Differentiation Of Cardiac Progenitor Cells Research Articles

TBX1 Represses Vegfr2 Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells.

The T-box transcription factor TBX1 has critical roles in maintaining proliferation and inhibiting differentiation of cardiac progenitor cells of the second heart field (SHF). Haploinsufficiency of the gene that encodes it is a cause of congenital heart disease. Here, we developed an embryonic stem (ES) cell-based model in which Tbx1 expression can be modulated by tetracycline. Using this model, we found that TBX1 down regulates the expression of VEGFR2, and we confirmed this finding in vivo during embryonic development. In addition, we found a Vegfr2 domain of expression, not previously described, in the posterior SHF and this expression is extended by loss of Tbx1. VEGFR2 has been previously described as a marker of a subpopulation of cardiac progenitors. Clonal analysis of ES-derived VEGFR2+ cells indicated that 12.5% of clones expressed three markers of cardiac lineage (cardiomyocyte, smooth muscle and endothelium). However, a pulse of Tbx1 expression was sufficient to increase the percentage to 20.8%. In addition, the percentage of clones expressing markers of multiple cardiac lineages increased from 41.6% to 79.1% after Tbx1 pulse. These results suggest that TBX1 plays a role in maintaining a progenitor state in VEGFR2+ cells.

Arsenite Disrupts Zinc-Dependent TGFβ2-SMAD Activity During Murine Cardiac Progenitor Cell Differentiation.

TGFβ2 (transforming growth factor-β2) is a key growth factor regulating epithelial to mesenchymal transition (EMT). TGFβ2 triggers cardiac progenitor cells to differentiate into mesenchymal cells and give rise to the cellular components of coronary vessels as well as cells of aortic and pulmonary valves. TGFβ signaling is dependent on a dynamic on and off switch in Smad activity. Arsenite exposure of 1.34 μM for 24-48 h has been reported to disrupt Smad phosphorylation leading to deficits in TGFβ2-mediated cardiac precursor differentiation and transformation. In this study, the molecular mechanism of acute arsenite toxicity on TGFβ2-induced Smad2/3 nuclear shuttling and TGFβ2-mediated cardiac EMT was investigated. A 4-h exposure to 5 μM arsenite blocks nuclear accumulation of Smad2/3 in response to TGFβ2 without disrupting Smad phosphorylation or nuclear importation. The depletion of nuclear Smad is restored by knocking-down Smad-specific exportins, suggesting that arsenite augments Smad2/3 nuclear exportation. The blockage in TGFβ2-Smad signaling is likely due to the loss of Zn(2+) cofactor in Smad proteins, as Zn(2+) supplementation reverses the disruption in Smad2/3 nuclear translocation and transcriptional activity by arsenite. This coincides with Zn(2+) supplementation rescuing arsenite-mediated deficits in cardiac EMT. Thus, zinc partially protects cardiac EMT from developmental toxicity by arsenite.

Medicinal Chemistry Approaches to Heart Regeneration.

Because of the minimal and clearly insufficient ability of the adult heart to regenerate after ischemic injury, there is a great opportunity to identify biological mechanisms, substances, and factors that enhance this process. Hence, innovative therapeutic management of heart failure following infarction requires a paradigm shift in pharmacotherapy. Spurred by tremendous progress in the field of stem cell and cardiac biology, several attractive approaches for regeneration of lost cardiomyocytes and supporting vasculature have emerged. Research in this area focuses on restoring the hearts' original function via proliferation and differentiation of cardiac progenitor cells, proliferation of pre-existing cardiomyocytes, and reprogramming of cardiac fibroblasts. In this review, we outline these principal strategies, putative biological targets or signaling pathways and chemical agents, with a focus on small molecules, to achieve therapeutic heart regeneration. We also point out the many remaining questions and challenges, particularly for translating in vitro discoveries to in vivo application.

Accumulation of Mitochondrial DNA Mutations Disrupts Cardiac Progenitor Cell Function and Reduces Survival

Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.

