Cardiac Mesenchymal Stem Cells Research Articles

Slow H2S-Releasing Donors and 3D Printable Arrays Cellular Models in Osteo-Differentiation of Mesenchymal Stem Cells for Personalized Therapies

The effects of the hydrogen sulfide (H2S) slow-releasing donor, named GSGa, a glutathione-conjugate water-soluble garlic extract, on human mesenchymal stem cells (hMSCs) in both bidimensional (2D) and three-dimensional (3D) cultures were investigated, demonstrating increased expression of the antioxidant enzyme HO-1 and decreased expression of the pro-inflammatory cytokine interleukin-6 (IL-6). The administration of the H2S donor can therefore increase the expression of antioxidant enzymes, which may have potential therapeutic applications in osteoarthritis (OA). Moreover, GSGa was able to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), but not of cardiac mesenchymal stem cells (cMSCs) in a 2D culture system. This result highlights the varying sensitivity of hMSCs to the H2S donor GSGa, suggesting that the induction of osteogenic differentiation in stem cells by chemical factors is dependent on the tissue of origin. Additionally, a 3D-printable mesenchymal stem cells–bone matrix array (MSCBM), designed to closely mimic the stiffness of bone tissue, was developed to serve as a versatile tool for evaluating the effects of drugs and stem cells on bone repair in chronic diseases, such as OA. We demonstrated that the osteogenic differentiation process in cMSCs can be induced just by simulating bone stiffness in a 3D system. The expression of osteocalcin, RUNX2, and antioxidant enzymes was also assessed after treating MSCs with GSGa and/or increasing the stiffness of the culture environment. The printability of the array may enable better customization of the cavities, enabling an accurate replication of real bone defects. This could optimize the BM array to mimic bone defects not only in terms of stiffness, but also in terms of shape. This culture system may enable a rapid screening of antioxidant and anti-inflammatory compounds, facilitating a more personalized approach to regenerative therapy.

Stem cell exosomes in the pathophysiology of cardiovascular diseases

This study focused on the current state of the therapeutic potential of extracellular vesicles, which depends on the methods of their isolation and composition and the characteristics of the vesicular and nonvesicular components. Myocardial damage, particularly as a result of acute myocardial infarction, leads to irreversible death of cardiomyocytes and sarcomeres and ultimately to heart failure. The adult heart has limited regenerative capacity; therefore, stimulation of endogenous repair and regenerative potentials using cell therapy has potential. Moreover, the benefit from the injection of stem cells and progenitor cells into the damaged myocardium is mediated by the factors they secrete. In particular, exosomes, nanosized secreted extracellular vesicles of endosomal origin, have become key signaling organelles in intercellular communication and are currently considered key regenerative components of the secretome of stem and progenitor cells. Exosomes released from cardiac embryonic and mesenchymal stem cells, resident stem and progenitor cells (including a specific subgroup of cardiosphere cells), induced pluripotent stem cells, and cardiomyocytes isolated from these cells have cardioprotective, immunomodulatory, and reparative abilities. The use of exosomes in the targeted transport of drugs in lipid-like nanocontainers and extracellular vesicles is another promising area. Because artificial drug carriers, including liposomes and lipid-based nanoparticles, are limited by potential toxicity, immunogenicity, and inability to target specific organs, exosomes hold good promise as potential drug carriers. Compounds can be transported both inside exosomes and on their surface. Secreted extracellular vesicles, particularly exosomes, can be considered a key functional component of the secretome of stem cells and cardiogenic progenitor cells (mesenchymal stem cells, endogenous cardiac progenitor cells, cardiospheres, bone marrow embryonic stem cells, and bone marrow induced pluripotent stem cells). They have demonstrated therapeutic efficacy in preclinical models in the study of cardiovascular pathology.

Review of electrospun microtube array membrane (MTAM)-a novel new class of hollow fiber for encapsulated cell therapy (ECT) in clinical applications.

