Cardiac Progenitor Cell Population Research Articles

Urokinase-Type Plasminogen Activator Receptor Regulates Prosurvival and Angiogenic Properties of Cardiac Mesenchymal Stromal Cells.

One of the largest challenges to the implementation of cardiac cell therapy is identifying selective reparative targets to enhance stem/progenitor cell therapeutic efficacy. In this work, we hypothesized that such a target could be an urokinase-type plasminogen activator receptor (uPAR)-a glycosyl-phosphatidyl-inositol-anchored membrane protein, interacting with urokinase. uPAR is able to form complexes with various transmembrane proteins such as integrins, activating intracellular signaling pathway and thus regulating multiple cell functions. We focused on studying the CD117+ population of cardiac mesenchymal progenitor cells (MPCs), expressing uPAR on their surface. It was found that the number of CD117+ MPCs in the heart of the uPAR-/- mice is lower, as well as their ability to proliferate in vitro compared with cells from wild-type animals. Knockdown of uPAR in CD117+ MPCs of wild-type animals was accompanied by a decrease in survival rate and Akt signaling pathway activity and by an increase in the level of caspase activity in these cells. That suggests the role of uPAR in supporting cell survival. After intramyocardial transplantation of uPAR(-) MPCs, reduced cell retention and angiogenesis stimulation were observed in mice with myocardial infarction model compared to uPAR(+) cells transplantation. Taken together, the present results appear to prove a novel mechanism of uPAR action in maintaining the survival and angiogenic properties of CD117+ MPCs. These results emphasize the importance of the uPAR as a potential pharmacological target for the regulation of reparative properties of myocardial mesenchymal progenitor cells.

Identification of SALL4 Expressing Islet-1+ Cardiovascular Progenitor Cell Clones.

The utilization of cardiac progenitor cells (CPCs) has been shown to induce favorable regenerative effects. While there are various populations of endogenous CPCs in the heart, there is no consensus regarding which population is ideal for cell-based regenerative therapy. Early-stage progenitor cells can be differentiated into all cardiovascular lineages, including cardiomyocytes and endothelial cells. Identifying an Islet-1+ (Isl-1+) early-stage progenitor population with enhanced stemness, multipotency and differentiation potential would be beneficial for the development of novel regenerative therapies. Here, we investigated the transcriptome of human neonatal Isl-1+ CPCs. Isl-1+ human neonatal CPCs exhibit enhanced stemness properties and were found to express Spalt-like transcription factor 4 (SALL4). SALL4 plays a role in embryonic development as well as proliferation and expansion of hematopoietic progenitor cells. SALL4, SOX2, EpCAM and TBX5 are co-expressed in the majority of Isl-1+ clones isolated from neonatal patients. The pre-mesendodermal transcript TFAP2C was identified in select Isl-1, SALL4, SOX2, EpCAM and TBX5 expressing clones. The ability to isolate and expand pre-mesendodermal stage cells from human patients is a novel finding that holds potential value for applications in regenerative medicine.

The Vascular Niche for Adult Cardiac Progenitor Cells.

Research on cardiac progenitor cell populations has generated expectations about their potential for cardiac regeneration capacity after acute myocardial infarction and during physiological aging; however, the endogenous capacity of the adult mammalian heart is limited. The modest efficacy of exogenous cell-based treatments can guide the development of new approaches that, alone or in combination, can be applied to boost clinical efficacy. The identification and manipulation of the adult stem cell environment, termed niche, will be critical for providing new evidence on adult stem cell populations and improving stem-cell-based therapies. Here, we review and discuss the state of our understanding of the interaction of adult cardiac progenitor cells with other cardiac cell populations, with a focus on the description of the B-CPC progenitor population (Bmi1+ cardiac progenitor cell), which is a strong candidate progenitor for all main cardiac cell lineages, both in the steady state and after cardiac damage. The set of all interactions should be able to define the vascular cardiac stem cell niche, which is associated with low oxidative stress domains in vasculature, and whose manipulation would offer new hope in the cardiac regeneration field.