Hoxb1 regulates proliferation and differentiation of second heart field progenitors in pharyngeal mesoderm and genetically interacts with Hoxa1 during cardiac outflow tract development

Outflow tract (OFT) anomalies are among the most common congenital heart defects found at birth. The embryonic OFT grows by the progressive addition of cardiac progenitors, termed the second heart field (SHF), which originate from splanchnic pharyngeal mesoderm. Development of the SHF is controlled by multiple intercellular signals and transcription factors; however the relationship between different SHF regulators remains unclear. We have recently shown that Hoxa1 and Hoxb1 are expressed in a sub-population of the SHF contributing to the OFT. Here, we report that Hoxb1 deficiency results in a shorter OFT and ventricular septal defects (VSD). Mechanistically, we show that both FGF/ERK and BMP/SMAD signaling, which regulate proliferation and differentiation of cardiac progenitor cells and OFT morphogenesis, are enhanced in the pharyngeal region in Hoxb1 mutants. Absence of Hoxb1 also perturbed SHF development through premature myocardial differentiation. Hence, the positioning and remodeling of the mutant OFT is disrupted. Hoxa1−/− embryos, in contrast, have low percentage of VSD and normal SHF development. However, compound Hoxa1−/−; Hoxb1+/− embryos display OFT defects associated with premature SHF differentiation, demonstrating redundant roles of these factors during OFT development. Our findings provide new insights into the gene regulatory network controlling SHF and OFT formation.

Abstract 305: Single Cell RNA Sequencing of Adult Cardiac c-kit+ Cells in a Murine Lineage Tracing Model

A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac regeneration and improve ventricular function after ischemic cardiac injury. However, recent studies have shown that despite these benefits, c-kit+ progenitor cells in the adult murine heart are more inclined to take on an endothelial rather than cardiomyocyte lineage. To better define the factors involved in early differentiation of these resident cardiac progenitor cells and to distinguish distinct cell subpopulations, we performed single cell RNA sequencing on c-kit+ cells from Kit-Cre lineage traced GFP reporter mice versus total mesenchymal cells from the heart that were CD31- and CD45-. Cells were isolated by cardiac digestion and FACS was performed, positively sorting for the c-kit+ lineage while negatively sorting for CD31 and CD45 to eliminate endothelial and leukocyte progenitor contamination, respectively. Following this isolation, cells were examined to determine GFP reporter status and then submitted for single cell RNA sequencing using the Fluidigm A1 system. Clustering of 654 genes from this data identified 6 distinct subpopulations indicating various stages of early differentiation among CD31- and CD45-negative cardiac interstitial cells. Furthermore, comparison of GFP+ c-kit cells to the total non-GFP mesenchymal cells identified 75 differentially expressed transcripts. These unique gene signatures may help parse the genes that underlie cellular plasticity in the heart and define the best molecular lineages for transdifferentiation into cardiac myocytes.

Abstract 11: Acquisition of Mitochondrial DNA Mutations Impairs Mitochondrial Function in Cardiac Progenitor Cells

Activation of cardiac progenitor cells (CPCs) are critical for effective repair in response to pathologic injury. Stem cell activation and commitment involve increased energy demand and mitochondrial biogenesis. We have previously shown that incubation of c-kit+ CPCs in differentiation medium led to expansion of the mitochondrial network and lineage commitment. CPC function is reduced with age but the underlying mechanism is still unclear. Mitochondria contain their own DNA (mtDNA) which accumulates mutations over time that can impair mitochondrial function. In this study, we investigated the effects of acquiring mtDNA mutations on CPC proliferation, survival, and differentiation. We utilized a mouse model in which a mutation in the mtDNA polymerase gamma (POLGm/m) leads to accumulation of mtDNA mutations, mitochondrial dysfunction, and accelerated aging. Isolated CPCs from hearts of 2-month old POLGm/m mice had reduced proliferation and were more susceptible to oxidative stress and chemotherapeutic agents compared to WT CPCs. Incubation in differentiation medium resulted in fewer lineage committed POLGm/m CPCs compared to WT. In addition, the POLGm/m CPCs failed to activate mitochondrial biogenesis and did not increase levels of proteins involved in mitochondrial oxidative phosphorylation. We measured mitochondrial respiration with the Seahorse XF Analyzer and found that POLGm/m CPCs had undetectable oxygen consumption but still generated similar amounts of ATP as WT CPCs. Interestingly, POLGm/m CPCs produced increased amounts of l-lactate and were more sensitive to 2-deoxyglucose treatment, suggesting that these cells rely on glycolysis for energy production. Both WT and POLGm/m CPCs downregulated expression of glycolytic enzymes during differentiation. However, POLGm/m CPCs failed to undergo the metabolic transition from glycolysis to OXPHOS, which led to activation of cell death during differentiation. These data demonstrate that mitochondria play a critical role in CPC function, and accumulation of mtDNA mutations impairs CPC function and reduces their repair potential.

