Bone marrow-derived side population cells are capable of functional cardiomyogenic differentiation

It has been reported that bone marrow (BM)-side population (SP) cells, with hematopoietic stem cell activity, can transdifferentiate into cardiomyocytes and contribute to myocardial repair. However, this has been questioned by recent studies showing that hematopoietic stem cells (HSCs) adopt a hematopoietic cell lineage in the ischemic myocardium. The present study was designed to investigate whether BM-SP cells can in fact transdifferentiate into functional cardiomyocytes. Phenotypically, BM-SP cells were 19.59% (+/-)9.00 CD14+, 8.22% (+/-)2.72 CD34+, 92.93% (+/-)2.68 CD44+, 91.86% (+/-)4.07 CD45+, 28.48% (+/-)2.24 c-kit+, 71.09% (+/-)3.67 Sca-1+. Expression of endothelial cell markers (CD31, Flk-1, Tie-2 and VEGF-A) was higher in BM-SP cells than whole BM cells. After five days of co-culture with neonatal cardiomyocytes, 7.2% (+/-)1.2 of the BM-SP cells expressed sarcomeric alpha-actinin as measured by flow cytometry. Moreover, BM-SP cells co-cultured on neonatal cardiomyocytes fixed to inhibit cell fusion also expressed sarcomeric alpha-actinin. The co-cultured BM-SP cells showed neonatal cardiomyocyte-like action potentials of relatively long duration and shallow resting membrane potential. They also generated calcium transients with amplitude and duration similar to those of neonatal cardiomyocytes. These results show that BM-SP cells are capable of functional cardiomyogenic differentiation when co-cultured with neonatal cardiomyocytes.     

Labeling of adult stem cells for in vivo-application in the human heart

Tissue regeneration with human hematopoietic or mesenchymal stem cells has become a fashionable research topic. In cardiology, intracoronary injection of adult stem cells has already been used for the treatment of human myocardial infarction and ischemic cardiomyopathy. The experimental background of such therapies, however, i.e. the potential of adult stem cells to regenerate myocardium through "transdifferentiation" of hematopoietic or mesenchymal stem cells into cardiomyocytes described in animal models, has recently been challenged by other experimental data. Nonetheless, clinical trials are continuing. This may be due to the fact that, in open-labeled pilot trials, a benefit of intracoronary injection of adult stem cells for the treatment of myocardial infarction has been described. As pilot trials may overemphasize the beneficial effects of intracoronary injection of bone marrow stem cells, controlled double-blinded randomised multicenter studies are warranted. Furthermore, a careful characterization of the cells involved in the proposed cardiac repair as well as in vivo-monitoring of such cells following intracoronary injection in humans might help to answer many essential questions linked to this important research topic. The latter requires biocompatible labeling. This review focuses on the technologies available for stem cell labeling and summarizes the arguments and contra-arguments to use these labeling technologies for application in humans.     

G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.     

Cell transplantation in heart failure management

Heart failure is becoming a major issue for public health in western countries and the effect of currently available therapies is limited. Therefore cell transplantation was developed as an alternative strategy to improve cardiac structure and function. This review describes the multiple cell types and clinical trials considered for use in this indication. Most studies have been developed in models of post-ischemic heart failure. The transplantation of fetal or neonatal cardiomyocytes has proven to be functionally successful, but ethical as well as immunological and technical reasons make their clinical use limited. Recent reports, however, suggested that adult autologous cardiomyocytes could be prepared from stem cells present in various tissues (bone marrow, vessels, adult heart itself, adipose tissue). Alternatively, endothelial progenitors originating from bone marrow or peripheral blood could promote the neoangiogenesis within the scar tissue. Hematopietic stem cells prepared from bone marrow or peripheral blood have been proposed but their differentiation ability seems limited. Finally, the transplantation of skeletal muscle cells (myoblasts) in the infarcted area improved myocardial function, in correlation with the development of skeletal muscle tissue in various animal models. The latter results paved the way for the development of a first phase I clinical trial of myoblast transplantation in patients with severe post-ischemic heart failure. It required the scale-up of human cell production according to good manufacturing procedures, started in june 2000 in Paris and was terminated in november 2001, and was followed by several others. The results were encouraging and prompted the onset of a blinded, multicentric phase II clinical trial for skeletal muscle cells transplantation. Meanwhile, phase I clinical trials also evaluate the safeness and efficacy of various cell types originating from the bone marrow or the peripheral blood. However, potential side effects related to the biological properties of the cells or the delivery procedures are being reported. High quality clinical trials supported by strong pre-clinical data will help to evaluate the role of cell therapy as a potential treatment for heart failure.     

