Evidence for fusion between cardiac and skeletal muscle cells

Cardiomyoplasty with skeletal myoblasts may benefit cardiac function after infarction. Recent reports indicate that adult stem cells can fuse with other cell types. Because myoblasts are "fusigenic" cells by nature, we hypothesized they might be particularly likely to fuse with cardiomyocytes. To test this, neonatal rat cardiomyocytes labeled with LacZ and green fluorescent protein (GFP) were cocultured with unlabeled C2C12 myoblasts. After 3 days, we observed a small population of skeletal myotubes that expressed LacZ and GFP, indicating cell fusion. To test whether such fusion occurred in vivo, LacZ-expressing C2C12 myoblasts were grafted into normal nude mouse hearts. At 2 weeks after grafting, cells at the graft-host interface expressed both LacZ and cardiac-specific myosin light chain 2v (MLC2v). To test more definitively whether fusion between skeletal and cardiac muscle could occur, we used a Cre/lox reporter system that activated LacZ only upon cell fusion. When neonatal cardiomyocytes from -myosin heavy chain promoter (-MHC)-Cre mice were cocultured with myoblasts from floxed-lacZ reporter mice, LacZ was activated in a subset of cells, indicating cell fusion occurred in vitro. Finally, we grafted the floxed-lacZ myoblasts into normal hearts of -MHC-Cre+ and -MHC-Cre- mice (n=5 each). Hearts analyzed at 4 days and 1 week after transplantation demonstrated activation of LacZ when the skeletal muscle cells were implanted into hearts of -MHC-Cre+ mice, but not after implantation into -MHC-Cre- mice. These data indicate that skeletal muscle cell grafting gives rise to a subpopulation of skeletal-cardiac hybrid cells with a currently unknown phenotype. The full text of this article is available online at http://circres.ahajournals.org.     

Differentiation of pluripotent embryonic stem cells into cardiomyocytes

Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies, ES cells can differentiate into derivatives of all 3 primary germ layers, including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart, such as atrial-like, ventricular-like, sinus nodal-like, and Purkinje-like cells, have been established. During differentiation, cardiac-specific genes as well as proteins, receptors, and ion channels are expressed in a developmental continuum, which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro "gain-of-function" or "loss-of-function" genetic studies. Recently, human ES cell lines have been established that can be used to investigate cardiac development and the function of human heart cells and to determine the basic strategies of regenerative cell therapy. This review summarizes the current state of ES cell-derived cardiogenesis and provides an overview of how genomic strategies coupled with this in vitro differentiation system can be applied to cardiac research.     

Skeletal muscle engraftment potential of adult mouse skin side population cells

Adult bone marrow and skeletal muscle have been shown to contain a subpopulation of cells, called side population (SP) cells, that can be isolated with the fluorescence-activated cell sorter. We used a similar method to identify SP cells in the skin of adult mice. These cells express surface markers similar to SP cells isolated from skeletal muscle, but differ from bone marrow SP cells and do not express hematopoietic markers. When transplanted into nonirradiated mdx mice, nuclei from donor skin SP cells are found within myofibers that express dystrophin. Thus, adult skin SP cells can engraft in dystrophic skeletal muscle even in the absence of total body irradiation.     

