Cardiac Precursor Research Articles

Cardiac cell lineages that form the heart.

Myocardial cells ensure the contractility of the heart, which also depends on other mesodermal cell types for its function. Embryological experiments had identified the sources of cardiac precursor cells. With the advent of genetic engineering, novel tools have been used to reconstruct the lineage tree of cardiac cells that contribute to different parts of the heart, map the development of cardiac regions, and characterize their genetic signature. Such knowledge is of fundamental importance for our understanding of cardiogenesis and also for the diagnosis and treatment of heart malformations.

Scrib:Rac1 interactions are required for the morphogenesis of the ventricular myocardium.

AimsThe organization and maturation of ventricular cardiomyocytes from the embryonic to the adult form is crucial for normal cardiac function. We have shown that a polarity protein, Scrib, may be involved in regulating the early stages of this process. Our goal was to establish whether Scrib plays a cell autonomous role in the ventricular myocardium, and whether this involves well-known polarity pathways.Methods and resultsDeletion of Scrib in cardiac precursors utilizing Scribflox mice together with the Nkx2.5-Cre driver resulted in disruption of the cytoarchitecture of the forming trabeculae and ventricular septal defects. Although the majority of mice lacking Scrib in the myocardium survived to adulthood, they developed marked cardiac fibrosis. Scrib did not physically interact with the planar cell polarity (PCP) protein, Vangl2, in early cardiomyocytes as it does in other tissues, suggesting that the anomalies did not result from disruption of PCP signalling. However, Scrib interacted with Rac1 physically in embryonic cardiomyocytes and genetically to result in ventricular abnormalities, suggesting that this interaction is crucial for the development of the early myocardium.ConclusionsThe Scrib–Rac1 interaction plays a crucial role in the organization of developing cardiomyocytes and formation of the ventricular myocardium. Thus, we have identified a novel signalling pathway in the early, functioning, heart muscle. These data also show that the foetus can recover from relatively severe abnormalities in prenatal ventricular development, although cardiac fibrosis can be a long-term consequence.

Ischaemic Stroke and occult cardiac abnormality-A Transthoracic Echocardiography based study

Background: Most of the cases of stroke are of ischemic origin. Various cardiac diseases have been shown to increase risk of stroke. Cerebral embolism derived from a diversity of cardiac disorders is responsible for H”20% of ischemic stroke. AF is the most powerful and treatable cardiac precursor of stroke. Cardiac abnormalities are important risk factors for stroke. A cardiac source of thromboembolism must be excluded in patients presenting with a definite embolic occlusion of a peripheral artery, or multiple thromboembolic episodes in diverse anatomical regions. These patients should undergo transthoracic echocardiography initially and transoesophageal echo if image quality is unsatisfactory because of obesity, lung disease or chest deformity.
 Methods: An descriptive and observational study done in Neurology unit, Chittagong Medical College hospital from January 2007 to December 2007.Patients presented primarily as ischemic stroke was examined to find out prevalence of occult cardiac abnormality and to find out the frequency of different type cardiac abnormality in ischemic stroke through echocardiography.
 Results: A total number of 265 patients were examined. Different types of cardiac findings, the maximum number are LV hypertrophy (23%) and followed by Diastolic dysfunction (17%), than multiple vulvular disease (8.5%). Small number of other abnormality found but no PFO and mitral valve prolapse identified in this study. In under 30yrs age group of cases all the abnormal findings found are valvular abnormality. They are mitral stenosis (2), multiple valvular disease (2) and mitral regurgitation (1). In 30 – 49 yrs age group different abnormal findings, most of which are also valvular lesions including aortic stenosis(2), multiple valvular disease(2) and mitral regurgitation(1). Intracardiac thrombus found in one case.In e”50- 69 yrs age group of patients, maximum abnormality found is LVH(7) followed by Diastolic dysfunction(4).Maximum abnormality found in e”70 yrs of age group is LVH(3) and next to it is distolic dysfunction(2).
 Interpretation: Echocardiography may provide important information on the cause of ischemic stroke. Taking into account a low rate of findings with direct impact on evidence-based therapeutic strategies, routine use of echocardiography is not warranted in all patients with stroke. In patients younger than 50 years, echocardiography has higher diagnostic yield and should routinely be performed.
 Bangladesh Journal of Neuroscience 2014; Vol. 30 (2): 84-89