Abstract 12803: Human IPSC-Derived Committed Cardiac Progenitor Cells Engraft and Differentiate Following Transendocardial Injections in a Swine Ischemic Heart Failure Model

Introduction: A hallmark of human myocardium is the inability to regenerate functional myocardium following myocardial infarction (MI) which can result in heart failure. In an effort to remuscularize infarcted myocardium, we tested a novel human iPSC-derived committed cardiac progenitor (CCP) cell population. Whether CCPs survive and differentiate to form grafts following transcatheter delivery in a clinically representative large animal model is unknown. Hypothesis: We hypothesized that transendocardial injection (TEI) of CCPs, combined with a cardiac fibroblast-derived matrix (CFM) particles as a cell retention agent leads to functional cardiac tissue grafts. Methods: Large MI following coronary artery balloon-occlusion reperfusion was induced in mini-swine that received a 4-drug immunosuppression regimen. 28 days later, TEI targeting the peri-infarct region was performed using combinations of 200 million CCPs with or without 50 mg CFM particles and Flexbumin control. 14 injections/pig were performed, and animals were followed for 4-8 weeks. Histopathology, continuous ECG recording and cardiac MRI were performed. Results: Human cell grafts were identified using Ku80+ human cell specific antibody in the initial n=11 evaluable CCP treated swine after 1 month (see representative image, Ku80+ human cells=green). The CCPs largely differentiate into TnI positive cardiomyocytes (red) in grafts observed at one month. Immunolabeling for a panel of cardiomyocyte markers confirmed the presence of human cardiomyocytes. Of n=29 initial pigs, no treatment-related safety issues were identified, such as post TEI pericardial effusion, ventricular tachyarrhythmias or tumors. Conclusions: TEI of CCPs, with and without CFM, resulted in grafts of human cardiomyocytes in the myocardium of immunosuppressed pigs, after 1 month post MI. Ongoing cardiac MRI analyses will determine the impact of CCP therapy on cardiac structure and function.

Tbx5 navigates through the labyrinth of adult cardiac regeneration

Abstract Background Heart failure is the major cause of death and morbidity in industrialized countries with an estimated 23 million people affected per year, representing 30% of all global deaths. Injury to the adult mammalian cardiac muscle, often leads to a heart attack due to irreversible loss of a large number of cardiomyocytes (CM) and other cardiac interstitial cells, creating an unmet need for identifying a cardiac progenitor cell (CPC) population for cardiac replenishment. In contrast, amphibians and neonatal rodents possess the ability to regenerate their heart upon injury. It has been suggested recently that idle cardiac regenerative mechanisms may be present in adult mammals, inhibited by exogenous cues, or lack of. Murine and human CPCs can be isolated through the expression of Pdgfra, Kdr, and our novel surface marker, Gfra2. In addition, the expression of the embryonic transcription factor TBX5, is paramount for differentiation towards a cardiomyocyte fate. Therefore, Tbx5-expressing CPCs could be an effective target for proof-of-concept studies in the heart repair field, inclined to pharmacological modulation in patients with ischemic heart disease. Purpose To characterise an adult Tbx5-expressing CPC population in the injured heart. Using a developmental approach to two adult heart injury murine models, we show that Tbx5-expressing CPC exist in the injured adult mammalian heart, with a molecular signature that strongly correlates with that of embryonic and neonatal CPCs. Methods A well-defined tamoxifen-induced Tbx5-Cre; Rosa26R-eYFP/eYFP transgenic mouse model was employed, where myocardial infarction (MI) was induced through reperfusion/ischemia or chemical injury. Cardiac cells expressing YFP+ cells were collected from the adult injured hearts five to seven days post-injury. Flow-cytometric and single-cell mRNA-seq analysis was performed in order to collect and compare those YFP+ cells to control adult uninjured cardiac interstitial cells, and early neonatal-derived CPCs. Results Immunohistochemical analysis indicated that YFP+ interstitial cells were mostly present in the injury site, but also in distal cardiac areas. Flow cytometric analysis of live cells pinpoint these YFP+ cells are part of a CPC-like population. Single-cell mRNA transcriptomic analysis revealed that YFP+ cells resemble early postnatal CPC. Yet, YFP+ cells never expressed CM markers in vivo, but they did differentiate into CM-like cells, in vitro. Conclusions Upon MI, the adult heart possess an interstitial cell population that transiently re-activates the pioneer cardiac embryonic transcription factor Tbx5. The Tbx5-expressing cell population transcriptomically resembles that of CPC, which could promote CM regeneration upon neonatal injury. We show that Tbx5 lies in the centre of a novel adult CPC population. The adult heart microenvironment may hinder mammalian CM regeneration through regulation of the Tbx5-induced cardiac gene program. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Hellenic Foundation for Research & Innovation

Effects of Cardiotoxins on Cardiac Stem and Progenitor Cell Populations.

