor cells in cardiac stem cell (CSC) niches, fulfilling various roles in cardiac renewal. Among these, the ability to ‘nurse’ CSCs in situ, both through direct physical contact (junctions) as well as at a distance, by paracrine signalling or through extracellular vesicles containing mRNA. We employed electron microscopy to identify junctions (such as gap or adherens junctions) in a co‐culture of cardiac TCs and CSCs. Gap junctions were observed between TCs, which formed networks, however, not between TCs and CSCs. Instead, we show that TCs and CSCs interact in culture forming heterocellular adherens junctions, as well as non‐classical junctions such as puncta adherentia and stromal synapses. The stromal synapse formed between TCs and CSCs (both stromal cells) was frequently associated with the presence of electron‐dense nanostructures (on average about 15 nm in length) connecting the two opposing membranes. The average width of the synaptic cleft was 30 nm, whereas the average length of the intercellular contact was 5 μm. Recent studies have shown that stem cells fail to adequately engraft and survive in the hostile environment of the injured myocardium, possibly as a result of the absence of the pro‐regenerative components of the secretome (paracrine factors) and/or of neighbouring support cells. Herein, we emphasize the similarities between the junctions described in co‐culture and the junctions identified between TCs and CSCs in situ. Reproducing a CSC niche in culture may represent a viable alternative to mono‐cellular therapies.     

In vitro cardiomyogenic differentiation of adipose‐derived stromal cells using transforming growth factor‐β1

Transplanting stem cells differentiated towards a cardiac lineage can regenerate cardiac muscle tissues to treat myocardial infarction. In this study, we tested the hypothesis that transforming growth factor‐β1 (TGF‐β1) induces cardiomyogenic differentiation of adipose‐ derived stromal cells (ADSCs) in vitro. Rat ADSCs were cultured with TGF‐β1 (10 ng ml^?1) for 2 weeks in vitro. ADSCs cultured without TGF‐β1 served as a control. The mRNA expression of cardiac‐specific gene was induced by TGF‐β1, while the control culture did not show cardiac‐specific gene expression. Immunocytochemical analyses showed that a small fraction of ADSCs cultured with TGF‐β1 for 2 weeks stained positively for cardiac myosin heavy chain (MHC) and α‐sarcomeric actin. Flow cytometric analyses showed that the proportion of cells expressing cardiac MHC increased with TGF‐β1. However, no mesenchymal differentiation (e.g., osteogenic and adipogenic differentiation) was detected other than cardiomyogenic differentiation. These results showed that TGF‐β1 induce ADSC cardiomyogenic differentiation in vitro, which could be useful for myocardial infarction stem cell therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Sca-1+ cardiac stem cells mediate acute cardioprotection via paracrine factor SDF-1 following myocardial ischemia/reperfusion

Background

Cardiac stem cells (CSCs) promote myocardial recovery following ischemia through their regenerative properties. However, little is known regarding the implication of paracrine action by CSCs in the setting of myocardial ischemia/reperfusion (I/R) injury although it is well documented that non-cardiac stem cells mediate cardioprotection via the production of paracrine protective factors. Here, we studied whether CSCs could initiate acute protection following global myocardial I/R via paracrine effect and what component from CSCs is critical to this protection.

Methodology/Principal Findings

A murine model of global myocardial I/R was utilized to investigate paracrine effect of Sca-1+ CSCs on cardiac function. Intracoronary delivery of CSCs or CSC conditioned medium (CSC CM) prior to ischemia significantly improved myocardial function following I/R. siRNA targeting of VEGF in CSCs did not affect CSC-preserved myocardial function in response to I/R injury. However, differentiation of CSCs to cardiomyocytes (DCSCs) abolished this protection. Through direct comparison of the protein expression profiles of CSCs and DCSCs, SDF-1 was identified as one of the dominant paracrine factors secreted by CSCs. Blockade of the SDF-1 receptor by AMD3100 or downregulated SDF-1 expression in CSCs by specific SDF-1 siRNA dramatically impaired CSC-induced improvement in cardiac function and increased myocardial damage following I/R. Of note, CSC treatment increased myocardial STAT3 activation after I/R, whereas downregulation of SDF-1 action by blockade of the SDF-1 receptor or SDF-1 siRNA transfection abolished CSC-induced STAT3 activation. In addition, inhibition of STAT3 activation attenuated CSC-mediated cardioprotection following I/R. Finally, post-ischemic infusion of CSC CM was shown to significantly protect I/R-caused myocardial dysfunction.

Conclusions/Significance

This study suggests that CSCs acutely improve post-ischemic myocardial function through paracrine factor SDF-1 and up-regulated myocardial STAT3 activation.     