Concise Review: The Role of C-kit Expressing Cells in Heart Repair at the Neonatal and Adult Stage

Ischemic heart disease is the number one cause of morbidity and mortality in the developed world due to the inability of the heart to replace lost myocytes. The cause of postinfarction myogenic failure has been a subject of intense scientific investigation and much controversy. Recent data indicate a brief perinatal developmental window exists during which postinfarction myogenesis, and substantial heart regeneration, occurs. By contrast, repair of an equivalent injury of the adult heart results in prominent revascularization without myogenesis. Here, we review recent experiments on neonatal postinjury myogenesis, examine the mechanistic hypotheses of dedifferentiation and precursor expansion, and discuss experiments indicating that postinfarction revascularization derives primarily from cardiac vascular precursors. These data have profound consequences for the understanding of human heart repair, as they address the long standing question as to whether human postinfarction myogenic failure is due to the loss of precursors existent at the neonatal stage or to a context-dependent inhibition of these precursors within the infarct, and suggest strategies for the recapitulation of neonatal myogenic capacity and the augmentation of revascularization.

Finding a niche for cardiac precursors.

Within an embryo, a region next to the developing heart provides a niche where cardiac precursor cells can increase in number before they contribute to the development of this organ.

Collier/OLF/EBF-Dependent Transcriptional Dynamics Control Pharyngeal Muscle Specification from Primed Cardiopharyngeal Progenitors

In vertebrates, pluripotent pharyngeal mesoderm progenitors produce the cardiac precursors of the second heart field as well as the branchiomeric head muscles and associated stem cells. However, the mechanisms underlying the transition from multipotent progenitors to distinct muscle precursors remain obscured by the complexity of vertebrate embryos. Using Ciona intestinalis as a simple chordate model, we show that bipotent cardiopharyngeal progenitors are primed to activate both heart and pharyngeal muscle transcriptional programs, which progressively become restricted to corresponding precursors. The transcription factor COE (Collier/OLF/EBF) orchestrates the transition to pharyngeal muscle fate both by promoting an MRF-associated myogenic program in myoblasts and by maintaining an undifferentiated state in their sister cells through Notch-mediated lateral inhibition. The latter are stem cell-like muscle precursors that form most of the juvenile pharyngeal muscles. We discuss the implications of our findings for the development and evolution of the chordate cardiopharyngeal mesoderm.

Anti-hypertrophic effects of oxytocin in rat ventricular myocytes

BackgroundOxytocin (OT) and functional OT receptor (OTR) are expressed in the heart and are involved in blood pressure regulation and cardioprotection. Cardiac OTR signaling is associated with atrial natriuretic peptide (ANP) and nitric oxide (NO) release. During the synthesis of OT, its precursor, termed OT-Gly-Lys-Arg (OT-GKR), is accumulated in the developing rat heart. Consequently, we hypothesized that an OT-related mechanism of ANP controls cardiomyocyte (CM) hypertrophy. MethodsThe experiments were carried out in newborn and adult rat CM cultures. The enhanced protein synthesis and increased CM volume were mediated by a 24-h treatment with endothelin-1 or angiotensin II. ResultsThe treatment of CM with OT or its abundant cardiac precursor, OT-GKR, revealed ANP accumulation in the cell peri-nuclear region and increased intracellular cGMP. Consequently, the CM hypertrophy was abolished by the treatment of 10nM OT or 10nM OT-GKR. The ANP receptor antagonist (anantin) and NO synthases inhibitor (l-NAME) inhibited cGMP production in CMs exposed to OT. STO-609 and compound C inhibition of anti-hypertrophic OT effects in CMs indicated the contribution of calcium–calmodulin kinase kinase and AMP-activated protein kinase pathways. Moreover, in ET-1 stimulated cells, OT treatment normalized the reduced Akt phosphorylation, prevented abundant accumulation of ANP and blocked ET-1-mediated translocation of nuclear factor of activated T-cells (NFAT) into the cell nuclei. ConclusioncGMP/protein kinase G mediates OT-induced anti-hypertrophic response with the contribution of ANP and NO. OT treatment represents a novel approach in attenuation of cardiac hypertrophy during development and cardiac pathology.