Abstract 394: Transient Bone Morphogenic Protein Antagonism Directs Differentiation of iPSCs into the Cardiac Neural Crest and cKit+ Myocardial Progenitor Lineages

Introduction: The capability of cKit+ cardiac progenitor cells (CPCs) to participate in cardiomyocyte regeneration remains controversial, despite basic and clinical studies supporting such a role. Hypothesis: A non-permissive cardiac milieu minimizes the generation of cardiomyocytes from CPCs. Methods: We lineage-traced CPCs using novel dual-recombinase responsive indicator mice (cKitCreERT2;Wnt1::Flpe;RC::Fela) and iPSCs derived from cKitCreERT2;IRG (iPSCKit) mice. Results: Intersectional genetic fate-mapping of cKitCreERT2;Wnt1:: Flpe;RC::Fela embryos supported that cKit marks Wnt1-expressing cardiac neural crest (CNC) progenitors, emerging at ~E9.5 and contributing a limited number of cardiomyocytes. To decipher the mechanisms underlying cardiomyocyte differentiation of CPCs, we lineage-traced CPCs during stage-specific cardiogenic differentiation of iPSCKit. Ascorbate treatment promoted differentiation of cKit+ iPSC-derived embryoid bodies (EBs) into Nkx2.5+ myocardium, 45.5%±6.7% of which co-expressed the Cre-reporter EGFP (n=154 EBs; 12 preparations), suggesting that CPCs encompass fully competent cardiomyogenic progenitors. Noggin (or Dorsomorphin), a BMP antagonist transiently expressed in the heart at E7.5-E8.5 but not during CNC invasion, directed the differentiation of iPSCkit-EBs into Mesp1+/Isl1+/Nkx2.5+ cardiac mesoderm progenitors (p≤0.0001). Remarkably, the same signaling pathway subsequently directed EBs into the cKit+/Wnt1+/Pax3+/Mitf-H+/Isl1+/Nkx2.5+ CNC lineage (p≤0.0001), while suppressing the generation of WT1+/Tbx18+ epicardium (p<0.05). Stage-specific induction of Cre-recombination delineated that iPSCkit-derived CPCs encompass Mesp1–/cKit+/Nkx2.5+ CNC progenitors which contributed EGFP+ CNC derivatives, including Nkx2-5+ cardiomyocytes, to 60.7%±7.3% of spontaneously beating EBs (n=147 EBs; 12 preparations). Conclusions: Collectively, our data show that CPCkit are fully competent CNC-derived cardiomyogenic progenitors, whose differentiation to cardiomyocytes is minimized by a latent Noggin-mediated signaling pathway. Therefore exploiting CPCkit therapeutically, provides an important strategy for maximizing myocardial regeneration.

MiRNA-204 drives cardiomyocyte proliferation via targeting Jarid2

ObjectivesIn mammals, the heart grows by hypertrophy but not proliferation of cardiomyocytes after birth. The paucity of cardiomyocyte proliferation limits cardiac regeneration in a variety of heart diseases. To explore the efficient strategies that drive cardiomyocyte proliferation, we employed in vitro and in vivo models to investigate the function of miRNA-204, which was demonstrated to regulate the proliferation and differentiation of human cardiac progenitor cells in our previous study. Methods and resultsmiRNA-204 overexpression markedly promoted cardiomyocyte proliferation in both neonatal and adult rat cardiomyocytes in vitro. Transgenic mice with the cardiac-specific overexpression of miRNA-204 exhibited excessive cardiomyocyte proliferation throughout the embryonic and adult stages, leading to a pronounced increase in ventricular mass. Accordingly, the cell cycle regulators, including Cyclin A, Cyclin B, Cyclin D2, Cyclin E, CDC2 and PCNA, were upregulated in miRNA-204 transgenic embryonic hearts. Furthermore, we demonstrated that miRNA-204 directly targeted Jarid2. Knockdown of Jarid2 mimicked the pro-proliferative effect of miRNA-204 overexpression on cultured rat cardiomyocytes, whereas enhanced expression of Jarid2 conferred the myocytes with substantial resistance to proliferation by miRNA-204 overexpression. ConclusionOur findings identify a conserved role for miRNA-204 in regulating cardiomyocyte proliferation by targeting the Jarid2 signaling pathway.