Activation of Cardiac Stem Cells in Myocardial Infarction

The development of heart failure caused by acute myocardial infarction is accompanied by massive necrotic death of cardiomyocytes in lesion areas and subsequent pathological myocardial remodeling. Traditionally, the possibility of heart reparation has been considered to be severely limited or absent in the postnatal period. Endogenous cardiac stem cells with a regenerative potential have recently been described, but the mechanisms of activation of these cells remain poorly understood. The aim of our work was to obtain cardiac stem cells from the ischemic area of the myocardium and compare their functional properties with stem cells isolated from the healthy area of the myocardium. RT-PCR was used to quantify the gene expression in cardiac stem cells. In addition, differentiated cells were stained for specific markers using immunocytochemical method. Cardiac stem cells originating from the infarction area had a higher proliferative potential and a greater propensity to migrate in comparison to the cells originated from a healthy myocardial area. The expression level of several specific markers of cardiogenic, osteogenic and adipogenic differentiation upon induction of corresponding differentiation was higher in the cells from the infarction area than in cells from the healthy myocardium. We conclude that myocardial ischemia activates the internal regenerative potential of cardiac stem cells.     

Sublethal Caspase Activation Promotes Generation of Cardiomyocytes from Embryonic Stem Cells

Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury, but which kind of stimuli is most important for cardiomyocyte regeneration is still unclear. Here we explore if apoptotic stimuli, manifested through caspase activation, influences cardiac progenitor up-regulation and cardiomyocyte differentiation. Using mouse embryonic stem cells as a cellular model, we show that sublethal activation of caspases increases the yield of cardiomyocytes while concurrently promoting the proliferation and differentiation of c-Kit⁺/α-actinin^low cardiac progenitor cells. A broad-spectrum caspase inhibitor blocked these effects. In addition, the caspase inhibitor reversed the mRNA expression of genes expressed in cardiomyocytes and their precursors. Our study demonstrates that sublethal caspase-activation has an important role in cardiomyocyte differentiation and may have significant implications for promoting cardiac regeneration after myocardial injury involving exogenous or endogenous cell sources.     

Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes

Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit⁺ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit⁺ CSCs.     

Apoptotic and autophagic pathways with relevant small‐molecule compounds,in cancer stem cells

Accumulating evidence demonstrates existence of cancer stem cells (CSCs), which are suspected of contributing to cancer cell self‐renewal capacity and resistance to radiation and/or chemotherapy. Including evasion of apoptosis and autophagic cell death, CSCs have revealed abilities to resist cell death, making them appealing targets for cancer therapy. Recently, molecular mechanisms of apoptosis and of autophagy in CSCs have been gradually explored, comparing them in stem cells and in cancer cells; distinct expression of these systems in CSCs may elucidate how these cells exert their capacity of unlimited self‐renewal and hierarchical differentiation. Due to their proposed ability to drive tumour initiation and progression, CSCs may be considered to be potentially useful pharmacological targets. Further, multiple compounds have been verified as triggering apoptosis and/or autophagy, suppressing tumour growth, thus providing new strategies for cancer therapy. In this review, we summarized regulation of apoptosis and autophagy in CSCs to elucidate how key proteins participate in control of survival and death; in addition, currently well‐studied compounds that target CSC apoptosis and autophagy are selectively presented. With increasing attention to CSCs in cancer therapy, researchers are now trying to find responses to unsolved questions as unambiguous as possible, which may provide novel insight into future anti‐cancer regimes.     

Telocytes and putative stem cells in ageing human heart

Tradition considers that mammalian heart consists of about 70% non‐myocytes (interstitial cells) and 30% cardiomyocytes (CMs). Anyway, the presence of telocytes (TCs) has been overlooked, since they were described in 2010 (visit www.telocytes.com ). Also, the number of cardiac stem cells (CSCs) has not accurately estimated in humans during ageing. We used electron microscopy to identify and estimate the number of cells in human atrial myocardium (appendages). Three age‐related groups were studied: newborns (17 days–1 year), children (6–17 years) and adults (34–60 years). Morphometry was performed on low‐magnification electron microscope images using computer‐assisted technology. We found that interstitial area gradually increases with age from 31.3 ± 4.9% in newborns to 41 ± 5.2% in adults. Also, the number of blood capillaries (per mm²) increased with several hundreds in children and adults versus newborns. CMs are the most numerous cells, representing 76% in newborns, 88% in children and 86% in adults. Images of CMs mitoses were seen in the 17‐day newborns. Interestingly, no lipofuscin granules were found in CMs of human newborns and children. The percentage of cells that occupy interstitium were (depending on age): endothelial cells 52–62%; vascular smooth muscle cells and pericytes 22–28%, Schwann cells with nerve endings 6–7%, fibroblasts 3–10%, macrophages 1–8%, TCs about 1% and stem cells less than 1%. We cannot confirm the popular belief that cardiac fibroblasts are the most prevalent cell type in the heart and account for about 20% of myocardial volume. Numerically, TCs represent a small fraction of human cardiac interstitial cells, but because of their extensive telopodes, they achieve a 3D network that, for instance, supports CSCs. The myocardial (very) low capability to regenerate may be explained by the number of CSCs, which decreases fivefold by age (from 0.5% to 0.1% in newborns versus adults).     

