Cardiac Progenitor Cells Research Articles

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.

Comparison of transplantation effects among cardiac progenitor cell types on the mitochondrial energy-producing ability after myocardial infarction in rats

Comparison of transplantation effects among cardiac progenitor cell types on the mitochondrial energy-producing ability after myocardial infarction in rats

Global secretome analysis of resident cardiac progenitor cells from wild-type and transgenic heart failure mice: Why ambience matters.

Resident cardiac progenitor cells (CPCs) have gained attention in cardiac regenerative medicine primarily due to their paracrine activity. In our current study we determined the role of pathological conditions such as heart failure on the autocrine-paracrine action of stem cell antigen-1 (Sca-1) expressing CPC. This comparative secretome profiling of Sca-1+ cells derived from transgenic heart failure (αMHC-cyclin-T1/Gαq overexpression [Cyc] cells) versus healthy (wild-type [Wt] cells) mice, achieved via mass-spectrometric quantification, enabled the identification of over 700 proteins. Our results demonstrate that the heart failure milieu caused a 2-fold enrichment of extracellular matrix proteins (ECM) like biglycan, versican, collagen XII, and angiogenic factors like heparan sulfate proteoglycan 2, plasminogen activator inhibitor 1 in the secretome. We further elucidated the direct influence of the secretome on the functional behavior of Sca-1 + cells via in vitro tube forming assay. Secreted factors present in the diseased milieu induced tube formation in Cyc cells (1.7-fold; p < 0.01) when compared with Wt cells after 24 hr of exposure. The presence of conditioned media moderately increased the proliferation of Cyc cells but had a more pronounced effect on Wt cells. Overall, these findings revealed global modifications in the secretory activity of adult Sca-1 + cells in the heart failure milieu. The secretion of ECM proteins and angiogenic factors, which are crucial for cardiac remodeling and recovery, was notably enriched in the supernatant of Cyc cells. Thus, during heart failure the microenvironment of Sca-1 + cells might favor angiogenesis and proliferation suggesting their potential to recover the damaged heart.

Notch signal pathway - therapeutic target for regulation of reparative processes in the heart.

Notch signaling pathway is a universal regulator of cell fate in embryogenesis and in maintaining the cell homeostasis of adult tissue. Through local cell-cell interactions, he controls neighboring cells behavior and determines their capacity for self-renewal, growth, survival, differentiation, and apoptosis. Recent studies have shown that the control of regenerative processes in the heart is also carried out with the participation of Notch system. At the heart of Notch regulates migration bone marrow progenitors and stimulates the proliferation of cardiomyocytes, cardiac progenitor cell activity, limits cardiomyocyte hypertrophy and fibrosis progression and stimulates angiogenesis. Notch signaling pathway may be regarded as a very promising target for the development of drugs for the stimulation of regeneration in the myocardium.

Cardiac Progenitors Induced from Human Induced Pluripotent Stem Cells with Cardiogenic Small Molecule Effectively Regenerate Infarcted Hearts and Attenuate Fibrosis.

Cardiac progenitor cells (CPCs) being multipotent offer a promising source for cardiac repair due to their ability to proliferate and multiply into cardiac lineage cells. Here, we explored a novel strategy for human CPCs generation from human induced pluripotent stem cells (hiPSCs) using a cardiogenic small molecule, isoxazole (ISX-9) and their ability to grow in the scar tissue for functional improvement in the infarcted myocardium. CPCs were induced from hiPSCs with ISX-9. CPCs were characterized by immunocytochemistry and RT-PCR. The CPC survival and differentiation in the infarcted hearts were determined by in vivo transplantation in immunodeficient mice following left anterior descending artery ligation and their effects were determined on fibrosis and functional improvement. ISX-9 simultaneously induced expression of cardiac transcription factors, NK2 homeobox 5, islet-1, GATA binding protein 4, myocyte enhancer factor-2 in hiPSCs within 3 days of treatment and successfully differentiated into three cardiac lineages in vitro. Messenger RNA and microRNA-sequencing results showed that ISX-9 targeted multiple cardiac differentiation, proliferation signaling pathways and upregulated myogenesis and cardiac hypertrophy related-microRNA. ISX-9 activated multiple pathways including transforming growth factor β induced epithelial-mesenchymal transition signaling, canonical, and non-canonical Wnt signaling at different stages of cardiac differentiation. CPCs transplantation promoted myoangiogenesis, attenuated fibrosis, and led to functional improvement in treated mice.