骨髓动员对骨髓造血干细胞在心肌梗死后心脏中定植及分化的影响

目的观察小鼠心肌梗死后骨髓造血干细胞在心脏内的分化及细胞因子的影响。方法C57/BL6小鼠60只分为骨髓动员组和对照组,先后行脾切除、骨髓移植（骨髓供体为增强绿色荧光蛋白转基因小鼠）、骨髓动员及建立心肌梗死模型。心肌梗死后3周将小鼠心脏取出并切片行组织学及激光共聚焦显微镜免疫荧光检查。结果骨髓动员可以增加EGFP阳性细胞在心脏中梗死区和边缘区的定植,但绝大多数EGFP阳性细胞都同时表达CD45。仅发现有极少数骨髓来源的心肌细胞、成纤维细胞及血管内皮细胞,且与骨髓动员无相关性。结论骨髓动员能够明显促进骨髓来源细胞定植入小鼠心脏的梗死区;极少数骨髓造血干细胞可以分化为心肌细胞,其数量远不足以修复梗死心肌及改善心功能;骨髓造血干细胞不参与梗死区疤痕形成的病理过程。     

Temporal and spatial characteristics of apoptosis in the infarcted rat heart

Following myocardial infarction (MI), tissue repair/remodeling occurs in both the infarcted and noninfarcted myocardium. Apoptosis has been demonstrated to play an important role in these processes. In the present study, we sought to determine the temporal and spatial characteristics of apoptosis in the infarcted heart as well as to identify cells undergoing programmed cell death at different stages of repair/remodeling and their relationship to the expression of anti-/pro-apoptotic genes following MI. Our study has shown that apoptosis appears in both infarcted and noninfarcted myocardium, and cells undergoing apoptosis depend on the stage of healing. In the infarcted myocardium, apoptosis contributes to the loss of cardiomyocytes during the early stage of healing, elimination of inflammatory cells during the inflammatory phase of healing, and reduction of myofibroblasts with the fibrogenic phase of repair in the infarcted myocardium. In noninfarcted myocardium, cardiomyocyte apoptosis was observed from day 3 to 28 postMI. Cardiac apoptosis following MI is correlated with the increase of Bax expression.     

Cell fusion contributes to the rescue of apoptotic cardiomyocytes by bone marrow cells

Cardiomyocyte apoptosis is an important contributor to the progressive cardiac dysfunction that culminates in congestive heart failure. Bone marrow cells (BMCs) restore cardiac function following ischaemia, and transplanted BMCs have been reported to fuse with cells of diverse tissues. We previously demonstrated that the myogenic conversion of bone marrow stromal cells increased nearly twofold when the cells were co‐cultured with apoptotic (TNF‐α treated) cardiomyocytes. We therefore hypothesized that cell fusion may be a major mechanism by which BMCs rescue cardiomyocytes from apoptosis. We induced cellular apoptosis in neonatal rat cardiomyocytes by treatment with hydrogen peroxide (H₂O₂). The TUNEL assay demonstrated an increase in apoptosis from 4.5 ± 1.3% in non‐treated cells to 19.0 ± 4.4% (P < 0.05) in treated cells. We subsequently co‐cultured the apoptotic cardiomyocytes with BMCs and assessed cell fusion using flow cytometry. Fusion was rare in the non‐treated control cardiomyocytes (0.3%), whereas H₂O₂ treatment led to significantly higher fusion rates than the control group (P < 0.05), with the highest rate of 7.9 ± 0.3% occurring at 25 μM H₂O₂. We found an inverse correlation between cell fusion and completion of cardiomyocyte apoptosis (R² = 0.9863). An in vivo mouse model provided evidence of cell fusion in the infarcted myocardium following the injection of BMCs. The percentage of cells undergoing fusion was significantly higher in mice injected with BMCs following infarction (8.8 ± 1.3%) compared to mice that did not undergo infarction (4.6 ± 0.6%, P < 0.05). Enhancing cell fusion may be one method to preserve cardiomyocytes following myocardial infarction, and this new approach may provide a novel target for cardiac regenerative therapies.     