bFGF基因修饰骨骼肌卫星细胞移植对心肌梗死兔心功能的影响

目的:观察bFGF基因修饰骨骼肌卫星细胞在心肌梗死区存活情况以及对心功能的影响。方法:分离培养兔骨骼肌卫星细胞,构建bFGF基因修饰骨骼肌卫星细胞。建立急性心肌梗死兔模型,随机分为骨骼肌卫星细胞组、bFGF基因修饰骨骼肌卫星细胞组和对照组,每组6只,在各组动物梗死心肌内分别注射骨骼肌卫星细胞、bFGF基因修饰骨骼肌卫星细胞及等量的细胞培养液。造模前及细胞移植4周后,心脏超声测定兔左室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)、短轴缩短率(FS)和射血分数(EF),观察移植4周后心脏病理切片中心肌梗死边缘区骨骼肌卫星细胞存活情况、bFGF表达情况及新生血管形成情况。结果:细胞移植4周后病理学检查提示骨骼肌卫星细胞在心肌梗死边缘区存活,bFGF基因修饰骨骼肌卫星细胞组可见大量EGFPbFGF融合蛋白表达。与对照组相比,骨骼肌卫星细胞组和bFGF基因修饰骨骼肌卫星细胞组心肌梗死边缘区微血管密度均有增加(78.3±5.2和98.5±8.6对25.2±4.6,P均0.05),且bFGF基因修饰骨骼肌卫星细胞组微血管密度较骨骼肌卫星细胞组明显增加(P0.05)。与造模前相比,移植4周后对照组和骨骼肌卫星细胞组均出现LVESD和LVEDD增大,FS和EF降低(P均0.05),bFGF基因修饰骨骼肌卫星细胞组各指标与造模前相比差异无统计学意义。移植4周后,骨骼肌卫星细胞组和bFGF基因修饰骨骼肌卫星细胞组LVESD及LVEDD均小于对照组,FS和EF均高于对照组(P均0.05);与骨骼肌卫星细胞组相比,bFGF基因修饰骨骼肌卫星细胞组LVESD及LVEDD减小,FS和EF升高(P均0.05)。结论:bFGF基因修饰骨骼肌卫星细胞自体移植可增加急性心肌梗死兔心肌梗死边缘区新生血管形成,改善心功能。     

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.     

Transdifferentiation of chicken embryonic cells into muscle cells by the 3'' untranslated region of muscle tropomyosin.

Transfection with a plasmid encoding the 3' untranslated region (3' UTR) of skeletal muscle tropomyosin induces chicken embryonic fibroblasts to express skeletal tropomyosin. Such cells become spindle shaped, fuse, and express titin, a marker of striated muscle differentiation. Skeletal muscle tropomyosin and titin organize in sarcomeric arrays. When the tropomyosin 3' UTR is expressed in osteoblasts, less skeletal muscle tropomyosin is expressed, and titin expression is delayed. Some transfected osteoblasts become spindle shaped but do not fuse nor organize these proteins into sarcomeres. Transfected cells expressing muscle tropomyosin organize muscle and nonmuscle isoforms into the same structures. Thus, the skeletal muscle tropomyosin 3' UTR induces transdifferentiation into a striated muscle phenotype in a cell-type-specific context.     

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p < 0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.     

Do stem cells in the heart truly differentiate into cardiomyocytes?

Chronic congestive heart failure (CHF) is a common consequence of heart muscle or valve damage and remains a major cause of morbidity and mortality worldwide. There are increasing interests to treat cardiac failure by stem cell-based therapy. Many types of stem cells or progenitor cells have been suggested for cellular therapy of heart failure. While stem cell-based therapy was initially thought to be achieved by transdifferentiation of stem cells into myocardial cells including cardiomyocytes it has become clear that this may be rather an infrequent event. Instead cardiac regeneration may result from vascular differentiation of stem cells or even from stem cell-mediated reverse remodelling. Thus the term stem cell-mediated cardiac regeneration covers the spectrum from stem cell transdifferentiation into cardiomyocytes to cell-mediated pharmacotherapy. In this review we revise stem cell-based cardiac regeneration both in experimental models and in clinical application. We have limited our discussion on some selected types of stem cells, with particular emphasis on their differentiation potential, current status and perspectives on their future applications.     

Development of heart muscle-cell diversity: a help or a hindrance for phenotyping embryonic stem cell-derived cardiomyocytes

Despite the advances in cardiovascular treatment, cardiac disease remains a major cause of morbidity in all industrialized countries. The extraordinary potential of (embryonic) stem cells for therapeutic purposes has revolutionized ideas about cardiac repair of diseased cardiac muscle to exciting stages. This, in turn, has challenged research on cardiac differentiation of stem cells. For instance, cultures of mouse embryonic stem cells quite easily differentiate into the cardiogenic lineage, as assessed by their potential to beat spontaneously. However, repair of impaired cardiac muscle by spontaneously beating cardiac muscle cells might impose severe risks upon a human patient. Therefore, it is of crucial importance to understand the mechanisms that underlie the development of the distinct cardiac muscle cell types of the adult mammalian heart. In this review we tried to relate cardiac morphogenesis to the development of unique molecular phenotypes of cardiomyocytes. This relationship will provide a framework to assess the significance of the molecular phenotypes that are observed in embryonic stem cell-derived cardiomyocytes (ESDCs). Although for the phenotyping of ESDCs a comparison should be made with the phenotypes of the developing heart, so far none of the currently available markers allow unequivocal assignment of subtypes.     

