Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy

Background. In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart. Methods. hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine. Results. hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes. Conclusion. The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16: 163-9.)     

Role of β-Catenin in Adult Cardiac Remodeling

The adult heart has a uniform cellular response to adapt to injury after infarct or increased wall stress in chronic hypertension: hypertrophy of adult cardiomyocytes increases muscle fiber mass while at the same time apoptosis of cardiomyocytes may lead to further loss of contractile mass. The existence and quantitative amount of endogenous cardiac regeneration is currently under intense dispute, no clear picture has yet emerged. Recently, cardiac precursor cells and the signaling pathways controlling their differentiation in the adult organ have come into focus. In heart development, β-catenin was identified to play a biphasic role in cardiomyocyte differentiation. While initially WNT/β-catenin activation is required to commit mesenchymal cells to the cardiac lineage, downregulation of β-catenin is needed for cardiomyocyte differentiation at later stages. Recent genetic data published by our lab suggest β-catenin downregulation to be beneficial for adult cardiac remodeling. Here we discuss these data in the context of β-catenin´s role in adult cardiomyocyte hypertrophy, apoptosis and possibly regeneration.     

Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis of cardiac muscle cells

Apoptosis of cardiac muscle cells contributes to the development of cardiomyopathy. Recent studies showed that insulin-like growth factor I (IGF-I) inhibits apoptosis of cardiac muscle cells and improves myocardial function in experimental heart failure. This study was carried out to elucidate the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the anti-apoptotic actions of IGF-I in cardiomyocytes and to explore whether expression of constitutively active PI 3-kinase can inhibit apoptosis in cardiomyocytes. Apoptosis of primary cardiomyocytes was induced by doxorubicin treatment and serum withdrawal. Transduction of cardiomyocytes with constitutively active PI 3-kinase specifically lead to serine phosphorylation of Akt, whereas phosphorylation of IGF-I receptor, IRS1/2 and p44/42 mitogen-activated protein kinase were not increased. In the cardiomyocytes transduced with constitutively active PI 3-kinase, activation of the pro-apoptotic caspase 3 was attenuated and fragmentation of DNA was reduced. Preincubating cells with PI 3-kinase inhibitor LY294002 was associated with loss of anti-apoptotic actions of IGF-I and PI 3-kinase. Neither IGF-I nor constitutively active PI 3-kinase lead to serine phosphorylation of Bad, suggesting that the anti-apoptotic effects of PI 3-kinase are not mediated through Bad phosphorylation in cardiac muscle cells. To determine whether activation of caspase 3 is sufficient to induce apoptosis in cardiomyocytes, an engineered TAT-caspase 3 protein was introduced to cardiomyocytes. Significant reduction of cell viability occurred in the cardiomyocytes transduced with active caspase 3, indicating that activation of caspase 3 is sufficient to cause cardiomyocyte death. These findings indicate the existence of an IGF-I receptor-PI 3-kinase-caspase 3 pathway in cardiomyocytes that plays an important role in the anti-apoptotic actions of IGF-I in heart. Moreover, these data suggest that modulation of PI 3-kinase activities may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in cardiomyopathy.     

EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

Eph receptor (Eph)‐ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4–ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5⁺ cardiac progenitor cells and in α‐MHC⁺ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5‐GFP⁺ cardiac progenitor cells, but not in α‐MHC‐GFP⁺ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL‐RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full‐length EphB4 or intracellular domain‐truncated EphB4 in EphB4‐null ES cells was established to investigate the role of EphB4‐forward signaling in ES cells. Interestingly, while full‐length EphB4 was able to restore the cardiac lineage development in EphB4‐null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor‐kinase‐dependent EphB4‐forward signaling plays a crucial role in the development of cardiac progenitor cells. J. Cell. Biochem. 116: 467–475, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Extrinsic regulation of cardiomyocyte differentiation of embryonic stem cells

Cardiovascular disease is one of leading causes of death throughout the U.S. and the world. The damage of cardiomyocytes resulting from ischemic injury is irreversible and leads to the development of progressive heart failure, which is characterized by the loss of functional cardiomyocytes. Because cardiomyocytes are unable to regenerate in the adult heart, cell-based therapy of transplantation provides a potential alternative approach to replace damaged myocardial tissue and restore cardiac function. A major roadblock toward this goal is the lack of donor cells; therefore, it is urgent to identify the cardiovascular cells that are necessary for achieving cardiac muscle regeneration. Pluripotent embryonic stem (ES) cells have enormous potential as a source of therapeutic tissues, including cardiovascular cells; however, the regulatory elements mediating ES cell differentiation to cardiomyocytes are largely unknown. In this review, we will focus on extrinsic factors that play a role in regulating different stages of cardiomyocyte differentiation of ES cells.     