Encapsulated cell therapy (ECT) shows significant potential for treating neurodegenerative disorders including Alzheimer's and Parkinson's, which currently lack curative medicines and must be managed symptomatically. This novel technique encapsulates functional cells with a semi-permeable membrane, providing protection while enabling critical nutrients and therapeutic substances to pass through. Traditional ECT procedures, on the other hand, pose difficulties in terms of cell survival and retrieval. We introduce the Microtube Array Membrane (MTAM), a revolutionary technology that solves these constraints, in this comprehensive overview. Microtube Array Membrane has distinct microstructures that improve encapsulated cells' long-term viability by combining the advantages of macro and micron scales. Importantly, the MTAM platform improves biosafety by allowing the entire encapsulated unit to be retrieved in the event of an adverse reaction. Our findings show that MTAM-based ECT has a great potential in a variety of illness situations. For cancer treatment, hybridoma cells secreting anti-CEACAM 6 antibodies inhibit triple-negative breast cancer cell lines for an extended period of time. In animal brain models of Alzheimer's disease, hybridoma cells secreting anti-pTau antibodies successfully reduce pTau buildup, accompanied by improvements in memory performance. In mouse models, MTAM-encapsulated primary cardiac mesenchymal stem cells dramatically improve overall survival and heart function. These findings illustrate the efficacy and adaptability of MTAM-based ECT in addressing major issues such as immunological isolation, cell viability, and patient safety. We provide new possibilities for the treatment of neurodegenerative illnesses and other conditions by combining the potential of ECT with MTAM. Continued research and development in this subject has a lot of promise for developing cell therapy and giving hope to people suffering from chronic diseases.

Abstract GS.102: Cd206 + Il-4rα + Macrophages Are Drivers Of Adverse Cardiac Remodeling In Ischemic Cardiomyopathy

Background: Cardiac macrophages are significantly expanded in chronic heart failure (HF); however, their role is unclear. We tested the hypothesis that CD206 + macrophages expressing interleukin (IL)-4Rα in the failing heart are key drivers of adverse left ventricular (LV) remodeling and ischemic cardiomyopathy. Methods and Results: Adult male C57BL/6 (WT) mice underwent non-reperfused myocardial infarction (MI) to induce HF, or sham operation, and cardiac macrophages were profiled using flow cytometry. As compared to sham hearts, both the frequency and absolute number of CD206 + macrophages steadily increased in post-MI hearts during the progression of LV remodeling and HF, such that at 8 w post-MI they comprised ~85% of macrophages. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2) - with nearly half expressing IL-4Rα, and correlated with LV dysfunction and fibrosis. IL-4-polarized bone marrow derived CD206 + macrophages exhibited marked upregulation of found in inflammatory zone (FIZZ)1 and induced FIZZ1-dependent myofibroblast differentiation of cardiac mesenchymal stem cells (cMSCs), in part related to DLL-4/Jagged1-Notch1 signaling. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], macrophages induced progressive LV dilation and dysfunction and augmented cardiac fibrosis over 4 weeks in naïve mice. In vivo IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206 + IL-4Rα + macrophages and induced reverse LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressing local and systemic inflammation. Lastly, alternatively activated CD206 + and CD163 + macrophages were significantly expanded in human failing hearts and correlated with cardiac fibrosis, with the majority of CD163 + macrophages expressing IL-4Rα and FIZZ3, the human homolog of FIZZ1. Conclusion: CD206 + IL-4Rα + macrophages proliferate and expand in the failing heart and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206 + macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.

Electrical Stimulation Increases the Secretion of Cardioprotective Extracellular Vesicles from Cardiac Mesenchymal Stem Cells.

Clinical trials have shown that electric stimulation (ELSM) using either cardiac resynchronization therapy (CRT) or cardiac contractility modulation (CCM) approaches is an effective treatment for patients with moderate to severe heart failure, but the mechanisms are incompletely understood. Extracellular vesicles (EV) produced by cardiac mesenchymal stem cells (C-MSC) have been reported to be cardioprotective through cell-to-cell communication. In this study, we investigated the effects of ELSM stimulation on EV secretion from C-MSCs (C-MSCELSM). We observed enhanced EV-dependent cardioprotection conferred by conditioned medium (CM) from C-MSCELSM compared to that from non-stimulated control C-MSC (C-MSCCtrl). To investigate the mechanisms of ELSM-stimulated EV secretion, we examined the protein levels of neutral sphingomyelinase 2 (nSMase2), a key enzyme of the endosomal sorting complex required for EV biosynthesis. We detected a time-dependent increase in nSMase2 protein levels in C-MSCELSM compared to C-MSCCtrl. Knockdown of nSMase2 in C-MSC by siRNA significantly reduced EV secretion in C-MSCELSM and attenuated the cardioprotective effect of CM from C-MSCELSM in HL-1 cells. Taken together, our results suggest that ELSM-mediated increases in EV secretion from C-MSC enhance the cardioprotective effects of C-MSC through an EV-dependent mechanism involving nSMase2.