As research and understanding of the cardiotoxic side-effects of anticancer therapy expands further and the affected patient population grows, notably the long-term survivors of childhood cancers, it is important to consider the full range of myocardial cell types affected. While the direct impacts of these toxins on cardiac myocytes constitute the most immediate damage, over the longer term, the myocardial ability to repair, or adapt to this damage becomes an ever greater component of the disease phenotype. One aspect is the potential for endogenous myocardial repair and renewal and how this may be limited by cardiotoxins depleting the cells that contribute to these processes. Clear evidence exists of new cardiomyocyte formation in adult human myocardium, along with the identification in the myocardium of endogenous stem/progenitor cell populations with pro-regenerative properties. Any effects of cardiotoxins on either of these processes will worsen long-term prognosis. While the role of cardiac stem/progenitor cells in cardiomyocyte renewal appears at best limited (although with stronger evidence of this process in response to diffuse cardiomyocyte loss), there are strong indications of a pro-regenerative function through the support of injured cell survival. A number of recent studies have identified detrimental impacts of anticancer therapies on cardiac stem/progenitor cells, with negative effects seen from both long-established chemotherapy agents such as, doxorubicin and from newer, less overtly cardiotoxic agents such as tyrosine kinase inhibitors. Damaging impacts are seen both directly, on cell numbers and viability, but also on these cells' ability to maintain the myocardium through generation of pro-survival secretome and differentiated cells. We here present a review of the identified impacts of cardiotoxins on cardiac stem and progenitor cells, considered in the context of the likely role played by these cells in the maintenance of myocardial tissue homeostasis.

Understanding Heart Field Progenitor Cells for Modeling Congenital Heart Diseases.

Heart development is a meticulously coordinated process that involves the specification of two distinct populations of cardiac progenitor cells, namely the first and the second heart field. Disruption of heart field progenitors can result in congenital heart defects. In this review, we aim to describe the signaling pathways and transcription factors that link heart field development and congenital heart disease. Single-cell transcriptomics, lineage-tracing mouse models, and stem cell-based in vitro modeling of cardiogenesis have significantly improved the spatiotemporal characterization of cardiac progenitors. Additionally, novel functional genomic studies have now linked more genetic variants with congenital heart disease. Dysregulation of cardiac progenitor cells causes malformations that can be lethal. Ongoing research will continue to shed light on cardiac morphogenesis and help us better understand and treat patients with congenital heart disease.

Differences in Mitochondrial Membrane Potential Identify Distinct Populations of Human Cardiac Mesenchymal Progenitor Cells.

Adult human cardiac mesenchymal progenitor cells (hCmPC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Even if the mechanisms have not yet been fully elucidated, the stem cell differentiation is guided by the mitochondrial metabolism; however, mitochondrial approaches to identify hCmPC with enhanced stemness and/or differentiation capability for cellular therapy are not established. Here we demonstrated that hCmPCs sorted for low and high mitochondrial membrane potential (using a lipophilic cationic dye tetramethylrhodamine methyl ester, TMRM), presented differences in energy metabolism from preferential glycolysis to oxidative rates. TMRM-high cells are highly efficient in terms of oxygen consumption rate, basal and maximal respiration, and spare respiratory capacity compared to TMRM-low cells. TMRM-high cells showed characteristics of pre-committed cells and were associated with higher in vitro differentiation capacity through endothelial, cardiac-like, and, to a lesser extent, adipogenic and chondro/osteogenic cell lineage, when compared with TMRM-low cells. Conversely, TMRM-low showed higher self-renewal potential. To conclude, we identified two hCmPC populations with different metabolic profile, stemness maturity, and differentiation potential. Our findings suggest that metabolic sorting can isolate cells with higher regenerative capacity and/or long-term survival. This metabolism-based strategy to select cells may be broadly applicable to therapies.

12-O-Tetradecanoylphorbol-13-acetate increases cardiomyogenesis through PKC/ERK signaling

12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6–8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.

Isolation, growth kinetics, and immunophenotypic characterization of adult human cardiac progenitor cells.