Snail modulates the assembly of fibronectin via α5 integrin for myocardial migration in zebrafish embryos

The Snail family member snail encodes a zinc finger-containing transcriptional factor that is involved in heart formation. Yet, little is known about how Snail regulates heart development. Here, we identified that one of the duplicated snail genes, snai1b, was expressed in the heart region of zebrafish embryos. Depletion of Snai1b function dramatically reduced expression of α5 integrin, disrupted Fibronectin layer in the heart region, especially at the midline, and prevented migration of cardiac precursors, resulting in defects in cardiac morphology and function in zebrafish embryos. Injection of α5β1 protein rescued the Fibronectin layer and then the myocardial precursor migration in snai1b knockdown embryos. The results provide the molecular mechanism how Snail controls the morphogenesis of heart during embryonic development.

An Optimized and Simplified System of Mouse Embryonic Stem Cell Cardiac Differentiation for the Assessment of Differentiation Modifiers

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation.

Disruption of G-Protein γ5 Subtype Causes Embryonic Lethality in Mice

Heterotrimeric G-proteins modulate many processes essential for embryonic development including cellular proliferation, migration, differentiation, and survival. Although most research has focused on identifying the roles of the various αsubtypes, there is growing recognition that similarly divergent βγ dimers also regulate these processes. In this paper, we show that targeted disruption of the mouse Gng5 gene encoding the γ5 subtype produces embryonic lethality associated with severe head and heart defects. Collectively, these results add to a growing body of data that identify critical roles for the γ subunits in directing the assembly of functionally distinct G-αβγ trimers that are responsible for regulating diverse biological processes. Specifically, the finding that loss of the G-γ5 subtype is associated with a reduced number of cardiac precursor cells not only provides a causal basis for the mouse phenotype but also raises the possibility that G-βγ5 dependent signaling contributes to the pathogenesis of human congenital heart problems.

Establishment of transgenic lines to monitor and manipulate Yap/Taz-Tead activity in zebrafish reveals both evolutionarily conserved and divergent functions of the Hippo pathway

To investigate the role of Hippo pathway signaling during vertebrate development transgenic zebrafish lines were generated and validated to dynamically monitor and manipulate Yap/Taz-Tead activity. Spatial and temporal analysis of Yap/Taz-Tead activity suggested the importance of Hippo signaling during cardiac precursor migration and other developmental processes. When the transcriptional co-activators, Yap and Taz were restricted from interacting with DNA-binding Tead transcription factors through expression of a dominant negative transgene, cardiac precursors failed to migrate completely to the midline resulting in strong cardia bifida. Yap/Taz-Tead activity reporters also allowed us to investigate upstream and downstream factors known to regulate Hippo signaling output in Drosophila. While Crumbs mutations in Drosophila eye disc epithelia increase nuclear translocation and activity of Yorkie (the fly homolog of Yap/Taz), zebrafish crb2a mutants lacked nuclear Yap positive cells and down-regulated Yap/Taz-Tead activity reporters in the eye epithelia, despite the loss of apical-basal cell polarity in those cells. However, as an example of evolutionary conservation, the Tondu-domain containing protein Vestigial-like 4b (Vgll4b) was found to down-regulate endogenous Yap/Taz-Tead activity in the retinal pigment epithelium, similar to Drosophila Tgi in imaginal discs. In conclusion, the Yap/Taz-Tead activity reporters revealed the dynamics of Yap/Taz-Tead signaling and novel insights into Hippo pathway regulation for vertebrates. These studies highlight the utility of this transgenic tool-suite for ongoing analysis into the mechanisms of Hippo pathway regulation and the consequences of signaling output.