Abstract 14940: Neonatal Cardiac Mesenchymal Stem Cells Target ERK/MAPK Signaling Pathway to Ameliorate Oxidative Stress and Inflammation in Acute Kidney Injury

Introduction: Failure of multidrug therapy and the multifactorial nature of kidney injury has paved the path way regenerative medicine with over 45 clinical trials using stem cells-based therapy underway. Neonatal cardiac mesenchymal stem cells (nMSC) are one of the most potent stem cells due to their secretome. HYPOTHESIS: SOD2 and anti-inflammatory miRNAs (miR-214 & 95p) in paracrine secretions (secretome) of nMSC provides renoprotection in a rodent model of glycerol-induced AKI. Methods: nMSC were generated from neonatal myocardium using enzymatic digestion and antibodies-based selection. Secretome was collected by conditioning nMSCs for 72 hours in serum free basal medium. Human kidney cells (HKC) were used for in vitro anti-oxidation assays using cisplatin. THP-1 cells were used for anti-inflammatory assessment. CD1 mice were used for glycerol-induced AKI model. Mice were subjected to AKI via IM glycerol (9mg/kg). nMSC-derived secretome was intravenously administered immediately after, or 4 hours post-glycerol. Blood urea nitrogen (BUN) and creatinine were analyzed in serum. Cell survival/KIM1 was assessed by immunohistology/FACS. Other experiments utilized cisplatin toxicity in HKC. Results: Administration of nMSC secretome (5 or 10mg/kg) at the same time or 4-hours post-glycerol administration significantly reduced serum creatinine and BUN in a glycerol induced AKI animal model. Caspase-9 and KIM1 expression was significantly decreased in tubular cells as compared to placebo at a dose of 10mg/kg. KIM-1 was significantly downregulated as compared to placebo following nMSC-secretome administration. Western blot analysis of HKC treated with cisplatin in presence of nMSC-secretome showed significant reduction in NFkb and pERK expression (p<0.05) at 200 μg/ml of nMSC-secretome. IL-8 secretion from macrophages was significantly decreased by nMSC-secretome. Data showing the role of miR-210 in renoprotection will also be shown. Conclusion: nMSC and nMSC-derived secretome provide a renoprotective effect in the setting of glycerol-induced AKI. The mechanism of this effect is driven, at least in part, by a reduction in NfκB and pERK related signaling.

Abstract 335: Hsf1 Regulates Exosome Biogenesis And Cargo Enrichment Of These Exosomes In Stem Cells Thereby Enhancing Their Potential Of Ischemic Heart Regeneration.

