Cardiac Progenitor Cell Population Research Articles

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.

Cited4 is related to cardiogenic induction and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis.

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2′-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration.

Characterization and analysis of long non-coding rna (lncRNA) in In Vitro- and Ex Vivo-derived cardiac progenitor cells.

Recent advancements in cell-based therapies for the treatment of cardiovascular disease (CVD) show continuing promise for the use of transplanted stem and cardiac progenitor cells (CPCs) to promote cardiac restitution. However, a detailed understanding of the molecular mechanisms that control the development of these cells remains incomplete and is critical for optimizing their use in such therapy. Long non-coding (lnc) RNA has recently emerged as a crucial class of regulatory molecules involved in directing a variety of critical biological processes including development, homeostasis and disease. As such, a rising body of evidence suggests that they also play key regulatory roles in CPC development, though many questions remain regarding the expression landscape and specific identity of lncRNA involved in this process. To address this, we performed whole transcriptome sequencing of two murine CPC populations–Nkx2-5 EmGFP reporter-sorted embryonic stem (ES) cell-derived and ex vivo, cardiosphere-derived–in an effort to characterize their lncRNA profiles and potentially identify novel CPC regulators. The resulting sequencing data revealed an enrichment in both CPC populations for a panel of previously-identified lncRNA genes associated with cardiac differentiation. Additionally, a total of 1,678 differentially expressed and as-of-yet unannotated, putative lncRNA genes were found to be enriched for in the two CPC populations relative to undifferentiated ES cells.

Effects of β-adrenergic receptor drugs on embryonic ventricular cell proliferation and differentiation and their impact on donor cell transplantation.

β-Adrenergic receptors (β-ARs) and catecholamines are present in rodents as early as embryonic day (E)10.5. However, it is not known whether β-AR signaling plays any role in the proliferation and differentiation of ventricular cells in the embryonic heart. Here, we characterized expression profiles of β-AR subtypes and established dose-response curves for the nonselective β-AR agonist isoproterenol (ISO) in the developing mouse ventricular cells. Furthermore, we investigated the effects of ISO on cell cycle activity and differentiation of cultured E11.5 ventricular cells. ISO treatment significantly reduced tritiated thymidine incorporation and cell proliferation rates in both cardiac progenitor cell and cardiomyocyte populations. The ISO-mediated effects on DNA synthesis could be abolished by cotreatment of E11.5 cultures with either metoprolol (a β1-AR antagonist) or ICI-118,551 (a β2-AR antagonist). In contrast, ISO-mediated effects on cell proliferation could be abolished only by metoprolol. Furthermore, ISO treatment significantly increased the percentage of differentiated cardiomyocytes compared with that in control cultures. Additional experiments revealed that β-AR stimulation leads to downregulation of Erk and Akt phosphorylation followed by significant decreases in cyclin D1 and cyclin-dependent kinase 4 levels in E11.5 ventricular cells. Consistent with in vitro results, we found that chronic stimulation of recipient mice with ISO after intracardiac cell transplantation significantly decreased graft size, whereas metoprolol protected grafts from the inhibitory effects of systemic catecholamines. Collectively, these results underscore the effects of β-AR signaling in cardiac development as well as graft expansion after cell transplantation.NEW & NOTEWORTHY β-Adrenergic receptor (β-AR) stimulation can decrease the proliferation of embryonic ventricular cells in vitro and reduce the graft size after intracardiac cell transplantation. In contrast, β1-AR antagonists can abrogate the antiproliferative effects mediated by β-AR stimulation and increase graft size. These results highlight potential interactions between adrenergic drugs and cell transplantation.

Cardiac progenitor cells for heart repair.

Stem cell therapy is being investigated as an innovative and promising strategy to restore cardiac function in patients with heart failure. Several stem cell populations, including adult (multipotent) stem cells from developed organs and tissues, have been tested for cardiac repair with encouraging clinical and pre-clinical results. The heart has been traditionally considered a post-mitotic organ, however, this view has recently changed with the identification of stem/progenitor cells residing within the adult heart. Given their cardiac developmental origins, these endogenous cardiac progenitor cells (CPCs) may represent better candidates for cardiac cell therapy compared with stem cells from other organs such as the bone marrow and adipose tissue. This brief review will outline current research into CPC populations and their cardiac repair/regenerative potential.