LRP5 regulates cardiomyocyte proliferation and neonatal heart regeneration by the AKT/P21 pathway

The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low‐density lipoprotein receptor‐related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac‐specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin‐dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.     

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.     

Effects of cannabinoid receptor type 2 on endogenous myocardial regeneration by activating cardiac progenitor cells in mouse infarcted heart

Cannabinoid receptor type 2(CB2)activation is recently reported to promote proliferation of some types of resident stem cells(e.g.,hematopoietic stem/progenitor cell or neural progenitor cell).Resident cardiac progenitor cell(CPC)activation and proliferation are crucial for endogenous cardiac regeneration and cardiac repair after myocardial infarction(MI).This study aims to explore the role and possible mechanisms of CB2receptor activation in enhancing myocardial repair.Our results revealed that CB2receptor agonist AM1241 can significantly increase CPCs by c-kit and Runx1 staining in ischemic myocardium as well as improve cardiomyocyte proliferation.AM1241 also decreased serum levels of MDA,TNF-αand IL-6 after MI.In addition,AM1241 can ameliorate left ventricular ejection fraction and fractional shortening,and reduce fibrosis.Moreover,AM1241 treatment markedly increased p-Akt and HO-1 expression,and promoted Nrf-2 nuclear translocation.However,PI3K inhibitor wortmannin eliminated these cardioprotective roles of AM1241.In conclusion,AM1241 could induce myocardial regeneration and improve cardiac function,which might be associated with PI3K/Akt/Nrf2 signaling pathway activation.Our findings may provide a promising strategy for cardiac endogenous regeneration after MI.     

Apelin/APJ 与胚胎心肌分化

胚胎发育中心脏祖细胞迁移至生心区并分化为心肌细胞是心脏形成的基础。研究心肌分化对了解心脏发育异常以及应用干细胞治疗缺血性心脏病具有重要意义。最近研究发现apelin/APJ信号通路与祖细胞的迁移及心肌分化有关。本文就apelin/APJ与胚胎心肌分化的关系做一综述。     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Regulation of cardiomyocyte proliferation during development and regeneration

The regulation of cardiomyocyte proliferation is important for heart development and regeneration. The proliferation patterns of cardiomyocytes are closely related to heart morphogenesis, size, and functions. The proliferation levels are high during early embryogenesis; however, mammalian cardiomyocytes exit the cell cycle irreversibly soon after birth. The cell cycle exit inhibits cardiac regeneration in mammals. On the other hand, cardiomyocytes of adult zebrafish and probably newts can proliferate after cardiac injury, and the hearts can be regenerated. Therefore, the ability to reproliferate determines regenerative ability. As in other cells, the relationship between proliferation and differentiation is very interesting, and is closely related to cardiac development, regeneration and homeostasis. In this review, these topics are discussed.     

Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair

Adult mammalian hearts respond to injury with scar formation and not with cardiomyocyte proliferation, the cellular basis of regeneration. Although cardiogenic progenitor cells may maintain myocardial turnover, they do not give rise to a robust regenerative response. Here we show that extracellular periostin induced reentry of differentiated mammalian cardiomyocytes into the cell cycle. Periostin stimulated mononucleated cardiomyocytes to go through the full mitotic cell cycle. Periostin activated alphaV, beta1, beta3 and beta5 integrins located in the cardiomyocyte cell membrane. Activation of phosphatidylinositol-3-OH kinase was required for periostin-induced reentry of cardiomyocytes into the cell cycle and was sufficient for cell-cycle reentry in the absence of periostin. After myocardial infarction, periostin-induced cardiomyocyte cell-cycle reentry and mitosis were associated with improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis. Thus, periostin and the pathway that it regulates may provide a target for innovative strategies to treat heart failure.     