Introduction: Paracrine mechanism of stem cell-based therapy holds importance. However, genes regulating the paracrine factors have not been elucidated in detail. Here in this study, we evaluated the role of HSF1 in exosome biogenesis and their composition in neonatal cardiac mesenchymal stem cells (nMSCs) in concert with ischemic heart generation. Hypothesis: HSF1 in nMSCs promotes production of exosomes, functional exosomal miRs cargo, and exosome acquisition by recipient cells. Methodology: nMSCs and adult MSCs (aMSCs) were respectively isolated from right atrial appendage of neonate (<1month) and adult ( > 40 years) patients. nMSCs were knocked down (KD) for HSF1 gene using CRISPR-CAS9 technique while HSF1 gene was overexpressed (OE) in aMSCs using HSF1 overexpressing lentiviral constructs. Exosome biogenesis was evaluated using nano site technique. We next evaluated effect of HSF1 KD and OE on miRs cargo in nMSCs and aMSCs respectively. Exosome acquisition by target cells was studied by mCherry labeling technique. The potential of nMSCs and aMSCs and their corresponding exosomes to repair rat ischemic heart was evaluated under the effect of HSF1 KD and OE. Results: HSF1-KD in nMSCs resulted in significant reduction in production of exosomes, downregulation of ESCRT complex genes and therapeutic miRs viz, miR-199a, mi590-3P and miR-146. In contrast, HSF-1 over expression in aMSCs statistically increased production of exosomes, ESCRT complex genes and of therapeutic miRs. Moreover, HSF-1 OE in aMSCs resulted in statistical downregulation of miR-122, known to induce apoptosis, and downregulation of p27, a known target of miR-590. We identified that potential of nMSCs to repair ischemic heart decreased statistically after HSF1-KD while that of aMSCs increased significantly after HSF1-OE. Finally, we identified exosomes from these stem cells were preferentially taken up by cells in injured myocardium and not by cells in non-injured one and that HSF1-OE increased the uptake by recipient cells while HSF1-KD had opposite effect. Conclusion: Together, our results demonstrate that HSF-1 plays an important role in exosome biogenesis, miRs cargo enrichment and uptake of exosomes by recipient cells.

Abstract 15471: Neonatal Mesenchymal Stem Cells Rejuvenate Cardiac Myocardium by Regulating Macrophage Polarization Revealed by Single Cell Sequencing After Ischemia-Reperfusion Injury in Rats

Introduction: Stem cells are an emerging therapy for cardiovascular disease. The immune system is modulated by stem cell administration, therefore we sought to characterize the immune environment following stem cell administration. Hypothesis: Neonatal cardiac mesenchymal stem cells (nMSC) improved heart function through an acute sterile immune response characterized by the temporal and regional of macrophages. Methods: nMSC were isolated from healthy neonatal myocardium. Brown Norway rats underwent ischemia-reperfusion injury (IRI) by temporary ligation of left anterior descending artery for 45 minutes. Following closure of the chest cavity, neonatal mesenchymal stem cells (nMSCs), dead nMSCs (dnMSCs), or placebo was administered by intra-coronary injection. At day five heart was collected for single cell suspensions. CD45 positive selection was performed and fed into the Chromium Single Cell Gene Expression workflow (Pleasanton, CA). Single cell sequencing analysis was completed using the Seurat package on RStudio v1.3.1093 (Boston, MA). Echocardiography was completed on post-operative days 7 and 28 for rats not sacrificed for single cell sequencing. Results: Our results demonstrate that nMSC treatment after IRI injury can significantly recover left ventricular ejection fraction. Single-cell sequencing analysis of CD45+ cells identified multiple unique CD68+ clusters. Notably, macrophage cluster 2 primarily consisted of cells from the nMSC treated group, whereas the macrophage cluster 1 was primarily cells from the dnMSC and placebo groups. Gene ontology analysis of macrophage cluster 2 demonstrated that this cluster primarily consisted of macrophages strongly associated with various aspects of the activated immune response, including lymphocyte activation and cytokine production. Conversely, macrophage cluster 1 gene ontology analysis revealed a weaker immune response signature. Conclusions: nMSCs are a promising therapeutic candidate for the treatment of myocardial infarction. The mechanism of this therapy is likely related to macrophage activation that further enhances the immune response in the impacted and at-risk myocardium following IRI injury.

Exosomes and Exosomal Cargos: A Promising World for Ventricular Remodeling Following Myocardial Infarction.

Exosomes are a pluripotent group of extracellular nanovesicles secreted by all cells that mediate intercellular communications. The effective information within exosomes is primarily reflected in exosomal cargos, including proteins, lipids, DNAs, and non-coding RNAs (ncRNAs), the most intensively studied molecules. Cardiac resident cells (cardiomyocytes, fibroblasts, and endothelial cells) and foreign cells (infiltrated immune cells, cardiac progenitor cells, cardiosphere-derived cells, and mesenchymal stem cells) are involved in the progress of ventricular remodeling (VR) following myocardial infarction (MI) via transferring exosomes into target cells. Here, we summarize the pathological mechanisms of VR following MI, including cardiac myocyte hypertrophy, cardiac fibrosis, inflammation, pyroptosis, apoptosis, autophagy, angiogenesis, and metabolic disorders, and the roles of exosomal cargos in these processes, with a focus on proteins and ncRNAs. Continued research in this field reveals a novel diagnostic and therapeutic strategy for VR.