Reprogramming autologous skeletal myoblasts to express cardiomyogenic function. Challenges and possible approaches

Cell transplantation therapy is emerging as a promising mode of treatment following myocardial infarction. Of the various cell types that can potentially be used for transplantation, autologous skeletal myoblasts appear particularly attractive, because this would avoid issues of immunogenicity, tumorigenesis, ethics and donor availability. Additionally, skeletal myoblasts display much higher levels of ischemic tolerance and graft survival compared to other cell types. There is some evidence for improvement in heart function with skeletal myoblast transplantation. However, histological analysis revealed that transplanted myoblasts do not transdifferentiate into functional cardiomyocytes in situ. This is evident by the lack of expression of cardiac-specific antigens, and the absence of intercalated disc formation. Instead, there is differentiation into myotubes that are not electromechanically coupled to neighboring cardiomyocytes. This could in turn limit the clinical efficacy of treatment. This review would therefore examine the various challenges faced in attempting to reprogram autologous skeletal myoblast to express cardiomyogenic function, together with the various possible strategies that could be employed to achieve this objective.     

胚胎干细胞移植到大鼠梗死心脏后的分化研究

目的研究胚胎下细胞在梗死心脏微环境下向心肌细胞、成纤维细胞的分化情况。方法将大鼠分为两组,梗死组为正常大鼠通过结扎左前降支(LAD)制备,对照组为正常大鼠。将4,6-二氨基(DAPI)标记的具有伞能分化能力的鼠胚胎干细胞(mESCs)注射人急性心肌梗死大鼠(18只)或对照绀大鼠(16只)的心脏,观察胚胎干细胞在休内的分化情况。结果 DAPI标记的移植mESCs在对照和梗死心脏均能成活并形成稳定的移植岛,同时在移植区有巨噬细胞浸润。mESCs移植2～4周后,心脏特异性肌钙蛋白T(cTnT)阳性的移植mESCs比例在正常心脏较梗死心脏高(2.67%±0.79%比1.06%±0.52%,P0.01),但4周后cTnT阳性的DAP1标记细胞在正常和梗死心脏的比例差异无统计学意义(1.17%±0.98%比1.07±1,02%,P0.05)。mESCs在埘照组和梗死组心脏都能分化为成纤维细胞。结论移植mESCs不仪能仔活,还可分化进入大鼠梗死心肌细胞。但是,梗死心脏的微环境不能选择性促进mESCs分化进入心肌细胞。     

Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts

Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte-specific marker staining was utilized to analyze hESC-derived cardiomyocyte fate in a rat ischemia/reperfused myocardium. Differentiated, constitutively green fluorescent protein (GFP)-expressing hESCs (hES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4 degrees C to Los Angeles (shipping time approximately 3 days). The cells were dissociated and a cell suspension (2 x 10(6) cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 min after left coronary artery occlusion in athymic nude rats. After 15 min of ischemia, the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2 x 10(6) cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures. Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO(2) at 37 degrees C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore, cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and demonstrated cross striations. At 4 weeks engrafted hESCs expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. No evidence of rejection was observed in nude rats as determined by inspection for lymphocytic infiltrate and/or giant cells. In contrast, hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate. hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers and exhibit sarcomeric structure and they were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.     