G9a histone methyltransferase inhibitor BIX01294 promotes expansion of adult cardiac progenitor cells without changing their phenotype or differentiation potential.

As a follow-up to our previous reports showing that the G9a histone methyltransferase-specific inhibitor BIX01294 enhances bone marrow cell cardiac potential, this drug was examined for its effects on cardiomyocytes and mouse cardiac progenitor cells (CPCs). Cardiomyocytes and cardiac explants were cultured ± BIX01294, and examined for changes in cardiac function, protein and gene expression. Additionally, enriched populations of CPCs, contained in the 'phase bright cell' component of explants, were harvested from non-treated and BIX01294-treated cardiac tissue, and assayed for differences in cell phenotype and differentiation potential. Mouse CPCs were cultured with rat cardiomyocytes to allow differentiation of the progenitors to be assayed using species-specific PCR primers. While BIX01294 had no discernible effect on myocyte function and sarcomeric organization, treatment with this drug significantly increased CPC proliferation, as indicated by enhanced MTT metabolization and BrdUrd incorporation (4.1- and 2.0-fold, respectively, P < 0.001) after 48 h labelling, and increased Ki67 expression (4.8-fold, P < 0.001) after 7 days culture. Heart explants exposed to BIX01294 generated 3.6-fold (P < 0.005) greater yields of CPCs by 2 weeks culture. Importantly, CPCs obtained from non-treated and BIX01294-treated cultures did not differ in phenotype or differentiation potential. These data indicate that BIX01294 can expand CPCs without undermining their capacity as cardiac progenitors, and suggest that this drug may have utility for generating large numbers of CPCs for cardiac repair.

Abstract 15068: Expression of PW1 Identifies a New Candidate Cardiac Progenitor Population in the Adult Heart

Rationale: Adult myocardium contains a rare population of cardiac progenitor cells (CPCs), recruited in response to tissue injury, which are of central importance for the cardiac microenvironment after cardiac injury. However, their phenotype characterization and function in physiological and pathological has not yet been reported. Objective: Based on the identification of a cell population with very high expression of PW1 in different tissues, our goal was to determine the role that PW1+ cells play in the heart in a murine myocardial infarction model. Methods and Results: In this study we show that high expression of PW1 in cardiac cells (PW1+ cells), identified a rare population, located in the epicardium and interstitium in resting heart, increased in the injured and border area in response to myocardial infarction (MI). Using PW1-reporter mice, we defined three populations of CPCs that include fraction I (PW1-ckit+), II (PW1+ c-kit+) and III (PW1+ckit-) based on differential expression of PW1 and c-kit. We found that MI led to an increase of all the fractions especially fraction II. Furthermore, isolated PW1+ cells showed clonogenic capacity, measured as fibroblastic colony-forming units frequency, and differentiation capacity to give rise in vitro to several cardiac lineages. The phenotypic characterization of fractions I, II and III has also been studied by utilizing comparative gene expression profile as a screening tool. Taken together, these data identify a novel PW1+ cardiac progenitor population with the potential to undergo differentiation into multiple cardiac lineages, suggesting their involvement in cardiac repair in normal and pathological conditions. Conclusion: Based on the comparative phenotype analysis, we could demonstrate that the expression of PW1 identified a pure population of CPCs, augmented following MI, with clonogenic and multipotent capacity, which is likely to provide a novel target for therapeutic approaches aimed at improving cardiac repair.

Cardiac Bmi1(+) cells contribute to myocardial renewal in the murine adult heart.