Inhibition of Wnt6 by Sfrp2 regulates adult cardiac progenitor cell differentiation by differential modulation of Wnt pathways

Wnt signaling has recently emerged as an important regulator of cardiac progenitor cell proliferation and differentiation, but the exact mechanisms by which Wnt signaling modulates these effects are not known. Understanding these mechanisms is essential for advancing our knowledge of cardiac progenitor cell biology and applying this knowledge to enhance cardiac therapy. Here, we explored the effects of Sfrp2, a canonical Wnt inhibitor, in adult cardiac progenitor cell (CPC) differentiation and investigated the molecular mechanisms involved. Our data show that Sfrp2 treatment can promote differentiation of CPCs after ischemia–reperfusion injury. Treatment of CPCs with Sfrp2 inhibited CPC proliferation and primed them for cardiac differentiation. Sfrp2 binding to Wnt6 and inhibition of Wnt6 canonical pathway was essential for the inhibition of CPC proliferation. This inhibition of Wnt6 canonical signaling by Sfrp2 was important for activation of the non-canonical Wnt/Planar Cell Polarity (PCP) pathway through JNK, which in turn induced expression of cardiac transcription factors and CPC differentiation. Taken together, these results demonstrate a novel role of Sfrp2 and Wnt6 in regulating the dynamic process of CPC proliferation and differentiation, as well as providing new insights into the mechanisms of Wnt signaling in cardiac differentiation.

MicroRNAs and Cardiac Regeneration.

The human heart has a limited capacity to regenerate lost or damaged cardiomyocytes after cardiac insult. Instead, myocardial injury is characterized by extensive cardiac remodeling by fibroblasts, resulting in the eventual deterioration of cardiac structure and function. Cardiac function would be improved if these fibroblasts could be converted into cardiomyocytes. MicroRNAs (miRNAs), small noncoding RNAs that promote mRNA degradation and inhibit mRNA translation, have been shown to be important in cardiac development. Using this information, various researchers have used miRNAs to promote the formation of cardiomyocytes through several approaches. Several miRNAs acting in combination promote the direct conversion of cardiac fibroblasts into cardiomyocytes. Moreover, several miRNAs have been identified that aid the formation of inducible pluripotent stem cells and miRNAs also induce these cells to adopt a cardiac fate. MiRNAs have also been implicated in resident cardiac progenitor cell differentiation. In this review, we discuss the current literature as it pertains to these processes, as well as discussing the therapeutic implications of these findings.

Generation of cardiac progenitor cells through epicardial to mesenchymal transition.

The epithelial to mesenchymal transition (EMT) is a biological process that drives the formation of cells involved both in tissue repair and in pathological conditions, including tissue fibrosis and tumor metastasis by providing cancer cells with stem cell properties. Recent findings suggest that EMT is reactivated in the heart following ischemic injury. Specifically, epicardial EMT might be involved in the formation of cardiac progenitor cells (CPCs) that can differentiate into endothelial cells, smooth muscle cells, and, possibly, cardiomyocytes. The identification of mechanisms and signaling pathways governing EMT-derived CPC generation and differentiation may contribute to the development of a more efficient regenerative approach for adult heart repair. Here, we summarize key literature in the field.

Molecular weight and concentration of heparin in hyaluronic acid-based matrices modulates growth factor retention kinetics and stem cell fate

Growth factors are critical for regulating and inducing various stem cell functions. To study the effects of growth factor delivery kinetics and presentation on stem cell fate, we developed a series of heparin-containing hyaluronic acid (HyA)-based hydrogels with various degrees of growth factor affinity and retention. To characterize this system, we investigated the effect of heparin molecular weight, fractionation, and relative concentration on the loading efficiency and retention kinetics of TGFβ1 as a model growth factor. At equal concentrations, high MW heparin both loaded and retained the greatest amount of TGFβ1, and had the slowest release kinetics, primarily due to the higher affinity with TGFβ1 compared to low MW or unfractionated heparin. Subsequently, we tested the effect of TGFβ1, presented from various heparin-containing matrices, to differentiate a versatile population of Sca-1+/CD45− cardiac progenitor cells (CPCs) into endothelial cells and form vascular-like networks in vitro. High MW heparin HyA hydrogels stimulated more robust differentiation of CPCs into endothelial cells, which formed vascular-like networks within the hydrogel. This observation was attributed to the ability of high MW heparin HyA hydrogels to sequester endogenously synthesized angiogenic factors within the matrix. These results demonstrate the importance of molecular weight, fractionation, and concentration of heparin on presentation of heparin-binding growth factors and their effect on stem cell differentiation and lineage specification.