Wnt/\u03b2-catenin-mediated signaling re-activates proliferation of matured cardiomyocytes

BackgroundThe Wnt/β-catenin signaling pathway plays an important role in the development of second heart field (SHF Isl1+) that gives rise to the anterior heart field (AHF) cardiac progenitor cells (CPCs) for the formation of the right ventricle, outflow tract (OFT), and a portion of the inflow tract (IFT). During early cardiogenesis, these AHF CPCs reside within the pharyngeal mesoderm (PM) that provides a microenvironment for them to receive signals that direct their cell fates. Here, N-cadherin, which is weakly expressed by CPCs, plays a significant role by promoting the adhesion of CPCs within the AHF, regulating β-catenin levels in the cytoplasm to maintain high Wnt signaling and cardioproliferation while also preventing the premature differentiation of CPCs. On the contrary, strong expression of N-cadherin observed throughout matured myocardium is associated with downregulation of Wnt signaling due to β-catenin sequestration at the cell membrane, inhibiting cardioproliferation. As such, upregulation of Wnt signaling pathway to enhance cardiac tissue proliferation in mature cardiomyocytes can be explored as an interesting avenue for regenerative treatment to patients who have suffered from myocardial infarction.MethodsTo investigate if Wnt signaling is able to enhance cellular proliferation of matured cardiomyocytes, we treated cardiomyocytes isolated from adult mouse heart and both murine and human ES cell-derived matured cardiomyocytes with N-cadherin antibody or CHIR99021 GSK inhibitor in an attempt to increase levels of cytoplasmic β-catenin. Immunostaining, western blot, and quantitative PCR for cell proliferation markers, cell cycling markers, and Wnt signaling pathway markers were used to quantitate re-activation of cardioproliferation and Wnt signaling.ResultsN-cadherin antibody treatment releases sequestered β-catenin at N-cadherin-based adherens junction, resulting in an increased pool of cytoplasmic β-catenin, similar in effect to CHIR99021 GSK inhibitor treatment. Both treatments therefore upregulate Wnt signaling successfully and result in significant increases in matured cardiomyocyte proliferation.ConclusionAlthough both N-cadherin antibody and CHIR99021 treatment resulted in increased Wnt signaling and cardioproliferation, CHIR99021 was found to be the more effective treatment method for human ES cell-derived cardiomyocytes. Therefore, we propose that CHIR99021 could be a potential therapeutic option for myocardial infarction patients in need of regeneration of cardiac tissue.

FGF10 Signaling in Heart Development, Homeostasis, Disease and Repair.

Essential muscular organ that provides the whole body with oxygen and nutrients, the heart is the first organ to function during embryonic development. Cardiovascular diseases, including acquired and congenital heart defects, are the leading cause of mortality in industrialized countries. Fibroblast Growth Factors (FGFs) are involved in a variety of cellular responses including proliferation, differentiation, and migration. Among the 22 human/mouse FGFs, the secreted FGF10 ligand through the binding of its specific receptors (FGFR1b and FGFR2b) and subsequent activation of downstream signaling is known to play essential role in cardiac development, homeostasis and disease. FGF10 is one of the major marker of the early cardiac progenitor cells and a crucial regulator of differentiated cardiomyocyte proliferation in the developing embryo. Increasing evidence support the hypothesis that a detailed understanding of developmental processes is essential to identify targets for cardiac repair and regeneration. Indeed the activation of resident cardiomyocyte proliferation together with the injection of cardiac progenitors represent the most promising therapeutical strategies for cardiac regenerative medicine. The recent findings showing that FGF10 promotes adult cardiomyocyte cell cycle reentry and directs stem cell differentiation and cell reprogramming toward the cardiogenic lineage provide new insights into therapeutical strategies for cardiac regeneration and repair.