Regulation of Endothelial Progenitor Cell Functions in Ischemic Heart Disease: New Therapeutic Targets for Cardiac Remodeling and Repair.

Ischemic heart disease (IHD) is the leading cause of morbidity and mortality worldwide. Ischemia and hypoxia following myocardial infarction (MI) cause subsequent cardiomyocyte (CM) loss, cardiac remodeling, and heart failure. Endothelial progenitor cells (EPCs) are involved in vasculogenesis, angiogenesis and paracrine effects and thus have important clinical value in alternative processes for repairing damaged hearts. In fact, this study showed that the endogenous repair of EPCs may not be limited to a single cell type. EPC interactions with cardiac cell populations and mesenchymal stem cells (MSCs) in ischemic heart disease can attenuate cardiac inflammation and oxidative stress in a microenvironment, regulate cell survival and apoptosis, nourish CMs, enhance mature neovascularization, alleviate adverse ventricular remodeling after infarction and enhance ventricular function. In this review, we introduce the definition and discuss the origin and biological characteristics of EPCs and summarize the mechanisms of EPC recruitment in ischemic heart disease. We focus on the crosstalk between EPCs and endothelial cells (ECs), smooth muscle cells (SMCs), CMs, cardiac fibroblasts (CFs), cardiac progenitor cells (CPCs), and MSCs during cardiac remodeling and repair. Finally, we discuss the translation of EPC therapy to the clinic and treatment strategies.

Cardiac Mesenchymal Stem Cells Promote Fibrosis and Remodeling in Heart Failure: Role of PDGF Signaling

Heart failure (HF) is characterized by progressive fibrosis. Both fibroblasts and mesenchymal stem cells (MSCs) can differentiate into pro-fibrotic myofibroblasts. MSCs secrete and express platelet-derived growth factor (PDGF) and its receptors. We hypothesized that PDGF signaling in cardiac MSCs (cMSCs) promotes their myofibroblast differentiation and aggravates post-myocardial infarction left ventricular remodeling and fibrosis. We show that cMSCs from failing hearts post-myocardial infarction exhibit an altered phenotype. Inhibition of PDGF signaling invitro inhibited cMSC-myofibroblast differentiation, whereas invivo inhibition during established ischemic HF alleviated left ventricular remodeling and function, and decreased myocardial fibrosis, hypertrophy, and inflammation. Modulating cMSC PDGF receptor expression may thus represent a novel approach to limit pathologic cardiac fibrosis in HF.

MBNL1 drives dynamic transitions between fibroblasts and myofibroblasts in cardiac wound healing.

Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response. Because transcriptome maturation by muscleblind-like 1 (MBNL1) promotes differentiated cell states, this study investigated whether tactical control of MBNL1 activity could alter myofibroblast activity and fibrotic outcomes. In healthy mice, cardiac fibroblast-specific overexpression of MBNL1 transitioned the fibroblast transcriptome to that of a myofibroblast and after injury promoted myocyte remodeling and scar maturation. Both fibroblast- and myofibroblast-specific loss of MBNL1 limited scar production and stabilization, which was ascribed to negligible myofibroblast activity. The combination of MBNL1 deletion and injury caused quiescent fibroblasts to expand and adopt features of cardiac mesenchymal stem cells, whereas transgenic MBNL1 expression blocked fibroblast proliferation and drove the population into a mature myofibroblast state. These data suggest MBNL1 is a post-transcriptional switch, controlling fibroblast state plasticity during cardiac wound healing.