Rat Cardiac progenitor cells and their application in cell therapy

cells and the subsequent heart failure. When the pharmacological approach no longer complies with the disease evolution, organ transplantation appears to be the only treatment able to rescue the patient life. Cell therapy promises to be clinically efficient and would allow circumventing many limitations of organ transplantation, such as organ low availability, major surgical procedures, high costs and longterm immunosuppression [1]. We designed porous Poly-Lactic Acid (PLLA) and Fibroin scaffolds to deliver CPCs in the heart, we isolated and characterized CPCs for the expression of c-Kit, MDR-1 and Sca-1 by flow cytometry, we tested their degree of differentiation in vitro studying the expression of all known rat sarcomeric proteins by real-time PCR and their differentiated morphology on Electron Microscopy samples [2]. A particular attention was given to the expression of microRNA, because their role in the differentiation process of cardiac precursors is emerging. We also tested the host reaction to scaffolds, CPCs, and CPCs/scaffolds. In vivo, almost all the used scaffolds induced a foreign body reaction in nude mouse and rats, but not in SCID mice. Cardiac stem cells a T cell-mediated immune response induced in nude mice, letting us suppose that, differently from Mesenchymal Stem Cells, they express MHC molecules on their surface. The degree of differentiation, the expression of ECM and integrin proteins, and the expression of several sarcomeric proteins were dependent on the type of scaffold and the polymer used.

Expression pattern of zcchc24 during early Xenopus development.

We report the expression pattern of a novel Xenopus laevis gene, zcchc24, which encodes a protein containing two zinc finger domains from the zf-CCHC and zf-3CxxC superfamilies. This protein shares >84% amino acid identity with its vertebrate homologues. During X. laevis embryonic development, zcchc24 is expressed at gastrula stages in the dorsal mesoderm, including the cardiac precursors region. During neurula stages, zcchc24 is expressed as two stripes in the dorsal region, more precisely, in the somitogenic mesoderm until the cardiac mesoderm. At early tailbud stages, zcchc24 continues to be expressed in these regions, but starts to be expressed in the migrating neural crest. Later, this gene is expressed in the head, branchial arches, heart and somites. The zinc finger domains present in Zcchc24 protein and its dynamic gene expression pattern suggest that Zcchc24 might be involved in the regulation of heart, somites and of branchial arch formation/patterning, namely in the regulation of apoptosis.

Sca1-Derived Cells Are a Source of Myocardial Renewal in the Murine Adult Heart

SummaryAlthough the mammalian heart is one of the least regenerative organs in the body, recent evidence indicates that the myocardium undergoes a certain degree of renewal to maintain homeostasis during normal aging. However, the cellular origin of cardiomyocyte renewal has remained elusive due to lack of lineage tracing experiments focusing on putative adult cardiac precursor cells. We have generated triple-transgenic mice based on the tet-cre system to identify descendants of cells that have expressed the stem cell marker Sca1. We found a significant and lasting contribution of Sca1-derived cells to cardiomyocytes during normal aging. Ischemic damage and pressure overload resulted in increased differentiation of Sca1-derived cells to the different cell types present in the heart. Our results reveal a source of cells for cardiomyocyte renewal and provide a possible explanation for the limited contribution of Sca1-derived cells to myocardial repair under pathological conditions.

Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left–right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer's vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left–right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads to coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left–right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects.

Common Expression of Stemness Molecular Markers and Early Cardiac Transcription Factors in Human Wharton's Jelly-Derived Mesenchymal Stem Cells and Embryonic Stem Cells