Application of stem cells in cardiology: where we are and where we are going

Heart disease including myocardial infarction and ischemia is associated with the irreversible loss of cardiomyocytes and vasculature, both via apoptosis or necrosis. However, the native capacity for the renewal and repair of myocardial tissue is inadequate as have been current therapeutic measures to prevent left ventricular remodeling. Cell transplantation has emerged as a potentially viable therapeutic approach to directly repopulate and repair the damaged myocardium. A detailed analysis and a vision for future progress in stem cell applications, both in research and clinical cardiology are presented in this review, highlighting the use of a wide spectrum of stem/progenitor cell types including embryonic or fetal stem cells, myoblasts, and adult bone marrow stem cells. An up-to-date comparison of donor cell-types used, and evaluation of the myocardial disorders that might be most amenable to stem cell therapy are discussed. The roles that myocardial cell fusion and transdifferentiation play in stem cell transplantation, the specific shortcomings of available technologies, and recommendations for practical ways that these concerns might be overcome, are also presented.     

Stable benefit of embryonic stem cell therapy in myocardial infarction

Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited information is available on their ultimate therapeutic value. We treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function, and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated with host myocardium within infarct regions. This resulted in a functional benefit within 3 wk that remained sustained over 12 wk of continuous follow-up and included a vigorous inotropic response to beta-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little scar, and a decrease in signs of myocardial necrosis. In contrast, sham-treated infarcted hearts exhibited ventricular cavity dilation and aneurysm formation, poor ventricular function, and a lack of response to beta-adrenergic stimulation. No evidence of graft rejection, ectopy, sudden cardiac death, or tumor formation was observed after therapy. These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in the infarcted heart.     

Repair of Ischemic Heart Disease with Novel Bone Marrow-Derived Multipotent Stem Cells

Congestive heart failure is a growing, worldwide epidemic. The major causes of heart failure are related to irreversible damage resulting from myocardial infarction (heart attack). The long-standing axiom has been that the myocardium has a limited capacity for self-repair or regeneration; and the irreversible loss of cardiac muscle and accompanying contraction and fibrosis of myocardial scar tissue, sets into play a series of events, namely, progressive ventricular remodeling of nonischemic myocardium that ultimately leads to progressive heart failure. The loss of cardiomyocyte survival cues is associated with diverse pathways for heart failure, underscoring the importance of maintaining the number of viable cardiomyocytes during heart failure progression. Currently, no medication or procedure used clinically has shown efficacy in replacing the myocardial scar with functioning contractile tissue. Therefore, given the major morbidity and mortality associated with myocardial infarction and heart failure, new approaches have been sought to address the principal pathophysiologic deficits responsible for these conditions, resulting from the loss of cardiomyocytes and viable blood vessels. Recently, the identification of stem cells from bone marrow capable of contributing to tissue regeneration has ignited significant interest in the possibility that cell therapy could be employed therapeutically for the repair of damaged myocardium. In this review, we will discuss the currently available bone marrow-derived stem progenitor cells for myocardial repair and focus on the advantages of using recently identified novel bone marrow-derived multipotent stem cells (BMSC)     

Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.     

The bifunctional SDF‐1‐AnxA5 fusion protein protects cardiac function after myocardial infarction

Stromal cell‐derived factor‐1 (SDF‐1) is a well‐characterized cytokine that protects heart from ischaemic injury. However, the beneficial effects of native SDF‐1, in terms of promoting myocardial repair, are limited by its low concentration in the ischaemic myocardium. Annexin V (AnxA5) can precisely detect dead cells in vivo. As massive cardiomyocytes die after MI, we hypothesize that AnxA5 can be used as an anchor to carry SDF‐1 to the ischaemic myocardium. In this study, we constructed a fusion protein consisting of SDF‐1 and AnxA5 domains. The receptor competition assay revealed that SDF‐1‐AnxA5 had high binding affinity to SDF‐1 receptor CXCR4. The treatment of SDF‐1‐AnxA5 could significantly promote phosphorylation of AKT and ERK and induce chemotactic response, angiogenesis and cell survival in vitro. The binding membrane assay and immunofluorescence revealed that AnxA5 domain had the ability to specifically recognize and bind to cells injured by hypoxia. Furthermore, SDF‐1‐AnxA5 administered via peripheral vein could accumulate at the infarcted myocardium in vivo. The treatment with SDF‐1‐AnxA5 attenuated cell apoptosis, enhanced angiogenesis, reduced infarcted size and improved cardiac function after mouse myocardial infarction. Our results suggest that the bifunctional SDF‐1‐AnxA5 can specifically bind to dead cells. The systemic administration of bifunctional SDF‐1‐AnxA5 effectively provides cardioprotection after myocardial infarction.     