IntroductionThe mammalian adult heart maintains a continuous, low cardiomyocyte turnover rate throughout life. Although many cardiac stem cell populations have been studied, the natural source for homeostatic repair has not yet been defined. The Polycomb protein BMI1 is the most representative marker of mouse adult stem cell systems. We have evaluated the relevance and role of cardiac Bmi1+ cells in cardiac physiological homeostasis.MethodsBmi1CreER/+;Rosa26YFP/+ (Bmi1-YFP) mice were used for lineage tracing strategy. After tamoxifen (TM) induction, yellow fluorescent protein (YFP) is expressed under the control of Rosa26 regulatory sequences in Bmi1+ cells. These cells and their progeny were tracked by FACS, immunofluorescence and RT-qPCR techniques from 5 days to 1 year.ResultsFACS analysis of non-cardiomyocyte compartment from TM-induced Bmi1-YFP mice showed a Bmi1+-expressing cardiac progenitor cell (Bmi1-CPC: B-CPC) population, SCA-1 antigen-positive (95.9 ± 0.4 %) that expresses some stemness-associated genes. B-CPC were also able to differentiate in vitro to the three main cardiac lineages. Pulse-chase analysis showed that B-CPC remained quite stable for extended periods (up to 1 year), which suggests that this Bmi1+ population contains cardiac progenitors with substantial self-maintenance potential. Specific immunostaining of Bmi1-YFP hearts serial sections 5 days post-TM induction indicated broad distribution of B-CPC, which were detected in variably sized clusters, although no YFP+ cardiomyocytes (CM) were detected at this time. Between 2 to 12 months after TM induction, YFP+ CM were clearly identified (3 ± 0.6 % to 6.7 ± 1.3 %) by immunohistochemistry of serial sections and by flow cytometry of total freshly isolated CM. B-CPC also contributed to endothelial and smooth muscle (SM) lineages in vivo.ConclusionsHigh Bmi1 expression identifies a non-cardiomyocyte resident cardiac population (B-CPC) that contributes to the main lineages of the heart in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0196-9) contains supplementary material, which is available to authorized users.

Predicting Variabilities in Cardiac Gene Expression with a Boolean Network Incorporating Uncertainty.

Gene interactions in cells can be represented by gene regulatory networks. A Boolean network models gene interactions according to rules where gene expression is represented by binary values (on / off or {1, 0}). In reality, however, the gene’s state can have multiple values due to biological properties. Furthermore, the noisy nature of the experimental design results in uncertainty about a state of the gene. Here we present a new Boolean network paradigm to allow intermediate values on the interval [0, 1]. As in the Boolean network, fixed points or attractors of such a model correspond to biological phenotypes or states. We use our new extension of the Boolean network paradigm to model gene expression in first and second heart field lineages which are cardiac progenitor cell populations involved in early vertebrate heart development. By this we are able to predict additional biological phenotypes that the Boolean model alone is not able to identify without utilizing additional biological knowledge. The additional phenotypes predicted by the model were confirmed by published biological experiments. Furthermore, the new method predicts gene expression propensities for modelled but yet to be analyzed genes.

Activated macrophages as a feeder layer for growth of resident cardiac progenitor cells.

The adult heart contains a population of cardiac progenitor cells (CPCs). Growing and collecting an adequate number of CPCs demands complex culture media containing growth factors. Since activated macrophages secrete many growth factors, we investigated if activated isolated heart cells seeded on a feeder layer of activated peritoneal macrophages (PM) could result in CPCs and if these, in turn, could exert cardioprotection in rats with myocardial infarction (MI). Heart cells of inbred Wistar rats were isolated by collagenase digestion and cultured on PM obtained 72h after intraperitoneal injection of 12ml thioglycollate. Cells (1×10(6)) exhibiting CPC phenotype (immunohistochemistry) were injected in the periphery of rat MI 10min after coronary artery occlusion. Control rats received vehicle. Three weeks later, left ventricular (LV) function (echocardiogram) was assessed, animals were euthanized and the hearts removed for histological studies. Five to six days after seeding heart cells on PM, spherical clusters composed of small bright and spherical cells expressing mostly c-Kit and Sca-1 antigens were apparent. After explant, those clusters developed cobblestone-like monolayers that expressed smooth muscle actin and sarcomeric actin and were successfully transferred for more than ten passages. When injected in the MI periphery, many of them survived at 21days after coronary ligature, improved LV ejection fraction and decreased scar size as compared with control rats. CPC-derived cells with cardiocyte and smooth muscle phenotypes can be successfully grown on a feeder layer of activated syngeneic PM. These cells decreased scar size and improved heart function in rats with MI.