The discovery of cardiac progenitor cells (CPCs) has raised expectations for the development of cell-based therapy of the heart.Although cell therapy is emerging as a novel treatment for heart failure, several issues still exist concerning an unambiguous definition of the phenotype of CPC types. There is a need to define and validate the methods for the generation of quality CPC populations used in cell therapy applications. Considering the critical roles of cardiac cell progenitors in cellular therapy, we speculate that long term culture might modulate the immunophenotypes of CPCs. Hence, a strategy to validate the isolation and cell culture expansion of cardiac cell populations was devised. Isolation of three subpopulations of human CPCs was done from a single tissue sample using explant, enzymatic isolation, and c-kit+ immunomagnetic sorting methods. The study assessed the effects of ex vivo expansion on proliferation, immunophenotypes, and differentiation of CPCs. Additionally, we report that an explant culture can take over 2 months to achieve similar cell yields, and cell sorting requires a much larger starting population to match this expansion time frame. In comparison, an enzymatic method is expected to yield equivalent quantities of CPCs in 2-3 weeks, notably at a significantly lower cost, which may intensify their use in therapeutic approaches. We determined that ex vivo expansion caused changes in cellular characteristics, and hence propose validated molecular signatures should be established to evaluate the impact of ex vivo expansion for a safe cell therapy product.

MiR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations.

MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent Nkx2.5 enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.

Disruption of mitochondrial membrane potential coupled with alterations in cardiac biomarker expression as early cardiotoxic signatures in human ES cell-derived cardiac cells.

Cardiotoxicity is one of the most significant reasons of attrition in drug development. The present study assessed the sensitivity of various endpoints for early monitoring of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiac cells, including precursors as well as mature cardiomyocytes, by correlating changes in cardiac biomarker expression. Directed differentiation was induced and cardiac progenitor cell (CPC) population were treated with cardiotoxic drugs, namely, doxorubicin (Dox) and paclitaxel (Pac), and with noncardiotoxic drug, namely penicillin G. To assess cardiac-specific toxicity, the changes in the expression of key markers of cardiac lineage, such as Nkx2.5, Tbx5, α-myosin heavy chain α-MHC, and cardiac troponin T, were studied using quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry (FC). The half-maximal inhibition in the expression of these cardiac markers was analyzed from the dose-response curves. We also assessed the half-maximal inhibition (IC50) in cardiac cells using propidium iodide dye (IC50 PI) and by measuring disruption in the mitochondrial membrane potential (IC50 MMP). We observed that the most sensitive marker was α-MHC in the case of both Dox and Pac, and the order of sensitivity of the various prediction assays was MMP > protein expression by FC > gene expression by qRT-PCR > cell viability by PI staining. The results could enrich the screening of drug-induced cardiotoxicity in vitro and propose disruption in MMP along with downregulation of α-MHC protein as a potential biomarker of predicting cardiotoxicity earlier during drug safety evaluation.

Discovering small molecules as Wnt inhibitors that promote heart regeneration and injury repair.

There are intense interests in discovering proregenerative medicine leads that can promote cardiac differentiation and regeneration, as well as repair damaged heart tissues. We have combined zebrafish embryo-based screens with cardiomyogenesis assays to discover selective small molecules that modulate heart development and regeneration with minimal adverse effects. Two related compounds with novel structures, named as Cardiomogen 1 and 2 (CDMG1 and CDMG2), were identified for their capacity to promote myocardial hyperplasia through expansion of the cardiac progenitor cell population. We find that Cardiomogen acts as a Wnt inhibitor by targeting β-catenin and reducing Tcf/Lef-mediated transcription in cultured cells. CDMG treatment of amputated zebrafish hearts reduces nuclear β-catenin in injured heart tissue, increases cardiomyocyte (CM) proliferation, and expedites wound healing, thus accelerating cardiac muscle regeneration. Importantly, Cardiomogen can alleviate the functional deterioration of mammalian hearts after myocardial infarction. Injured hearts exposed to CDMG1 display increased newly formed CMs and reduced fibrotic scar tissue, which are in part attributable to the β-catenin reduction. Our findings indicate Cardiomogen as a Wnt inhibitor in enhancing injury-induced CM proliferation and heart regeneration, highlighting the values of embryo-based small molecule screens in discovery of effective and safe medicine leads.