Cell therapy for myocardial infarction

The review discusses cell therapy; one of the most promising approaches to myocardial infarction treatment. The possibility to use cell material of various origins is analyzed. The review sums up data on the application of fetal and neonatal cardiomyocytes, myoblasts, bone marrow mononuclear fraction, hematopoietic and mesenchymal stem cells (MSX) as cell therapy agents. The conclusion is made that MSC are promising cell material for myocardial infarction therapy. MSC are able to migrate to the injured area, differentiate into myocardial lineage. They produce a wide range of factors that stimulate angiogenesis and increase viability of cells, including cardiomyocytes.     

Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion

Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion hybrids, but almost exclusively in response to injuries or inflammation.     

Myocardial neovascularization by bone marrow angioblasts results in cardiomyocyte regeneration

The primary cardiac response to ischemic insult is cardiomyocyte hypertrophy, which initiates a genetic program culminating in apoptotic myocyte loss, progressive collagen replacement, and heart failure, a process termed cardiac remodeling. Although a few cardiomyocytes at the peri-infarct region can proliferate and regenerate after injury, no approaches are known to effectively induce endogenous cardiomyocytes to enter the cell cycle. We recently isolated, in human adult bone marrow, endothelial progenitor cells, or angioblasts, that migrate to ischemic myocardium, where they induce neovascularization and prevent myocardial remodeling. Here we show that increasing the number of angioblasts trafficking to the infarct zone results in dose-dependent neovascularization with development of progressively larger-sized capillaries. This results in sustained improvement in cardiac function by mechanisms involving protection against apoptosis and, strikingly, induction of proliferation/regeneration of endogenous cardiomyocytes. Our results suggest that agents that increase myocardial homing of bone marrow angioblasts could effectively induce endogenous cardiomyocytes to enter the cell cycle and improve functional cardiac recovery.     

胚胎干细胞的心脏应用

心肌梗死期间死亡的心肌细胞将由没有收缩功能的疤痕组织替代,因而极可能引起心力衰竭。对治疗心衰来说,修复死亡或损伤的心肌以及改善心功能仍面临着极大挑战。干细胞移植已在近年来的实验中用于修复损失的心肌。本文总结了近期在心肌损伤动物中实施胚胎干细胞移植的实验结果,并着重介绍对这类特定细胞的研究进展。胚胎干细胞取源于早期哺乳类胚胎的胚芽细胞,属于多功能干细胞。这类细胞具有长期增殖而不分化的能力,或台色够在培养过程中分化成包括心肌细胞在内的所有特殊体细胞。由于胚胎干细胞具有极大的增殖和分化为成熟组织的能力,它们可能成为一种潜在的很有实用价值的细胞来源,可用于对病态心脏的功能心肌再生的细胞治疗。新近的研究表明,在心肌梗死动物模型中,心肌内移植胚胎干细胞或由其分化成的心肌样细胞,能导致已损伤心肌的再生,并改善心脏功能。另外,在病毒性心肌炎小鼠中,静脉输入胚胎干细胞可明显提高生存率和减轻心肌损伤。有关人类胚胎干细胞在体外分化成心肌细胞以及这些细胞的特性,近来已有报道。然而,要在临床能应用人类胚胎干细胞或由其分化成的心肌细胞来治疗晚期心脏疾病,还必须越过大量的伦理、法律和科学上的障碍。     

Structural remodelling of cardiomyocytes in the border zone of infarcted rabbit heart