Abstract 18698: Identification of a Novel Cardiac Progenitor Population Expressing Pw1/peg3 in the Murine Heart

The myocardium responds to injury by recruiting cardiac progenitor cells (CPCs) to the injured tissue to promote cardiac repair. Although different classes of CPCs have been identified, their contribution in physiological and pathological conditions remains unclear. PW1 gene has recently been proposed as a marker of resident adult stem and progenitor cell populations in several adult tissues. Our goal was to characterize and determine the role of PW1+ population in the heart. Here, we employ immunostaining and fluorescence-activated cell sorting (FACS) analysis in PW1-reporter mouse to perform qualitative and quantitative analyses of PW1+ population in the heart. We first found that PW1+ cells are mainly located in the epicardium and myocardial interstitium of normal hearts. The average percentage of PW1+ cells, as assessed by FACS, was 1.56±1.41%. A subset of PW1+ cells also co-express other CPC markers such as Sca-1 (52±22%) or PDGFR1α (43±14%). In contrast, a very small proportion of PW1+ cells co-express c-kit (6±5%). To investigate the contribution of PW1+ cells in pathological conditions, we then performed myocardial infarction (MI) by LAD ligation in PW1-reporter mice. We found that MI resulted in a 3-fold increase in the number of PW1+ cells in infarcted mice compared with sham-operated groups, at 1 week post-MI (1.16%±0.47% in sham versus 3.43%±0.82 in MI). This population preferentially localized in the injured myocardium and border area. PW1+ cells were isolated by FACS from the whole infarcted heart from PW1-reporter mice. In vitro differentiation assays reveal that purified PW1+ cells are multipotent and can spontaneously differentiate into smooth muscle cells, endothelial cells and cardiomyocyte-like cells. Taken together, our data identify a novel PW1+ cardiac progenitor population with the potential to undergo differentiation into multiple cardiac lineages, suggesting their involvement in cardiac repair in normal and pathological conditions. The discovery of a novel population of cardiac progenitor cells, augmented following MI and with cardiogenic potential, provides a novel target for therapeutic approaches aimed at improving cardiac regeneration.

COMPARATIVE CHARACTERIZATION OF CARDIAC ATRIAL PROGENITOR CELL POPULATIONS FOR USE IN CELL THERAPY

Endogenous cardiac progenitor cells (CPC) have the potential to regenerate damaged tissue following myocardial infarction and restore contractility; however, their insufficient numbers hinders the replacement of the massive loss of cardiomyocytes. In vitro expansion of various CPC populations has been proposed, based on surface markers9 expression and isolation techniques, but doubt remains as to which is the optimal population for regenerative therapy. Here we compare expression of stem cell and early cardiac genes in CPC populations to that in differentiating embryonic stem cells (ESCs). CPCs were isolated from adult mouse atria and expanded in adherent culture as CDCs[A] (via explant-derived cells, cardiosphere formation for 3 days); CDCs[B] (as CDCs[A] with cardiospheres kept for 5 days); Coll[C] (after collagenase &amp; trypsin digestion); Coll[D] (as Coll[C] with a 5 day cardiosphere step). Gene-expression was assessed using RT-PCR and compared to ESCs at day 4 or day 7 of cardiac differentiation for cardiac and stem cell genes or to CDCs[A] for mesenchymal/fibroblast markers. All populations had lower expression of Oct4 and Sox2 than the differentiating ESCs. CDCs[A] also expressed lower levels of early cardiac genes, whereas expression of early cardiac genes in CDCs[B] and Coll[C] was comparable to day 4 ESCs. c-Kit expression was significantly higher in Coll[C] than in CDCs[A] or CDCs[B] and was further increased after cardiosphere formation in Coll[D], however expression of Sca1 and CD105 was increased in CDCs[B]. Thus these progenitors resemble ESCs before day 7 of differentiation but expression of c-kit or Sca-1 can be manipulated during expansion.

Different types of cultured human adult cardiac progenitor cells have a high degree of transcriptome similarity.

Different types of cultured human adult cardiac progenitor cells have a high degree of transcriptome similarity.