Tumor necrosis factor alpha induces platelet‐derived growth factor receptor expression in cardiac mesenchymal stem cells that guides their myofibroblast differentiation

Heart failure (HF) is a global health problem. Analogous to inflammation, failing hearts also exhibit progressive fibrosis. Stem cells hold significant promise for ischemic heart diseases. However, there has not been clinical success so far. Understanding the effect of failing myocardium on stem cells is thus essential for devising efficient cell-based therapies. In addition to fibroblasts, mesenchymal stem cells (MSCs) can also differentiate into pro-fibrotic myofibroblasts. Platelet derived growth factors (PDGF) and PDGF-receptors (PDGFRs) are known regulators of fibrosis. We have discovered that cardiac MSCs (cMSCs) from failing hearts exhibit increased PDGFRβ (Rβ) expression and PDGFR-dependent myofibroblast differentiation. We hypothesize that inflammatory microenvironment in failing hearts promotes cMSC-Rβ expression and their subsequent myofibroblast differentiation. Wild type mouse cMSCs (Sca1+CD31-DDR2-) when exposed to pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) significantly increased their Rβ-expression that is mediated by JNK and Erk MAPK-activation but not NF-kB activation. More importantly, TNF-induced Rβ-expression in cMSCs is independent of Rβ-promoter transactivation. Evaluation of Rβ-promoter identified a putative CpG island which when deleted significantly increased basal Rβ-promoter activity suggesting role of DNA-methylation in modulating TNF-induced Rβ-expression in cMSCs. DNA-methylation is known to correlate directly with promotor activation and gene expression. Indeed, compared to HF cMSCs (with increased Rβ-expression), CpG island within Rβ-promoter in sham cMSCs (with decreased Rβ-expression) is predominantly methylated, and acute TNF exposure (24h) promotes its de-methylation that correlates with increased Rβ-expression. We conclude that regulating cMSC-localized Rβ expression could therapeutically be used to prevent cMSC-myofibroblast differentiation and attenuate overt fibrotic responses in HF.

Cardiac Mesenchymal Stem Cells Promote Fibrosis and Remodeling in Heart Failure: Role of PDGF Signaling

Objective: To characterize cardiac mesenchymal stem cells (cMSCs) and define the role of platelet derived growth factor receptor (PDGFR) signaling on cMSC-myofibroblast differentiation. To evaluate the in vivo effects of PDGFR inhibition on the outcomes of post-myocardial infraction (MI) cardiac remodeling, function and inflammation. Background: Heart failure (HF) is characterized by chronic inflammation, fibrosis, and activation of pro-fibrotic myofibroblasts. PDGF is a known regulator of fibrosis while PDGFR is a marker of MSCs. In addition to fibroblasts, tissue MSCs also differentiate into myofibroblasts; however, the contribution of cMSCs and cMSC-localized PDGFR to fibrosis in the failing heart is unknown. Methods: Flow cytometry was used to quantitate cMSCs in failing hearts. Multiple in vitro experiments were used to characterize cMSCs. Permanent coronary ligation in mice was used to induce myocardial infarction and failure while echocardiography was used asses cardiac function. Results: We demonstrate that; 1) cMSCs temporally expand post-MI and acquire a pro-inflammatory and a pro-fibrotic cell fate, 2) pro-inflammatory macrophages promote a PDGFR-dependent pro-fibrotic myofibroblast differentiation fate specifically in HF-derived cMSCs, and that, 3) inhibition of PDGFR signaling in vivo attenuates post-MI LV remodeling and function. These in vivo effects occurred concomitantly with decreased myocardial fibrosis, myocyte hypertrophy and reduced myocardial infiltration of pro-inflammatory macrophages. Conclusion: Our findings suggest that restoring or maintaining a normal cMSC phenotype by modulating PDGFR expression and/or activation may represent a novel approach for both combating pathological fibrosis in the failing heart as well as potentially enhancing cardiac repair upon exogenous cMSC transplantation in HF.