Notch activation enhances lineage commitment and protective signaling in cardiac progenitor cells.

Phase I clinical trials applying autologous progenitor cells to treat heart failure have yielded promising results; however, improvement in function is modest, indicating a need to enhance cardiac stem cell reparative capacity. Notch signaling plays a crucial role in cardiac development, guiding cell fate decisions that underlie myocyte and vessel differentiation. The Notch pathway is retained in the adult cardiac stem cell niche, where level and duration of Notch signal influence proliferation and differentiation of cardiac progenitors. In this study, Notch signaling promotes growth, survival and differentiation of cardiac progenitor cells into smooth muscle lineages in vitro. Cardiac progenitor cells expressing tamoxifen-regulated intracellular Notch1 (CPCeK) are significantly larger and proliferate more slowly than control cells, exhibit elevated mTORC1 and Akt signaling, and are resistant to oxidative stress. Vascular smooth muscle and cardiomyocyte markers increase in CPCeK and are augmented further upon ligand-mediated induction of Notch signal. Paracrine signals indicative of growth, survival and differentiation increase with Notch activity, while markers of senescence are decreased. Adoptive transfer of CPCeK into infarcted mouse myocardium enhances preservation of cardiac function and reduces infarct size relative to hearts receiving control cells. Greater capillary density and proportion of vascular smooth muscle tissue in CPCeK-treated hearts indicate improved vascularization. Finally, we report a previously undescribed signaling mechanism whereby Notch activation stimulates CPC growth, survival and differentiation via mTORC1 and paracrine factor expression. Taken together, these findings suggest that regulated Notch activation potentiates the reparative capacity of CPCs in the treatment of cardiac disease.

Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation

Vertebrate heart formation is a spatiotemporally regulated morphogenic process that initiates with bilaterally symmetric cardiac primordial cells migrating toward the midline to form a linear heart tube. The heart tube then elongates and undergoes a series of looping morphogenesis, followed by expansions of regions that are destined to become primitive heart chambers. During the cardiac morphogenesis, cells derived from the first heart field contribute to the primary heart tube, and cells from the secondary heart field, cardiac neural crest, and pro-epicardial organ are added to the heart tube in a precise spatiotemporal manner. The coordinated addition of these cells and the accompanying endocardial cushion morphogenesis yield the atrial, ventricular, and valvular septa, resulting in the formation of a four-chambered heart. Perturbation of progenitor cells' deployment and differentiation leads to a spectrum of congenital heart diseases. Two of the genes that were recently discovered to be involved in cardiac morphogenesis are Numb and Numblike. Numb, an intracellular adaptor protein, distinguishes sibling cell fates by its asymmetric distribution between the two daughter cells and its ability to inhibit Notch signaling. Numb regulates cardiac progenitor cell differentiation in Drosophila and controls heart tube laterality in Zebrafish. In mice, Numb and Numblike, the Numb family proteins (NFPs), function redundantly and have been shown to be essential for epicardial development, cardiac progenitor cell differentiation, outflow tract alignment, atrioventricular septum morphogenesis, myocardial trabeculation, and compaction. In this review, we will summarize the functions of NFPs in cardiac development and discuss potential mechanisms of NFPs in the regulation of cardiac development.

Activation of Cardiogenic Fate of C‐kit+ Cardiac Progenitor Cells in Doxorubicin Induced Cardiotoxicity

Cardiac regeneration is partly regulated by c‐kit+ cardiac progenitor cells (CPC). Doxorubicin (DOX) is an effective anticancer chemotherapeutic which induces cardiotoxicity. To what extent c‐kit+ CPCs are activated in response to DOX treatment and their fate remains unknown. Here, we used the following genetic mouse models to characterize the contribution of c‐kit+ CPCs to cardiac repair mechanisms after DOX; c‐kit Cre‐IRES‐GFP mice to mark all cells that currently express the kit locus, c‐kit MerCreMer(MCM) cross‐bred to R26‐GFP reporter mice to mark CPC derived cells using inducible genetic lineage tracing, and c‐kit MCM cross‐bred to R26‐mT/mG double fluorescent reporter mice to differentiate de novo cardiomyocyte from cell fusion. Two‐month‐old mice were treated with DOX (20 mg/kg cumulative dose). Echocardiography one week after DOX injection showed significantly depressed cardiac function accompanied by cardiomyocyte apoptosis using immunohistochemistry for cleaved caspase 3. Furthermore, DOX activates expression of GATA4 specifically in CPCs. Presumably as a consequence of increased Gata4 expression, genetic lineage tracing of CPCs showed a significant increase (25‐fold) in cardiomyocyte formation, mostly newly formed. Interestingly, this was not at the expense of endothelial fate commitment. In conclusion, DOX enhances c‐kit+ CPC cardiomyocyte commitment and differentiation. We are currently unraveling the underlying molecular pathways to enhance the myogenic potential of c‐kit+ CPCs to repair injured hearts.