At present, there are still significant barriers that impede the clinical use of hESCs and iPS cells, including ethics, immunorejection, tumorigenesis from hESCs, and teratoma formation from iPS cells. It is therefore necessary to search for alternative sources of stem cells. WJ-MSCs originate from embryonic epiblasts and possess properties intermediate between hESCs and adult stem cells. However, the stemness properties of molecules in WJ-MSCs remain unclear compared to those of hESCs. In the present study, we isolated WJ-MSCs by a nonenzymatic method. Further, using microarray analysis by Affymetrix GeneChip and functional network analyses, we determined the degree of expression of stemness genes exhibited by the Human Stem Cell Pluripotency array. We also defined a wide range of stem cell gene expression in the WJ-MSCs in comparison with hESCs. At the same time, the definitive markers of early cardiac precursor cells and more committed progenitors were further characterized in WJ-MSCs. Our results demonstrated for the first time that WJ-MSCs had significant expression of undifferentiated human embryonic stem cell core markers, such as SOX2, NANOG, LIN28, SSEA1, SSEA3, SSEA4, KLF4, c-MYC, CRIPTO, and REX1, with a relatively lower level of expression than in hESCs. We also found WJ-MSCs have high expression of early cardiac transcription factors, such as Flk-1, Isl-1, and Nkx2.5. Functional analysis revealed signature genes of WJ-MSCs with specific roles involved in immune, cytoskeletal, and chemokine regulation, cell adhesion, and cell signaling. Our study indicated that there is a significant overlap between the stemness genes expressed by hESCs and WJ-MSCs. WJ-MSCs harbor a true stem cell population and are promising cells for stem cell-based therapies.

The developmental origin of postnatal cardiac regeneration

Recent studies have shown that postnatal mammalian hearts undergo cardiomyocyte refreshment. However, evidence is lacking for the cell origin of these postnatal cardiomyogenesis. We first confirmed that myocardial injury triggered the expression of embryonic cardiogenesis genes at the external part of the heart. To determine whether cardiac lineage-specific progenitor cells exist in the postnatal mammalian heart and have cardiomyogenic potential, we created coronary artery ligation on the Nkx2.5 enhancer-eGFP transgenic mice. The number of Nkx2.5 enhancer-eGFP cells was increasing after myocardial injury and peaked at 2 weeks post injury. The activated eGFP+ cells expressed cardiac precursor markers. The eGFP signaling was not expressed in mature cardiomyocytes, hematopoietic cells or fibroblasts. These activated Nkx2.5 enhancer-eGFP cells would differentiate into striated cardiomyocytes in vitro. We further confirmed that cardiac progenitor cells contributed directly to postnatal cardiomyogenesis by lineage tracing using inducible Cre system. To trace the developmental origin of the activated Nkx2.5 enhancer-eGFP cells, we created different lineage-Cre/Nkx2.5 enhancer-eGFP/ ROSA26 reporter triple transgenic mice. The post-injury Nkx2.5 enhancer-eGFP+ cells originated from the embryonic epicardial cells, but did not originate from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells, and perinatal/ postnatal epicardial cells. Taken together, our data confirmed that cardiac-lineage progenitor cells, which originated from embryonic epicardium-derived cells, contributed to postnatal mammamian cardiomyogenesis. The discovery of cardiomyogenic cell population in the postnatal heart enables the future cell therapy for cardiac regeneration.

Abstract 013: Role for β-Arrestins in Stem/Precursor Cell Function and Cardiac Regeneration

Chronic heart failure after myocardial injury (MI) is characterized by an extensive loss of myocytes due to considerable cell death. Bone marrow derived stem cells (BMSCs) can transdifferentiate and show potential for regenerating the myocardium after MI. Stem cell mobilization, egress from the bone marrow and recruitment to the site of injury can be regulated by signals through G protein coupled receptors (GPCRs). βArrestins have signalling and scaffolding functions and act as downstream regulators of GPCR desensitization and endocytosis. We explored the potential role for βArrestins in cardiac precursor cell function, concentrating on BMSCs. Using knockout (KO) mice, we investigated the role βArrestin1 (βArr1) and βArrestin2 (βArr2), their modulation of regenerative competence of BMSCs and their contribution to cardiac repair after ischemic injury. in vitro, we observed that BM derived cells devoid of either βArr1 or βArr2 are slower to proliferate, colonize and migrate, compared to wild type (WT) BM cells. We also observed elevated cell death in βArr2 deficient cells following oxidative stress. Additionally, the number of cKit+ stem cells, thought to be potential cardiac precursor cells, was significantly lower in the BM and blood of βArr KO vs WT. Similarly, BM and blood of the chimeras contained fewer and less viable cardiac stem/precursor cells pre and post MI, compared to WT transplanted controls. In our in vivo study, we carried out BM transplants to determine whether the βArrs may be involved in cardiac repair. WT mice were irradiated and received BM transplants from WT, βArr1 KO or βArr2 KO mice. Following BM reconstitution, mice underwent MI and their recovery was monitored. Interestingly, chimeric mice with βArr1 and βArr2 KO BM had significantly inferior outcomes, including significantly decreased post MI survival with βArr2 KO BM and both βArr chimeras had significantly lower cardiac function post MI than mice receiving WT BM. Histology revealed that both chimeras developed larger infarcts and hypertrophy at an faster rate. We conclude that βArrs play a novel role downstream of GPCR desensitization in cardiac progenitor cells in BM and appear to be critically involved in the heart’s response to ischemic injury via cardiac repair and regeneration.