Abstract 16827: miR-34a Expression in Cardiac Neonatal Mesenchymal Stem Cells is Essential for Heart Regeneration

Advancing chronological age negatively impacts the functional activity of stem cell-based therapies. Despite the early promise stem cell mediated differentiation into selected cellular lineages when injected in the chosen organ, stem cell’s functional unit is the secretome that is directly modulated by chronological aging. However, the precise mechanism driving the secretome potency is unknown. Here we show a single miRNA, influenced by aging, drives the potency of the stem cell secretome at multiple cellular levels by increasing exosome production and directly increasing the levels of independent stem cell paracrine factors. We show differential miRNA microarray analysis of cardiac neonatal mesenchymal stem cells (nMSCs) and adult MSCs (aMSCs) identified miR34a among the top ten differentially expressed miRNAs present in aMSCs but not nMSCs. We demonstrated that miR34a singly inhibits the aMSC’s regenerative abilities in the acute myocardial infarct model by decreasing the secretome potency when compared to nMSCs in knockdown studies of miR-34a. Moreover, overexpression of miR-34a in nMSCs reversed the strong regenerative properties of nMSCs by decreasing their secretome potency. Mechanistically, miR-34a regulates the secretome potency firstly by decreasing Heat Shock Factor 1 (HSF1) levels that directly decreases exosome production and alters the exosome cargo to a less-regenerative phenotype, and secondly by directly inhibiting transcriptional levels of critical stem cell paracrine factors. These results implicate the miR34a-HIF1 pathway as a critical pathway controlling the secretome of MSCs impacted by advancing chronological age. Our findings further advance miRNA-based therapeutic approaches to cardiac repair and heart regeneration by indicating how to improve the quality of secretome of transplanted stem cells.

Abstract 16596: Immune Evasion by Human Neonatal Cardiac Mesenchymal Stem Cells Demonstrates Therapeutic Application in Myocardial Infarction

Immune rejection of transplanted stem cells is a major stumbling block in designing effective therapy for myocardial infarction. Human neonatal cardiac mesenchymal stem cells (nMSCs) showed superior cardiac functional recovery compared to adult MSCs in immune competent rat MI model. However, molecular mechanisms underlying immune evasion by transplanted nMSCs in the infarcted myocardium remain unexplored. In this investigation, we demonstrate for the first-time the expression, regulation and function of CD47 in human nMSCs and its novel mechanism of immune evasion increases its regenerative potential in rat MI model. Transplanted nMSCs showed significant increased cell retention, reduced phagocytosis and CD68 + cells compared to aMSCs in rat MI model. Comparative proteomic analysis by LC-MS/MS on nMSCs and aMSCs showed that CD47 higher in nMSCs. Increased CD47 expression in nMSCs inhibited phagocytosis compared to aMSCs in vitro and in vivo . Further, CD47 blockade in nMSCs using anti-CD47, siRNA and shRNA lentiviral based approaches increased in vitro , in vivo phagocytosis, CD68 + cells and reduced cell retention and MI recovery in vivo . Microarray analysis and validation showed miR-34a was significantly higher in aMSCs than nMSCs. To unravel CD47 regulation in aMSCs, we performed target scan analysis that predicted miR-34a binds on CD47. Further, miR-34a over expression in nMSCs reduced CD47 expression, increased in vitro and in vivo phagocytosis and reduced cell retention and MI recovery. In conclusion, increased CD47 expression in nMSCs inhibit phagocytosis by CD47/SIRPα immune regulatory axis to demonstrates its immune evasion potential and its therapeutic applications in myocardial infarction.

Abstract 17093: Human Neonatal Cardiac Mesenchymal Stem Cells Demonstrates Superior Cardiac Regenerative Abilities Compared to Other Stem Cells