Elektrophysiologische Eigenschaften von Stammzellen

New concepts for treatment of myocardial infarction include the implantation of adult stem cells for regeneration of damaged muscle tissue. Several clinical trials have demonstrated a small, but significant improvement of ventricular function. Transdifferentiation of stem cells into cardiomyocytes, formation of new vessels and paracrine factors have been discussed as putative mechanisms for the therapeutic effect. Several types of stem cells have been used clinically including myoblasts derived from skeletal muscle satellite cells, bone marrow-derived stem cells or blood-derived mononuclear progenitor cells. In addition, multiple organs were shown to contain a small number of stem cells that could differentiate into cardiomyocytes.Embryonic stem cells differentiate into spontaneously beating cells that have varying electrophysiological properties. Their action potentials resemble those of cardiac pacemaker cell, atrial or ventricular myocytes (Figure 1) suggesting true differentiation into cardiomyocytes. Beating cells derived from a newly described population of skeletal muscle-derived cells ("skeletal precursors of cardiomyocytes" [SPOCs]) also exhibit spontaneous action potentials, however, unlike cardiac pacemaker cells, their electrical activity is suppressed with the sodium channel blocker tetrodotoxin (Figure 2). Undifferentiated bone marrow-derived mesenchymal stem cells are not electrically excitable. Nevertheless, they express functional ion channels like L-type Ca(2+) channels, albeit not in every cell. Co-culturing stem cells with neonatal rat ventricular myocytes induces good electrical contacts between cells via gap junction formation. Excitatory wave fronts spread evenly in the co-culture. By contrast, gap junctions fail to form when myoblasts are co-cultured with neonatal cardiomyocytes and reentry arrhythmias develop. This pathomechanism could serve as an explanation for the enhanced clinical risk of arrhythmia after transplantation of myoblasts into the infarcted hearts.     

Human embryonic stem cell-derived cardiomyocytes and cardiac repair in rodents

Cell transplantation may restore heart function in disease associated with loss or dysfunction of cardiomyocytes. Recently, Laflamme et al reported an improvement in cardiac function in immunodeficient rats 4 weeks after coronary artery ligation and injection of human embryonic stem cell-derived cardiomyocytes (hESC-CMs). We have recently carried out a comparable study transplanting hESC-CMs to the hearts of mice with myocardial infarction. Our findings were similar up to the 4-week time point, with significant improvements in cardiac function. However, our follow-up was longer, and, at 3 months, the difference between mice receiving cardiomyocytes and those receiving other cells was no longer significant. Hypothesizing that the improvement observed by Laflamme et al may have been more likely to be sustained long term because the grafts in their study appeared larger, we injected 3 times as many cells. Although this resulted in a significantly increased graft size, we again observed a functional improvement at 1 month but not at 3 months. Our results show that midterm data in these kinds of experiments must be interpreted with caution and longer-term follow-up is essential to draw conclusions on the efficacy of cardiac cell transplantation. Furthermore, our findings demonstrate the unlikely success of merely generating and injecting more cells of the same type to increase functional improvement.     

Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation

OBJECTIVES: We report histological analysis of hearts from patients with end-stage heart disease who were transplanted with autologous skeletal myoblasts concurrent with left ventricular assist device (LVAD) implantation. BACKGROUND: Autologous skeletal myoblast transplantation is under investigation as a means to repair infarcted myocardium. To date, there is only indirect evidence to suggest survival of skeletal muscle in humans. METHODS: Five patients (all male; median age 60 years) with ischemic cardiomyopathy, refractory heart failure, and listed for heart transplantation underwent muscle biopsy from the quadriceps muscle. The muscle specimen was shipped to a cell isolation facility where myoblasts were isolated and grown. Patients received a transplant of 300 million cells concomitant with LVAD implantation. Four patients underwent LVAD explant after 68, 91, 141, and 191 days of LVAD support (three transplant, one LVAD death), respectively. One patient remains alive on LVAD support awaiting heart transplantation. RESULTS: Skeletal muscle cell survival and differentiation into mature myofibers were directly demonstrated in scarred myocardium from three of the four explanted hearts using an antibody against skeletal muscle-specific myosin heavy chain. An increase in small vessel formation was observed in one of three patients at the site of surviving myotubes, but not in adjacent tissue devoid of engrafted cells. CONCLUSIONS: These findings represent demonstration of autologous myoblast cell survival in human heart. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results establish the feasibility of myoblast transplants for myocardial repair in humans.