Stable Phenotype and Function of Immortalized Lin−Sca-1+ Cardiac Progenitor Cells in Long-Term Culture: A Step Closer to Standardization

Putative cardiac progenitor cells (CPCs) have been identified in the myocardium and are regarded as promising candidates for cardiac cell-based therapies. Although two distinct populations of CPCs reached the clinical setting, more detailed studies are required to portray the optimal cell type and therapeutic setting to drive robust cell engraftment and cardiomyogenesis after injury. Owing to the scarcity of the CPCs and the need for reproducibility, the generation of faithful cellular models would facilitate this scrutiny. Here, we evaluate whether immortalized Lin(-)Sca-1(+) CPCs (iCPC(Sca-1)) represent their native-cell counterpart, thereby constituting a robust in vitro model system for standardized investigation in the cardiac field. iCPC(Sca-1) were established in vitro as plastic adherent cells endowed with robust self-renewal capacity while preserving a stable phenotype in long-term culture. iCPC(Sca-1) differentiated into cardiomyocytic-, endothelial-, and smooth muscle-like cells when subjected to appropriate stimuli. The cell line consistently displayed features of Lin(-)Sca-1(+) CPCs in vitro, as well as in vivo after intramyocardial delivery in the onset of myocardial infarction (MI). Transplanted iCPC(Sca-1) significantly attenuated the functional and anatomical alterations caused by MI while promoting neovascularization. iCPC(Sca-1) are further shown to engraft, establish functional connections, and differentiate in loco into cardiomyocyte- and vasculature-like cells. These data validate iCPC(Sca-1) as an in vitro model system for Lin(-)Sca-1(+) progenitors and for systematic dissection of mechanisms underlying CPC subsets engraftment/differentiation in vivo. Moreover, iCPC(Sca-1) can be regarded as a ready-to-use CPCs source for pre-clinical bioengineering studies toward the development of novel strategies for restoration of the damaged myocardium.

Identification and characterization of a postnatal Nkx2-5 enhancer positive cardiac progenitor cell population after myocardial infarction

Identification and characterization of a postnatal Nkx2-5 enhancer positive cardiac progenitor cell population after myocardial infarction

Murine Cardiosphere-Derived Cells Are Impaired by Age but Not by Cardiac Dystrophic Dysfunction

To be clinically relevant as a therapy for heart failure, endogenous progenitor cells must be isolated and expanded from aged and/or diseased tissue. Here, we investigated the effect of age and cardiac impairment resulting from lack of dystrophin on murine cardiosphere-derived cells (CDCs). CDCs were isolated and expanded from atrial biopsies from wild-type mice aged 1.5, 6, 18, and 24 months and from mdx mice aged 6 and 18 months. Cardiac function was measured in mdx mice and age-matched wild-type mice using high-resolution cine magnetic resonance imaging. CDCs could be isolated and expanded from all mice, however, the number of cells obtained, and their regenerative potential, decreased with age, as demonstrated by decreased expression of stem cell markers, c-kit and Sca-1, and decreased cell proliferation, migration, clonogenicity, and differentiation. Six-month-old mdx mice showed right ventricular (RV) dilation and reduced RV ejection fraction (EF) in comparison to wild-type mice. Older mdx mice displayed significant RV and left ventricular dilation and decreased EF in both ventricles, compared with age-matched wild-type mice. Mdx mouse hearts contained significantly more fibrotic tissue than age-matched wild-type mouse hearts. However, CDCs isolated from mice aged 6 and 18 months had the same number and regenerative potential from mdx mice and age-matched wild-type mice. Thus, the cardiac progenitor cell population is impaired by age but is not substantially altered by the progressive deterioration in function of the dystrophic heart.

Cardiac progenitor cells from the left atrial appendage may originate from a resident non-hematopoietic myeloid progenitor population