The Wnt inhibitor Dkk1 is required for maintaining the normal cardiac differentiation program in Xenopus laevis

Wnt proteins can activate different intracellular signaling pathways. These pathways need to be tightly regulated for proper cardiogenesis. The canonical Wnt/β-catenin inhibitor Dkk1 has been shown to be sufficient to trigger cardiogenesis in gain-of-function experiments performed in multiple model systems. Loss-of-function studies however did not reveal any fundamental function for Dkk1 during cardiogenesis. Using Xenopus laevis as a model we here show for the first time that Dkk1 is required for proper differentiation of cardiomyocytes, whereas specification of cardiomyocytes remains unaffected in absence of Dkk1. This effect is at least in part mediated through regulation of non-canonical Wnt signaling via Wnt11. In line with these observations we also found that Isl1, a critical regulator for specification of the common cardiac progenitor cell (CPC) population, acts upstream of Dkk1.

Isolation and Characterization of Cardiac Progenitor Cells.

Cardiac progenitor cells (CPCs) are gaining interest as a therapeutic option for the treatment of the heart. Due to the limited pool of CPCs residing in the heart, it is essential to isolate and expand the CPCs in vitro. Here we describe the protocol for isolation and culture of the heterogeneous population of CPCs from right atrial appendage and left ventricular tissue collected from patients undergoing on-pump coronary artery bypass graft surgery for the treatment of ischemic heart disease. Our protocol is developed to simultaneously isolate, culture, and characterize the CPCs from both atrial and ventricular tissues. We also describe the protocol for flow cytometry and immunohistochemical characterization of the isolated CPCs.

Cardiosphere-derived cells suppress allogeneic lymphocytes by production of PGE2 acting via the EP4 receptor

Cardiosphere-derived cells (CDCs) are a cardiac progenitor cell population, which have been shown to possess cardiac regenerative properties and can improve heart function in a variety of cardiac diseases. Studies in large animal models have predominantly focussed on using autologous cells for safety, however allogeneic cell banks would allow for a practical, cost-effective and efficient use in a clinical setting. The aim of this work was to determine the immunomodulatory status of these cells using CDCs and lymphocytes from 5 dogs. CDCs expressed MHC I but not MHC II molecules and in mixed lymphocyte reactions demonstrated a lack of lymphocyte proliferation in response to MHC-mismatched CDCs. Furthermore, MHC-mismatched CDCs suppressed lymphocyte proliferation and activation in response to Concanavalin A. Transwell experiments demonstrated that this was predominantly due to direct cell-cell contact in addition to soluble mediators whereby CDCs produced high levels of PGE2 under inflammatory conditions. This led to down-regulation of CD25 expression on lymphocytes via the EP4 receptor. Blocking prostaglandin synthesis restored both, proliferation and activation (measured via CD25 expression) of stimulated lymphocytes. We demonstrated for the first time in a large animal model that CDCs inhibit proliferation in allo-reactive lymphocytes and have potent immunosuppressive activity mediated via PGE2.

Abstract 202: Low Mitochondrial Membrane Potential Identified a Novel Population of Human Cardiac Progenitor Cells with the Capacity for Myocardial Repair

As resident cardiac stem/progenitor cells (CPCs) stay in the hypoxia niche, have less metabolic activity and lower mitochondrial membrane potential, the aim of this study was to characterize a new subset of CPCs with lower mitochondrial membrane potential and examine their potential for myocardial repair. Following the live cell staining with a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) at a final concentration of 25 nm, a novel subset of hCPCs with the lowest ~3% TMRM fluorescence were sorted from right atrial appendage derived cells (RAACs) in patients with cardiomyopathy, and named as ΔΨ m low -hCPCs, which are self-renewable, and characterized as positive (>90%) for CD90, CD44, CD140b, CD172a, however, negative (<5%) for CD34, CD45, CD54, CD117, CD133. The increased expression of pluripotent genes (LIN28, NANOG, & OCT3/4) and cardiac lineage genes (GATA4, MEF2C, MYOCD, & NKX2.5), as well as the decreased expression of cell cycle inhibitor (CDKN2A & CDKN1A) were observed in ΔΨ m low -hCPCs comparing with the non-sorted RAACs. Interestingly, ΔΨ m low -hCPCs exclusively express heme oxygenase-1 (HO-1). And CPCs positive for HO-1 and NKX2.5/GATA4 were found in the intact human heart tissue sections. ΔΨ m low -hCPCs are able to differentiate into all three cardiac lineage cells in both in vitro and in vivo conditions, and display superior ability for survival, proliferation and release of cytoprotective cytokines compared to the RAACs. At 35 days, compared with the vehicle and control cell groups, transplantation of ΔΨ m low -hCPCs significantly improve the cardiac structure and function following myocardial infarction in the immunodeficient mouse model, as indicated by the increased left ventricular (LV) ejection fraction, increased positive and negative LV dP/dt, reduced LV remodeling, and reduced infarct size. The immunohistochemistry studies showed the significant increased cardiac proliferation, newly formed cardiomyocytes, angiogenesis, and decreased cell apoptosis in the ΔΨ m low -hCPCs treated group. In summary, we identified a new hCPC population that might represent a primitive progenitor cell population within human hearts. The existence of these cells opens new opportunities for myocardial repair.