The secretome of myocardial telocytes modulates the activity of cardiac stem cells

Telocytes (TCs) are interstitial cells that are present in numerous organs, including the heart interstitial space and cardiac stem cell niche. TCs are completely different from fibroblasts. TCs release extracellular vesicles that may interact with cardiac stem cells (CSCs) via paracrine effects. Data on the secretory profile of TCs and the bidirectional shuttle vesicular signalling mechanism between TCs and CSCs are scarce. We aimed to characterize and understand the in vitro effect of the TC secretome on CSC fate. Therefore, we studied the protein secretory profile using supernatants from mouse cultured cardiac TCs. We also performed a comparative secretome analysis using supernatants from rat cultured cardiac TCs, a pure CSC line and TCs‐CSCs in co‐culture using (i) high‐sensitivity on‐chip electrophoresis, (ii) surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry and (iii) multiplex analysis by Luminex‐xMAP. We identified several highly expressed molecules in the mouse cardiac TC secretory profile: interleukin (IL)‐6, VEGF, macrophage inflammatory protein 1α (MIP‐1α), MIP‐2 and MCP‐1, which are also present in the proteome of rat cardiac TCs. In addition, rat cardiac TCs secrete a slightly greater number of cytokines, IL‐2, IL‐10, IL‐13 and some chemokines like, GRO‐KC. We found that VEGF, IL‐6 and some chemokines (all stimulated by IL‐6 signalling) are secreted by cardiac TCs and overexpressed in co‐cultures with CSCs. The expression levels of MIP‐2 and MIP‐1α increased twofold and fourfold, respectively, when TCs were co‐cultured with CSCs, while the expression of IL‐2 did not significantly differ between TCs and CSCs in mono culture and significantly decreased (twofold) in the co‐culture system. These data suggest that the TC secretome plays a modulatory role in stem cell proliferation and differentiation.     

Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.     

Vascular endothelial growth factor promotes cardiac stem cell migration via the PI3K/Akt pathway

VEGF is a major inducer of angiogenesis. However, the homing role of VEGF for cardiac stem cells (CSCs) is unclear. In in vitro experiments, CSCs were isolated from the rat hearts, and a cellular migration assay was performed using a 24-well transwell system. VEGF induced CSC migration in a concentration-dependent manner, and SU5416 blocked this. Western blot analysis showed that the phosphorylated Akt was markedly increased in the VEGF-treated CSCs and that inhibition of pAkt activity significantly attenuated the VEGF-induced the migration of CSCs. In in vivo experiments, rat heart myocardial infarction (MI) was induced by left coronary artery ligation. One week after MI, the adenoviral vector expressing hVEGF165 and LacZ genes were injected separately into the infarcted myocardium at four sites before endomyocardial transplantation of 2 × 10⁵ PKH26 labeled CSCs (50 μL) at atrioventricular groove. One week after CSC transplantation, RT-PCR, immunohistochemical staining, Western blot, and ELISA analysis were performed to detect the hVEGF mRNA and protein. The expression of hVEGF mRNA and protein was significantly increased in the infarcted and hVEGF165 transfected rat hearts, accompanied by an enhanced PI3K/Ak activity, a greater accumulation of CSCs in the infarcted region, and an improvement in cardiac function. The CSC accumulation was inhibited by either the VEGF receptor blocker SU5416 or the PI3K/Ak inhibitor wortmannin. VEGF signaling may mediate the migration of CSCs via activation of PI3K/Akt.     

Identification of cardiac stem cells within mature cardiac myocytes

Cardiac stem cells are described in a number of mammalian species including humans. Cardiac stem cell clusters consisting of both lineage-negative and partially committed cells are generally identified between contracting cardiac myocytes. In the present study, c-kit⁺, Sca⁺, and Isl1⁺ stem cells were revealed to be located inside the sarcoplasm of cardiac myocytes in myocardial cell cultures derived from newborn, 20-, and 40-day-old rats. Intracellularly localized cardiac stem cells had a coating or capsule with a few pores that opened into the host cell sarcoplasm. The similar structures were also identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats. The results from this study provide direct evidence for the replicative division of encapsulated stem cells, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple transient amplifying cells following the capsule rupture. In conclusion, functional cardiac stem cells can reside not only exterior to but also within cardiomyocytes.     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.