Human cardiomyocytes undergo enhanced maturation in embryonic stem cell-derived organoid transplants

Human cardiomyocytes (CM) differentiated from pluripotent stem cells (PSCs) are relatively immature when generated in two-dimensional (2D) in vitro cultures, which limits their biomedical applications. Here, we devised a strategy to enhance maturation of human CM in vitro by assembly of three-dimensional (3D) cardiac organoids (CO) containing human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs), endothelial cells (ECs), and mesenchymal stem cells (MSCs). In contrast to corresponding 2D cultures, 3D CO not only developed into structures containing spontaneously beating CM, but also showed enhanced maturity as indicated by increased expressions of sarcomere and ion channel genes and reduced proliferation. Heterotopic implantation of CO into the peritoneal cavity of immunodeficient mice induced neovascularization, and further stimulated upregulation of genes coding for the contractile apparatus, Ca2+ handling and ion channel proteins. In addition, CM in implanted CO were characterized by a more mature ultrastructure compared to CM implanted without CO support. Functional analysis revealed the presence of working cardiomyocytes in both in vivo and ex ovo chorioallantoic membrane implanted CO. Our results demonstrate that cultivation in 3D CO and subsequent heterotopic implantation enhance maturation of CM towards an adult-like phenotype. We reason that CO-derived CM represent an attractive source for drug discovery and other biomedical applications.

Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

During the phylotypic period, embryos from different genera show similar gene expression patterns, implying common regulatory mechanisms. Here we set out to identify enhancers involved in the initial events of cardiogenesis, which occurs during the phylotypic period. We isolate early cardiac progenitor cells from zebrafish embryos and characterize 3838 open chromatin regions specific to this cell population. Of these regions, 162 overlap with conserved non-coding elements (CNEs) that also map to open chromatin regions in human. Most of the zebrafish conserved open chromatin elements tested drive gene expression in the developing heart. Despite modest sequence identity, human orthologous open chromatin regions recapitulate the spatial temporal expression patterns of the zebrafish sequence, potentially providing a basis for phylotypic gene expression patterns. Genome-wide, we discover 5598 zebrafish-human conserved open chromatin regions, suggesting that a diverse repertoire of ancient enhancers is established prior to organogenesis and the phylotypic period.

Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential

Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5+ cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20+ cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.

Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

Formation and segregation of cell lineages forming the heart have been studied extensively but the underlying gene regulatory networks and epigenetic changes driving cell fate transitions during early cardiogenesis are still only partially understood. Here, we comprehensively characterize mouse cardiac progenitor cells (CPCs) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing and transposase-accessible chromatin profiling (ATAC-seq). By leveraging on cell-to-cell transcriptome and chromatin accessibility heterogeneity, we identify different previously unknown cardiac subpopulations. Reconstruction of developmental trajectories reveal that multipotent Isl1+ CPC pass through an attractor state before separating into different developmental branches, whereas extended expression of Nkx2-5 commits CPC to an unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states critically depending on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution.

Cardiac Progenitor Cell\u2013Derived Extracellular Vesicles Reduce Infarct Size and Associate with Increased Cardiovascular Cell Proliferation

Cell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs) results in an increased ejection fraction, but survival and integration are low. Therefore, paracrine factors including extracellular vesicles (EVs) are likely to contribute to the beneficial effects. We investigated the contribution of EVs by transplanting hCPCs with reduced EV secretion. Interestingly, these hCPCs were unable to reduce infarct size post-MI. Moreover, injection of hCPC-EVs did significantly reduce infarct size. Analysis of EV uptake showed cardiomyocytes and endothelial cells primarily positive and a higher Ki67 expression in these cell types. Yes-associated protein (YAP), a proliferation marker associated with Ki67, was also increased in the entire infarcted area. In summary, our data suggest that EV secretion is the driving force behind the short-term beneficial effect of hCPC transplantation on cardiac recovery after MI.

Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac 19F MRS/MRI and temporal tracking of progenitor cells

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and 19F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENEHD-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine 19F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold 19F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac 19F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with 19F MRI.

Atorvastatin protects cardiac progenitor cells from hypoxia-induced cell growth inhibition via MEG3/miR-22/HMGB1 pathway.