Purpose: We recently identified three distinct cardiac progenitor cell (CPC) populations from the left atrial appendages (LAAs) of adult murine hearts. These populations have distinct phenotypes and characteristics. Type A CPCs are c-kit+/Sca-1+, Type B CPCs are c-kit+/CD45+, and more mature Type C CPCs can be derived from Type B CPCs in vitro. The purpose of this study was to analyze the embryologic origin and the connection between the different CPC populations. Methods: We performed a whole-genome expression array to the cell sorted Type A, Type B and Type C CPC populations. The LAAs were analyzed using immunohistochemistry. Tissue culture derived cells were analyzed using immunostaining and FACS. Neural crest lineage tracing was performed with Pax3-cre eYFP reporter mice. We investigated the spontaneous cardiac differentiation process from Type B to Type C using live microscopy and FACS. Results: Transcriptome comparison demonstrated a significant up-regulation of the neural crest differentiation –pathway genes in Type A and C CPCs. Type B CPCs expressed a gene profile, which matches that of non-hematopoietic, resident macrophage –like cells found in other tissues. Pax3 lineage tracing demonstrated traced cells in the subepicardial zone of the LAA, co-stained with F4/80 macrophage marker and c-kit. Closely attached were epicardially lined F4/80+ cells with no eYFP signal. In tissue culture, eYFP signal was strongest in the Type B CPCs and lower in the Type A CPCs. Fibroblasts in the culture were eYFP negative. The CD45 expression in the Type B CPCs was stable (∼90% of the cells) up to five passages folllowed by a rapid decline of CD45, c-kit and Flk-1 expression. In live microscopy, formation of giant Sca-1+ and Gata-4+ cells by cell fusion was followed by asymmetric divisions creating Type C CPCs, which expressed cardiac myosin heavy chain and atrial natriuretic factor. The transcriptome was significantly skewed toward a cardiomyocyte phenotype with the spontaneous differentiation, although this differentiation was not complete, as seen by electron microscopy. The partially differentiated Type C CPC population could be grown for >60 passages. Conclusion: Our results suggest that rather than having distinct embryonic origins, the different types of CPCs in the LAA originate from a resident, non-hematopoietic myeloid progenitor cell population. The neural crest differentiation pathway can be activated in these cells during adulthood, possibly in cardiac stress situations, enabling differentiation to cardiac lineage with a novel mechanism consisting of cell fusion and asymmetric division.

Role of the myeloid zinc finger protein 1 (Mzf1) in an Nkx2.5 enhancer positive cardiac progenitor cell population - modulation of cardiogenesis

Purpose: The understanding of cardiac differentiation of pluripotent stem cells (PSCs) and the underlying mechanisms of embryonic cardiac development is crucial for a future efficient cardiac regenerative therapy. We investigated the role of the transcription factor (TF) Mzf1 during differentiation of PSC into cardiac progenitor cells (CPCs). Methods: The activity of Mzf1 was analyzed on an Nkx2.5 cardiac enhancer (CE) by luciferase reporter assays in several cell lines. EMSA and ChIP assays evaluated DNA-protein interaction. Gene expression of Mzf1 was compared in GFP+ (CPCs) and GFP- differentiated Nkx2.5 CE-eGFP ES cells and in the analogous embryonic populations of the corresponding transgenic mouse line. The direct influence of Mzf1-overexpression on CPCs was investigated by differentiating Nkx2.5 CE-eGFP ES cells carrying the dox-inducible Mzf1-cDNA. The frequency of CPCs (GFP+) was determined by FACS and gene expression was confirmed by qRT-PCR. Results: Luciferase assays identified Mzf1 as a potential activator of the Nkx2.5 CE in 293, H9c2 and HL-1 cells. Binding of Mzf1 protein to the Nkx2.5 CE was confirmed for several predicted Mzf1 binding sites by EMSA in vitro and by ChIP in differentiated murine ES cells in vivo. An up to 70-fold enhancement of Mzf1 expression in FACS sorted eGFP+ CPCs was detected after differentiation of Nkx2.5 CE-eGFP ES cells on day 5 and 7. Similarly, FACS sorting of eGFP+ cells of E9.5 embryos of the Nkx2.5 CE-eGFP transgenic mouse line yielded a more than 80-fold overexpression of Mzf1 in the respective population. In contrast, neither in vitro differentiated cardiomyocytes (CMs) (from αMHC-Cre mT/mG transgenic ES cells) nor in vivo CMs (sorted from αMHC-Cre mT/mG mice) revealed a similar overexpression of Mzf1. Permanent Mzf1 overexpression till day 8 of differentiation implicated a clear inhibitory effect on CPCs. This was validated by the significant decrease of GFP+ cells after FACS sorting (p<0.001) and a corresponding downregulation of cardiac gene expression (e.g. Nkx2.5, Tbx5, Tnnt2, αActinin, p<0.001). Endodermal marker Sox17, however, was upregulated (p<0.01) while overexpression of Mzf1 starting at day 5 of differentiation lead to an increase in GFP+ CPCs (p<0.05) and concomitant upregulation of cardiac gene expression (e.g. Nkx2.5, Tbx5, αActinin, p<0.05). Conclusion: The understanding of the regulatory network of vertebrate heart development is fundamental for the development of cardiac regenerative strategies. Our results reveal a novel role of Mzf1 as a modulatory TF in early cardiogenesis.