Cardiomyocyte dedifferentiation, as detected in hibernating myocardium of chronic ischemic patients, is one of the characteristics seen at the border of myocardial infarcts in small and large animals. Our objectives were to study in detail the morphological changes occurring at the border zone of a rabbit myocardial infarction and its use as model for hibernating myocardium. Ligation of the left coronary artery (LAD) was performed on rabbit hearts and animals were sacrificed at 2, 4, 8 and 12 weeks post-infarction. These hearts together with a non-infarcted control heart were perfusion-fixed and tissue samples were embedded in epoxy resin. Hibernating cardiomyocytes were mainly distributed in the non-infarcted region adjacent to the border zone of infarcted myocardium but only in a limited number. In the border zone itself vacuolated cardiomyocytes surrounded by fibrotic tissue were frequently observed. Ultrastructural analysis of these vacuolated cells revealed the presence of a basal lamina inside the vacuoles adjacent to the surrounding membrane, the presence of pinocytotic vesicles and an association with cisternae of the sarcoplasmatic reticulum. Myocyte quantitative analyses revealed a gradual increase in vacuolar area/total cell area ratio and in collagen fibril deposition inside the vacuoles from 2 to 12 weeks post-infarction. Related to the remote zone, the increase in cell width of myocytes located in and adjacent to the border zone demonstrated cellular hypertrophy. These results indicate the occurrence of cardiomyocyte remodelling mechanisms in the border zone and adjacent regions of infarcted myocardium. It is suggested that the vacuoles represent plasma membrane invaginations and/or dilatations of T-tubular structures.     

Occurrence of a terminal vascularisation after experimental myocardial infarction

Physiological data indicate a residual vascularisation within ischemic myocardial regions where necrosis of most cells have been reported to occur after myocardial infarction. We therefore studied, by means of immunohistochemistry, computer-assisted morphometry, and electron microscopy, the terminal vascularisation in correlation to cardiomyocytes in ten canine hearts 1 and 3 weeks after occlusion of the left anterior descending (LAD) coronary artery. In comparison to non-infarcted myocardium we found the following alterations in infarcted myocardium: (1) the area density of cardiomyocytes decreased from 98% (control) to 7.9% (1 week after occlusion) and to 2.7% (3 weeks after occlusion); (2) the number of capillaries was diminished to 11.6% and to 2.6%; respectively; (3) smooth muscle α-actin was induced in endothelial (EC) cells of the microvessels; and (4) terminal resistance vessels increased 11-fold and 20-fold in number, respectively. Our findings confirm the necrosis of the vast majority of cardiomyocytes and capillaries within the first 3 weeks after myocardial infarction. Besides a small number of capillaries, many terminal resistance vessels, however, seem to persist in the scarring infarcted tissue. The occurrence of these microvessels is supposed to be important for the granulation tissue as well as for the control and regulation of a residual blood supply during scar formation.     

Myogenic reprogramming of bone marrow derived cells in a W⁴¹Dmd(mdx) deficient mouse model

Lack of expression of dystrophin leads to degeneration of muscle fibers and infiltration of connective and adipose tissue. Cell transplantation therapy has been proposed as a treatment for intractable muscle degenerative disorders. Several reports have demonstrated the ability of bone-marrow derived cells (BMDC) to contribute to non-haematopoietic tissues including epithelium, heart, liver, skeletal muscle and brain following transplantation by means of fusion and reprogramming. A key issue is the extent to which fusion and reprogramming can occur in vivo, particularly under conditions of myogenic deterioration.To investigate the therapeutic potential of bone marrow transplantation in monogenetic myopathy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into non-irradiated c-kit receptor-deficient (W?1) mdx mice. This model allows BMDC reconstitution in the absence of irradiation induced myeloablation. We provide the first report of BMDC fusion in a W?1Dmd(mdx) deficient mouse model.In the absence of irradiation induced injury, few GFP+ cardiomyocytes and muscle fibres were detected 24 weeks post BMT. It was expected that the frequency of fusion in the hearts of W?1Dmd(mdx) mice would be similar to frequencies observed in infarcted mice. Although, it is clear from this study that individual cardiomyocytes with monogenetic deficiencies can be rescued by fusion, it is as clear that in the absence of irradiation, the formation of stable and reprogrammed fusion hybrids occurs, with the current techniques, at very low levels in non-irradiated recipients.     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.