The effects of calcium channel blockade on proliferation and differentiation of cardiac progenitor cells

Cardiogenesis depends on a tightly regulated balance between proliferation and differentiation of cardiac progenitor cells (CPCs) and their cardiomyocyte descendants. While exposure of early mouse embryos to Ca²⁺ channel antagonists has been associated with abnormal cardiac morphogenesis, less is known about the consequences of Ca²⁺ channel blockade on proliferation and differentiation of CPCs at the cellular level. Here we showed that at embryonic day (E) 11.5, the murine ventricles express several L-type and T-type Ca²⁺ channel isoforms, and that the dihydropyridine Ca²⁺ channel antagonist, nifedipine, blunts isoproterenol induced increases in intracellular Ca²⁺. Nifedipine mediated Ca²⁺ channel blockade was associated with a reduction in cell cycle activity of E11.5 CPCs and impaired assembly of the cardiomyocyte contractile apparatus. Furthermore, in cell transplantation experiments, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs and cardiomyocytes) was associated with smaller graft sizes compared to vehicle treated control animals. These data suggest that intracellular Ca²⁺ is a critical regulator of the balance between CPC proliferation and differentiation and demonstrate that interactions between pharmacological drugs and transplanted cells could have a significant impact on the effectiveness of cell based therapies for myocardial repair.     

Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca²⁺ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.     

Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells

Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.     

Telocytes in exercise‐induced cardiac growth

Exercise can induce physiological cardiac growth, which is featured by enlarged cardiomyocyte cell size and formation of new cardiomyocytes. Telocytes (TCs) are a recently identified distinct interstitial cell type, existing in many tissues and organs including heart. TCs have been shown to form a tandem with cardiac stem/progenitor cells in cardiac stem cell niches, participating in cardiac regeneration and repair. Although exercise‐induced cardiac growth has been confirmed as an important way to promote cardiac regeneration and repair, the response of cardiac TCs to exercise is still unclear. In this study, 4 weeks of swimming training was used to induce robust healthy cardiac growth. Exercise can induce an increase in cardiomyocyte cell size and formation of new cardiomyocytes as determined by Wheat Germ Lectin and EdU staining respectively. TCs were identified by three immunofluorescence stainings including double labelling for CD34/vimentin, CD34/platelet‐derived growth factor (PDGF) receptor‐α and CD34/PDGF receptor‐β. We found that cardiac TCs were significantly increased in exercised heart, suggesting that TCs might help control the activity of cardiac stem/progenitor cells, cardiomyocytes or endothelial cells. Adding cardiac TCs might help promote cardiac regeneration and renewal.     

The role of stem cells in cardiac regeneration

After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. Cell transplantation has emerged as a potential therapy and stem cells may be an important and powerful cellular source. Embryonic stem cells can differentiate into true cardiomyocytes, making them in principle an unlimited source of transplantable cells for cardiac repair, although immunological and ethical constraints exist. Somatic stem cells are an attractive option to explore for transplantation as they are autologous, but their differentiation potential is more restricted than embryonic stem cells. Currently, the major sources of somatic cells used for basic research and in clinical trials originate from the bone marrow. The differentiation capacity of different populations of bone marrow-derived stem cells into cardiomyocytes has been studied intensively. The results are rather confusing and difficult to compare, since different isolation and identification methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. A newly identified cell population isolated from cardiac tissue, called cardiac progenitor cells, holds great potential for cardiac regeneration. Here we discuss the potential of the different cell populations and their usefulness in stem cell based therapy to repair the damaged heart.     

Cyclosporin-A potently induces highly cardiogenic progenitors from embryonic stem cells

Though cardiac progenitor cells should be a suitable material for cardiac regeneration, efficient ways to induce cardiac progenitors from embryonic stem (ES) cells have not been established. Extending our systematic cardiovascular differentiation method of ES cells, here we show efficient and specific expansion of cardiomyocytes and highly cardiogenic progenitors from ES cells. An immunosuppressant, cyclosporin-A (CSA), showed a novel effect specifically acting on mesoderm cells to drastically increase cardiac progenitors as well as cardiomyocytes by 10-20 times. Approximately 200 cardiomyocytes could be induced from one mouse ES cell using this method. Expanded progenitors successfully integrated into scar tissue of infracted heart as cardiomyocytes after cell transplantation to rat myocardial infarction model. CSA elicited specific induction of cardiac lineage from mesoderm in a novel mesoderm-specific, NFAT independent fashion. This simple but efficient differentiation technology would be extended to induce pluripotent stem (iPS) cells and broadly contribute to cardiac regeneration.