Stem cell populations in the heart and the role of Isl1 positive cells

Cardiac progenitor cells are multipotent stem cells isolated from both embryonic and adult hearts in several species and are able to differentiate at least into smooth muscle cells, endothelial cells and cardiomyocytes. The embryonic origin of these cells has not yet been demonstrated, but it has been suggested that these cells may derive from the first and secondary heart fields and from the neural crest. In the last decade, two different populations of cardiac progenitor or stem cells have been identified and isolated, i.e., the Islet1 positive (Isl1+) and c-Kit positive (c-Kit+)/Stem Cell Antigen-1 positive (Sca-1+) cells. Until 2012, these two populations have been considered two separate entities with different roles and a different origin, but new evidence now suggests a connection between the two populations and that the two populations may represent two subpopulations of a unique pool of cardiac stem cells, derived from a common immature primitive cell. To find a common consensus on this concept is very important in furthering the application of stem cells to cardiac tissue engineering.

231 ISOLATION OF HUMAN C-KIT+ CARDIAC PROGENITOR CELLS TO IMPROVE OUR UNDERSTANDING OF CARDIAC TRANSCRIPTIONAL REGULATION

<h3>Introduction</h3> It was previously believed that the heart was a terminally differentiated organ, with limited or no regenerative capacity. However, over the last decade compelling evidence suggests that the heart does undergo regeneration with a limited population of resident progenitor cells able to replenish cardiomyocytes lost due to injury throughout adult life. The therapeutic manipulation of resident cardiac progenitor cells could transform outcomes in patients with heart failure if these cells can be harnessed for myocardial regeneration. In recent years, the search for cardiac progenitor candidates has gathered momentum, and several populations of resident cardiac progenitor cells have now been isolated in rat, mouse, dog, porcine and human hearts using a variety of stem cell markers. With a variety of cardiac progenitor populations now described it is important that we fully understand the properties and differentiation potential of these progenitor populations. <h3>Methods</h3> We describe the isolation of c-kit+ potential cardiac progenitor cells from atrial appendages of patients undergoing coronary artery bypass or valve replacement surgery (n=6) and from human fetal hearts (n=6). C-kit+ magnetic activated cell sorting (MACS) was used to isolate c-kit+ cells that were expanded in culture. Immunostaining and fluorescent activated cell sorting (FACS) were used to assess the expression of a variety of markers. Cardiomyocyte and endothelial differentiation techniques were used to assess the differentiation potential the cardiac progenitors. Single-cell gene expression techniques also allowed us to detect variations among cells in seemingly homogenous populations. <h3>Results</h3> These populations were CD45-, CD34-, but positive for CD146, CD105, KDR and the early cardiac transcription factors Mef2c, GATA4, Nkx2.5 and Isl1. The c-kit+ populations were able to differentiate <i>in vitro</i> to cardiomyocytes and endothelial cells. To improve our understanding of the dynamic networks of transcriptional regulation in cardiac progenitor cells both fetal and adult c-kit+ cardiac progenitors were used as a tool for gene profiling to identify key genetic differences between the populations. This allowed us to detectoverlapping and non-overlapping expression patterns and thus provide novel insights into the complex networks of transcriptional regulation in cardiac progenitor cells. <h3>Conclusion</h3> Understanding the transcriptional regulation of cardiac progenitor cells will identify novel approaches for cellular reprogramming and permit us to develop robust methods for isolating and expanding these cells for clinical application in the treatment of patients with heart failure.