Heart failure (HF) induced by ischemia myocardial infarction (MI) is one of the major causes of morbidity and mortality all around the world. Atorvastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, has been demonstrated to benefit patients with ischemic or non-ischemic-induced HF, but the mechanism is still poorly understood. Increasing evidence indicates that lncRNAs play important role in variety of human disease. However, the role and underlying molecular mechanisms remain largely unclear. In our work, we applied 0.5% O2 to generate a hypoxia cardiac progenitor cell (CPC) model. Then, CCK8 and EdU assays were employed to investigate the role of atorvastatin in hypoxia CPC cell model. We found that hypoxia inhibits CPC viability and proliferation through modulating MEG3 expression, while atorvastatin application can protect CPCs from hypoxia-induced injury through inhibiting MEG3 expression. Then, we demonstrated that repression of MEG3 inhibited the hypoxia-induced injury of CPCs and overexpression of MEG3 inhibited the protective effect of atorvastatin in the hypoxia-induced injury of CPCs. Furthermore, our study illustrated that atorvastatin played its role in CPC viability and proliferation by modulating the expression of HMGB1 through the MEG3/miR-22 pathway. Our study, for the first time, uncovered the molecular mechanism of atorvastatin's protective role in cardiomyocytes under hypoxia condition, which may provide an exploitable target in developing effective therapy drugs for MI patients.

Mitochondrial Dysfunction and Senescence of Human Cardiac Progenitor Cells Are Prevented by Hypoxic Culture

Rationale: Therapeutic potential of c-Kit+ cardiac progenitor cells (CPCs) is modest in autologous cell therapy clinical trials and could benefit from optimization to maximize functional activity. Early proliferation arrest limits expansion potential of human CPCs in vitro. In contrast, self-renewal is preserved and mitochondrial reactive oxidative species (mtROS) minimized within the CPC hypoxic niche in vivo. Expansion under oxygen tension comparable to the hypoxic niche may therefore improve CPC reparative function.

Cardiac Progenitor Cell Fate in Embryonic and Neonatal Environments

Cardiac Progenitor Cell Fate in Embryonic and Neonatal Environments

A Bioprinted Cardiac Patch Composed of Cardiac-Specific Extracellular Matrix and Progenitor Cells for Heart Repair.

Congenital heart defects are present in 8 of 1000 newborns and palliative surgical therapy has increased survival. Despite improved outcomes, many children develop reduced cardiac function and heart failure requiring transplantation. Human cardiac progenitor cell (hCPC) therapy has potential to repair the pediatric myocardium through release of reparative factors, but therapy suffers from limited hCPC retention and functionality. Decellularized cardiac extracellular matrix hydrogel (cECM) improves heart function in animals, and human trials are ongoing. In the present study, a 3D-bioprinted patch containing cECM for delivery of pediatric hCPCs is developed. Cardiac patches are printed with bioinks composed of cECM, hCPCs, and gelatin methacrylate (GelMA). GelMA-cECM bioinks print uniformly with a homogeneous distribution of cECM and hCPCs. hCPCs maintain >75% viability and incorporation of cECM within patches results in a 30-fold increase in cardiogenic gene expression of hCPCs compared to hCPCs grown in pure GelMA patches. Conditioned media from GelMA-cECM patches show increased angiogenic potential (>2-fold) over GelMA alone, as seen by improved endothelial cell tube formation. Finally, patches are retained on rat hearts and show vascularization over 14 d in vivo. This work shows the successful bioprinting and implementation of cECM-hCPC patches for potential use in repairing damaged myocardium.

Triggering Endogenous Cardiac Repair and Regeneration via Extracellular Vesicle-Mediated Communication.

