Comparative proteomic analysis of nuclear and cytoplasmic compartments in human cardiac progenitor cells

Clinical trials evaluating cardiac progenitor cells (CPC) demonstrated feasibility and safety, but no clear functional benefits. Therefore a deeper understanding of CPC biology is warranted to inform strategies capable to enhance their therapeutic potential. Here we have defined, using a label-free proteomic approach, the differential cytoplasmic and nuclear compartments of human CPC (hCPC). Global analysis of cytoplasmic repertoire in hCPC suggested an important hypoxia response capacity and active collagen metabolism. In addition, comparative analysis of the nuclear protein compartment identified a significant regulation of a small number of proteins in hCPC versus human mesenchymal stem cells (hMSC). Two proteins significantly upregulated in the hCPC nuclear compartment, IL1A and IMP3, showed also a parallel increase in mRNA expression in hCPC versus hMSC, and were studied further. IL1A, subjected to an important post-transcriptional regulation, was demonstrated to act as a dual-function cytokine with a plausible role in apoptosis regulation. The knockdown of the mRNA binding protein (IMP3) did not negatively impact hCPC viability, but reduced their proliferation and migration capacity. Analysis of a panel of putative candidate genes identified HMGA2 and PTPRF as IMP3 targets in hCPC. Therefore, they are potentially involved in hCPC proliferation/migration regulation.

Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

Intracoronary Delivery of Porcine Cardiac Progenitor Cells Overexpressing IGF-1 and HGF in a Pig Model of Sub-Acute Myocardial Infarction

Human cardiac progenitor cells (hCPC) are considered a good candidate in cell therapy for ischemic heart disease, demonstrating capacity to improve functional recovery after myocardial infarction (MI), both in small and large preclinical animal models. However, improvements are required in terms of cell engraftment and efficacy. Based on previously published reports, insulin-growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) have demonstrated substantial cardioprotective, repair and regeneration activities, so they are good candidates to be evaluated in large animal model of MI. We have validated porcine cardiac progenitor cells (pCPC) and lentiviral vectors to overexpress IGF-1 (co-expressing eGFP) and HGF (co-expressing mCherry). pCPC were transduced and IGF1-eGFPpos and HGF-mCherrypos populations were purified by cell sorting and further expanded. Overexpression of IGF-1 has a limited impact on pCPC expression profile, whereas results indicated that pCPC-HGF-mCherry cultures could be counter selecting high expresser cells. In addition, pCPC-IGF1-eGFP showed a higher cardiogenic response, evaluated in co-cultures with decellularized extracellular matrix, compared with native pCPC or pCPC-HGF-mCherry. In vivo intracoronary co-administration of pCPC-IGF1-eGFP and pCPC-HFG-mCherry (1:1; 40 × 106/animal), one week after the induction of an MI model in swine, revealed no significant improvement in cardiac function.

Strong as a Hippo’s Heart: Biomechanical Hippo Signaling During Zebrafish Cardiac Development

The heart is comprised of multiple tissues that contribute to its physiological functions. During development, the growth of myocardium and endocardium is coupled and morphogenetic processes within these separate tissue layers are integrated. Here, we discuss the roles of mechanosensitive Hippo signaling in growth and morphogenesis of the zebrafish heart. Hippo signaling is involved in defining numbers of cardiac progenitor cells derived from the secondary heart field, in restricting the growth of the epicardium, and in guiding trabeculation and outflow tract formation. Recent work also shows that myocardial chamber dimensions serve as a blueprint for Hippo signaling-dependent growth of the endocardium. Evidently, Hippo pathway components act at the crossroads of various signaling pathways involved in embryonic zebrafish heart development. Elucidating how biomechanical Hippo signaling guides heart morphogenesis has direct implications for our understanding of cardiac physiology and pathophysiology.

Imatinib mesylate induces necroptotic cell death and impairs autophagic flux in human cardiac progenitor cells

The receptor tyrosine kinase inhibitor imatinib mesylate has improved patient cancer survival rates but has been linked to long-term cardiotoxicity. This study investigated the effects of imatinib on cell viability, apoptosis, autophagy and necroptosis in human cardiac progenitor cells in vitro. After 24 hours, imatinib significantly reduced cell viability (75.9±2.7% vs. 100.0±0.0%, n=5, p<0.05) at concentrations comparable to peak plasma levels (10 μM). Further investigation showed no increase in caspase 3 or 7 activation. Imatinib also significantly reduced the fluorescence of cells stained with TMRM (74.6±6.5% vs. 100.0±0.0%, n=5, p<0.05), consistent with mitochondrial depolarization. Imatinib increased lysosome and autophagosome content relative to the control, as indicated by changes in acridine orange fluorescence (46.0±5.4% vs. 9.0±3.0, n=7, p<0.001) and expression of LAMP2 (2.4±0.3 fold, n=3, p<0.05) after 24 hours treatment. Although imatinib increased the expression of proteins associated with autophagy, it also impaired the autophagic flux, as demonstrated by the proximity ligation assay staining for LAMP2 (lysosome marker) and LC3II (autophagosome marker), with control cells showing 11.3±2.1 puncta per cell and 48 hours of imatinib treatment reducing the visible puncta to 2.7±0.7 per cell (n=10, p<0.05). Cell viability was partially recovered by autophagosome inhibition by wortmannin, with a 91.8±8.2% (n=5, p>0.05) increase in viability after imatinib and wortmannin co-treatment. Imatinib-induced necroptosis was associated with an 8.5±2.5-fold increase in activation of mixed lineage kinase domain-like pseudokinase. Imatinib-induced toxicity was rescued by RIP1 inhibition relative to the control; 88.6±3.0% vs. 100.0±0.0% (n=4, p>0.05). In summary, imatinib applied to human cardiac progenitor cells depolarizes mitochondria and induces cell death through necroptosis, which can be recovered by inhibition of RIP1, with an additional partial role for autophagy in the cell death pathway. These data provide two possible targets for co-therapies to address imatinib-induced long-term cardiotoxicity.

Silk-based Matrices and C-kit Positive Cardiac Progenitor Cells for a Cardiac Organoid: Study of an in Vivo Model

In recent years, there has been a shift from tissue engineering to the production of organoids. The latter are useful tools to study many biochemical aspects and cellular reactions while avoiding the excessive use of laboratory animals. Organoids are very interesting tools because they can replicate the cellular and extracellular environment of an organ and retain some of the properties of the organ itself. However, without an adequate network of vessels, cell masses not only fail to grow, but they may exhibit an area of necrosis, indicating a lack of oxygen and nutrients. For this reason, scientific researchers are looking for ways to create organoids that can also mimic the vascular network of the organ from which they originate. One possibility is to implant the organoids in immunocompromised animals.In the present study, we generated cardiac organoids ex vivo by seeding tyrosine protein kinase kit (c-kit)-positive cardiac progenitor cells (CPC cells) from fresh rat hearts into a rat collagen I gel. We then implanted these patches into immunosuppressed animals and compared the suitability of different silk fibroin scaffolds with three different geometries. We demonstrated that CPC cells were destroyed by CD3+ lymphocytes, that the porous and partially oriented scaffolds induced a consistent foreign body response compared to the electrospun meshes, and that CPC cells were degraded by a T-cell-mediated immune response, although the latter may be suitable for generating rat cardiac organoids.