Cardiac tissue inhibitor of matrix metalloprotease 4 dictates cardiomyocyte contractility and differentiation of embryonic stem cells into cardiomyocytes: Road to therapy

BackgroundTIMP4 (Tissue Inhibitors of Matrix Metalloprotease 4), goes down in failing hearts and mice lacking TIMP4 show poor regeneration capacity after myocardial infarction (MI). This study is based on our previous observation that administration of cardiac inhibitor of metalloproteinase (~TIMP4) attenuates oxidative stress and remodeling in failing hearts. Therefore, we hypothesize that TIMP4 helps in cardiac regeneration by augmenting contractility and inducing the differentiation of cardiac progenitor cells into cardiomyocytes. MethodsTo validate this hypothesis, we transfected mouse cardiomyocytes with TIMP4 and TIMP4-siRNA and performed contractility studies in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. We evaluated the calcium channel gene serca2a (sarcoplasmic reticulum calcium ATPase2a) and mir122a which tightly regulates serca2a to explain the changes in contractility. We treated mouse embryonic stem cells with cardiac extract and cardiac extract minus TIMP4 (using TIMP4 monoclonal antibody) to examine the effect of TIMP4 on differentiation of cardiac progenitor cells. ResultsContractility was augmented in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. There was elevated expression of serca2a in the TIMP4 transformed myocytes and down regulation of mir122a. The cells treated with cardiac extract containing TIMP4 showed cardiac phenotype in terms of Ckit+, GATA4+ and Nkx2.5 expression. ConclusionThis is a novel report suggesting that TIMP4 augments contractility and induces differentiation of progenitor cells into cardiac phenotype. In view of the failure of MMP9 inhibitors for cardiac therapy, TIMP4 provides an alternative approach, being an indigenous molecule and a natural inhibitor of MMP9.

Atrial natriuretic peptide inhibits cell cycle activity of embryonic cardiac progenitor cells via its NPRA receptor signaling axis.

The biological effects of atrial natriuretic peptide (ANP) are mediated by natriuretic peptide receptors (NPRs), which can either activate guanylyl cyclase (NPRA and NPRB) or inhibit adenylyl cyclase (NPRC) to modulate intracellular cGMP or cAMP, respectively. During cardiac development, ANP serves as an early maker of differentiating atrial and ventricular chamber myocardium. As development proceeds, expression of ANP persists in the atria but declines in the ventricles. Currently, it is not known whether ANP is secreted or the ANP-NPR signaling system plays any active role in the developing ventricles. Thus the primary aims of this study were to 1) examine biological activity of ANP signaling systems in embryonic ventricular myocardium, and 2) determine whether ANP signaling modulates proliferation/differentiation of undifferentiated cardiac progenitor cells (CPCs) and/or cardiomyocytes. Here, we provide evidence that ANP synthesized in embryonic day (E)11.5 ventricular myocytes is actively secreted and processed to its biologically active form. Notably, NPRA and NPRC were detected in E11.5 ventricles and exogenous ANP stimulated production of cGMP in ventricular cell cultures. Furthermore, we showed that exogenous ANP significantly decreased cell number and DNA synthesis of CPCs but not cardiomyocytes and this effect could be reversed by pretreatment with the NPRA receptor-specific inhibitor A71915. ANP treatment also led to a robust increase in nuclear p27 levels in CPCs compared with cardiomyocytes. Collectively, these data provide evidence that in the developing mammalian ventricles ANP plays a local paracrine role in regulating the balance between CPC proliferation and differentiation via NPRA/cGMP-mediated signaling pathways.

Derivation of cardiac progenitor cells from embryonic stem cells.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

Functional cardiomyocytes derived from Isl1 cardiac progenitors via Bmp4 stimulation.

As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs) could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs) could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs) may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP) expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of Isl1 expressing cardiac progenitors, which could lead to enhancement of cardiac differentiation and engraftment, holding a significant therapeutic value for cardiac repair in the future.