Nox2 and Nox4 influence neonatal c-kit+ cardiac precursor cell status and differentiation

Redox status has emerged as critical in modulating stemness and lineage commitment in several precursor cell types. However, a role for redox genes, specifically NADPH oxidases (Nox), in cardiac precursor cells (CPCs) has not been established. We tested whether CPCs marked by type III receptor tyrosine kinase c-kit (c-kit(+)) exhibit a unique NADPH oxidase signature that confers precursor status and whether alterations in this profile are functionally linked to changes in lineage specification. Dihydroethidium (DHE) microfluorography indicated reduced basal reactive oxygen species (ROS) formation within early postnatal c-kit(+) CPCs. Real-time quantitative PCR revealed downregulation of ROS generator Nox2 and its subunit p67(phox) in c-kit(+) CPCs under basal conditions but upregulation of Nox2 and Nox4 over the course of differentiation. Adenoviral silencing of Nox2 and Nox4 increased expression of CPC markers c-kit and Flk-1 and blunted smooth and cardiac muscle differentiation, respectively, while overexpression of Nox2 and Nox4 significantly reduced c-kit expression. These changes were accompanied by altered expression of transcription factors regulating cardiac lineage commitment, Gata6 and Gata4, and cytokine transforming growth factor (TGF)-β1. Similar to other precursor cell types, RT(2)Profiler PCR Arrays revealed that c-kit(+) CPCs also exhibit enhanced antioxidant capacity at the mRNA level. In conclusion, we report that c-kit(+) CPCs demonstrate reduced Nox2 expression and ROS levels and that increases in Nox2 and Nox4 influence their differentiation into mature cells. We speculate that ROS generators Nox2 and Nox4, along with the antioxidant genes identified by PCR Arrays, may be novel targets in CPCs that could prove useful in cell-based therapy of the heart.

Embryonic Stem Cell–Derived CD166 + Precursors Develop Into Fully Functional Sinoatrial-Like Cells

A cell-based biological pacemaker is based on the differentiation of stem cells and the selection of a population displaying the molecular and functional properties of native sinoatrial node (SAN) cardiomyocytes. So far, such selection has been hampered by the lack of proper markers. CD166 is specifically but transiently expressed in the mouse heart tube and sinus venosus, the prospective SAN. We have explored the possibility of using CD166 expression for isolating SAN progenitors from differentiating embryonic stem cells. We found that in embryonic day 10.5 mouse hearts, CD166 and HCN4, markers of the pacemaker tissue, are coexpressed. Sorting embryonic stem cells for CD166 expression at differentiation day 8 selects a population of pacemaker precursors. CD166+ cells express high levels of genes involved in SAN development (Tbx18, Tbx3, Isl-1, Shox2) and function (Cx30.2, HCN4, HCN1, CaV1.3) and low levels of ventricular genes (Cx43, Kv4.2, HCN2, Nkx2.5). In culture, CD166+ cells form an autorhythmic syncytium composed of cells morphologically similar to and with the electrophysiological properties of murine SAN myocytes. Isoproterenol increases (+57%) and acetylcholine decreases (-23%) the beating rate of CD166-selected cells, which express the β-adrenergic and muscarinic receptors. In cocultures, CD166-selected cells are able to pace neonatal ventricular myocytes at a rate faster than their own. Furthermore, CD166+ cells have lost pluripotency genes and do not form teratomas in vivo. We demonstrated for the first time the isolation of a nonteratogenic population of cardiac precursors able to mature and form a fully functional SAN-like tissue.