A variety of paracrine signals create networks within the myocardium and mediate intercellular communications. Indeed, paracrine stimulation of the endogenous regenerative program of the heart, mainly based on resident cardiac progenitor cell (CPC) activation together with cardiomyocyte proliferation, has become increasingly relevant for future cardiac medicine. In the last years, it has been shown that extracellular vesicles (EV), including exosomes (Ex), are powerful conveyors of relevant biological effects. EV have been proposed not only as promising therapeutic tool for triggering cardiac regeneration and improving repair, but also as means of better understanding the physiological and pathological relationships between specific cardiac components, including cardiomyocytes and fibroblasts. Actually, EV from different kinds of exogenous stem cells have been shown to mediate beneficial effects on the injured myocardium. Moreover, endogenous cells, like CPC can instruct cardiovascular cell types, including cardiomyocytes, while cardiac stromal cells, especially fibroblasts, secrete EV that modulate relevant aspects of cardiomyocyte biology, such as hypertrophy and electrophysiological properties. Finally, cardiomyocytes too may release EV influencing the function of other cardiac cell types. Therefore, EV-based crosstalk is thought to be important in both physiology and pathology, being involved in the responses of the heart to noxious stimuli. In this review we will discuss the role of EV in both regulating cardiac homeostasis and driving heart regeneration. In particular, we will address their role in: (i) providing cardio-protection and enhancing cardiac repair mechanisms; (ii) CPC biology; and (iii) influencing adult cardiomyocyte behavior.

Trypanosoma cruziNeurotrophic Factor Facilitates Cardiac Repair in a Mouse Model of Chronic Chagas Disease

Most patients acutely infected with Trypanosoma cruzi undergo short-term structural and functional cardiac alterations that heal without sequelae. By contrast, in patients whose disease progresses to chronic infection, irreversible degenerative chronic Chagas cardiomyopathy (CCC) may develop. To account for the contrast between cardiac regeneration in high-parasitism acute infection and progressive cardiomyopathy in low-parasitism CCC, we hypothesized that T. cruzi expresses repair factors that directly facilitate cardiac regeneration. We investigated, as one such repair factor, the T. cruzi parasite-derived neurotrophic factor (PDNF), known to trigger survival of cardiac myocytes and fibroblasts and upregulate chemokine chemokine C-C motif ligand 2, which promotes migration of regenerative cardiac progenitor cells (CPCs). Using in vivo and in vitro models of Chagas disease, we tested whether T. cruzi PDNF promotes cardiac repair. Quantitative PCR and flow cytometry of heart tissue revealed that stem-cell antigen-1 (Sca-1+) CPCs expand in acute infection in parallel to parasitism. Recombinant PDNF induced survival and expansion of ex vivo CPCs, and intravenous administration of PDNF into naïve mice upregulated mRNA of cardiac stem-cell marker Sca-1. Furthermore, in CCC mice, a 3-week intravenous administration of PDNF protocol induced CPC expansion and reversed left ventricular T-cell accumulation and cardiac remodeling including fibrosis. Compared with CCC vehicle-treated mice, which developed severe atrioventricular block, PDNF-treated mice exhibited reduced frequency and severity of conduction abnormalities. Our findings are in support of the novel concept that T. cruzi uses PDNF to promote mutually beneficial cardiac repair in Chagas disease. This could indicate a possible path to prevention or treatment of CCC.

Hypoxia induces hypomethylation of the HMGB1 promoter via the MAPK/DNMT1/HMGB1 pathway in cardiac progenitor cells.

Apoptosis is involved in the death of cardiac progenitor cells (CPCs) after myocardial infarction (MI) in the heart. The loss of CPCs results in infarct scar and further deterioration of the heart function. Though stem cell-based therapy provides an effective approach for heart function recovery after MI, the retention of CPCs in the infarcted area of the heart is the main barrier that limits its promising therapy. Therefore, the underlying mechanisms of CPC apoptosis in hypoxia are important for the development of new therapeutic targets for MI patients. In this work, we found that the expression of high-mobility group box 1(HMGB1) was upregulated in CPCs under hypoxia conditions. Further study demonstrated that HMGB1 was regulated by DNA methyltransferases 1 (DNMT1) via changing the methylation state of CpGs in the promoter of HMGB1 in CPCs during hypoxia process. Additionally, mitogen-activated protein kinase (MAPK) signaling pathway was found to be involved in regulating DNMT1/HMGB1-mediated CPC apoptosis in hypoxia process. In conclusion, our findings demonstrate a novel regulatory mechanism for CPC apoptosis and proliferation under hypoxia conditions, which may provide a new therapeutic approach for MI patients.