C-Kit Cardiac Progenitor Cell Based Cell Sheet Improves Vascularization and Attenuates Cardiac Remodeling following Myocardial Infarction in Rats.

The adult heart contains small populations of multipotent cardiac progenitor cells (CPC) that present a convenient and efficient resource for treatment of myocardial infarction. Several clinical studies of direct CPC delivery by injection have already been performed but showed low engraftment rate that limited beneficial effects of procedure. «Cell sheet» technology has been developed to facilitate longer retention of grafted cells and show new directions for cell-based therapy using this strategy. In this study we hypothesized that СPC-based cell sheet transplantation could improve regeneration after myocardial infarction. We demonstrated that c-kit+ CPC were able to form cell sheets on temperature-responsive surfaces. Cell sheet represented a well-organized structure, in which CPC survived, retained ability to proliferate, expressed progenitor cell marker Gata-4 formed connexin-43+ gap junctions, and were surrounded by significant amount of extracellular matrix proteins. Transplantation of cell sheets after myocardial infarction resulted in CPC engraftment as well as their proliferation, migration, and differentiation; cell sheets also stimulated neovascularization and cardiomyocyte proliferation in underlining myocardium and ameliorated left ventricular remodeling. Obtained data strongly supported potential use of CPC sheet transplantation for repair of damaged heart.

MiR34 inhibition induces human heart progenitor proliferation

MiR34 involvement in myocardial injury repair and ageing has been well documented in mouse model. Our aim was to establish whether the inhibition of miR34 expression through locked nucleic acid (LNA) could be used as a pharmacological intervention to enhance human heart repair. Cardiac progenitor cells were obtained by right atrial specimen collection during intraoperative procedures. Our analysis revealed a direct correlation between miR34 expression and patient age, and its silencing by LNA promoted the cardiac progenitor growth rate up to twofold ( ± 0.8). Our results confirmed the relevance of miR34a in human heart ageing, as previously demonstrated in mouse. Moreover, the decrease of miR34 expression in the cardiac progenitor cell population indicates its role in maintaining an undifferentiated status and consequently in a lower proliferation rate with the involvement of genes such as Notch-1, Numb, and p63.

Glycosylated Cell Surface Markers for the Isolation of Human Cardiac Progenitors.

The aim of stem cell therapy after cardiac injury is to replace damaged cardiac tissue. Human cardiac progenitor cells (CPCs) represent an interesting cell population for clinical strategies to treat cardiac disease and human CPC-specific antibodies would aid in the clinical implementation of cardiac progenitor-based cell therapy. However, the field of CPC biology suffers from the lack of human CPC-specific markers. Therefore, we raised a panel of monoclonal antibodies (mAb) against CPCs. Of this panel of antibodies, we show that mAb C1096 recognizes a progenitor-like population in the fetal and adult human heart and partially colocalize with reported CPC populations in vitro. Furthermore, mAb C1096 can be used to isolate a multipotent progenitor population from human heart tissue. Interestingly, the two lead candidates, mAb C1096 and mAb C19, recognize glycosylated residues on PECAM1 (platelet and endothelial cell adhesion molecule 1) and GRP78, respectively, and de-N-glycosylation significantly abolishes their binding. Thereby, this report describes new clinically applicable antibodies against human CPCs, and for the first time demonstrates the importance of glycosylated residues as CPCs specific markers.