Stem cells transplantation is being explored as an effective therapy for heart diseases. However, majority of stem cell therapies for adult patients with myocardial infarction (MI) had mixed and inconsistent results implying chronological age may influence the effectiveness of regenerative therapies. Therefore, herein, we performed a head-to-head comparison between different, well-studied stem cell types to identify the superior regenerative cell type using rodent MI model.After our standard characterization for each stem cell type (FACS for cell surface markers), 1 million neonatal Cardiac Mesenchymal Stem cells (nMSCs), adult MSCs (aMSCs), adult derived cardiosphere derived cells (aCDCs), umbilical cord derived cells (UCBCs), Bone Marrow derived Mesenchymal Stem cells (BM-MSCs), or cell-free Iscove Modified Dulbecco Medium (IMDM as placebo control) were injected into athymic rat myocardial infarct model. Although all the tested groups significantly improved ejection fraction, nMSCs outperformed other stem cells in cardiac functional recovery. Additionally, nMSCs also showed significant increased cardiac functional recovery compared to aMSCs in wild type rat MI model. Mason trichrome staining with heart sections revealed that decreased fibrosis was evident on nMSCs injection compared to aMSCs in both athymic and wild type rat MI model. Myocardial sections from rats received nMSCs showed significantly reduced M1 macrophages (inflammatory) and increased M2 macrophages (anti-inflammatory) compared with sections from rats having received aMSCs and IMDM control. Pro and anti-inflammatory cytokines analyzed on sera collected on day 2 and 7 revealed that anti-inflammatory cytokine (IL10) was significantly increased and inflammatory cytokines (IL4 and IL12) reduced in nMSCs compared to aMSCs transplanted MI rat model.In conclusion, nMSCs demonstrated superior functional abilities, reduced fibrosis, inflammatory cells and cytokines compared to all the other cell types and with aMSCs demonstrating that nMSCs is an ideal stem cell type for therapeutic application in myocardial infarction.

Abstract 16769: Intravenous Injection of Neonatal Cardiac Mesenchymal Stem Cells is a Viable Alternative Route to Treat Acute Myocardial Infarction

Introduction: Cardiovascular disease is major cause of morbidity and mortality around the world and major health care burden indeed. Ischemic heart disease (IHD) and myocardial ischemia (MI) are most devastating cardiovascular disease. Multiple stem cell/ cardiac progenitor cell therapy has been reported previously to treat cardiovascular disease safely. However randomized clinical trials with adult cardiac progenitor cells or cardiosphere-derived cells unable to show long-term efficacy. We have our unique source of human neonatal cardiac tissue derived neonatal cardiac mesenchymal stem cells (nMSCs). Systemic administration is preferred route of stem cell delivery in order to consecutive dosage for most of the clinical trials. We hypothesized that nMSCs have unique proteome profile, which supports their survival, migration and homing. They home to the injured myocardium when administered intravenously (IV) to a wild type male rat subjected to MI. Methods: This model was created in 6-week-old Brown-Norway Rats. Rats were subjected to an anterior myocardial Infraction (MI) by permanent LAD ligation. The rats were treated with nMSCs (1^10 6 cells/Kg, 5^10 6 cells/Kg and 10^10 6 cells/Kg) along with placebo and sham, which are delivered intravenously by tail vein injection. Rats are once again treated with nMSCs/placebo 4 days after MI. Baseline echocardiography is performed 24 hours after MI. Results: LVEF was significantly higher in the nMSC-treated group than in the placebo group. Other parameters, including fractional shortening (FS) and decreased end-systolic volume (ESV), were also significantly improved when compared with the placebo group, and other LV functional parameter, including cardiac output/body weight and posterior wall thickness tended towards improvement of remodeled heart. Conclusions: Twice intravenous administration of nMSCs for MI attenuate the progressive deterioration of left ventricle and adverse remodeling of rat heart.

Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates.

The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1β (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1β or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.

GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L-mediated OPA1 processing.

Growth differentiation factor 11 (GDF11) has been shown to promote stem cell activity, but little is known about the effect of GDF11 on viability and therapeutic efficacy of cardiac mesenchymal stem cells (MSCs) for cardiac injury. To understand the roles of GDF11 in MSCs, mouse heart‐derived MSCs were transduced with lentiviral vector carrying genes for both GDF11 and green fluorescent protein (GFP) (MSCsLV‐GDF11) or cultured with recombinant GDF11 (MSCsrGDF11). Either MSCsrGDF11 or MSCs LV‐GDF11 displayed less cell apoptosis and better paracrine function, as well as preserved mitochondrial morphology and function under hypoxic condition as compared with control MSCs. GDF11 enhanced phosphorylation of Smad2/3, which upregulated expression of YME1L, a mitochondria protease that balances OPA1 processing. Inhibitors of TGF‐β receptor (SB431542) or Smad2/3 (SIS3) attenuated the effects of GDF11 on cell viability, mitochondrial function, and expression of YME1L. Transplantation of MSCsGDF11 into infarct heart resulted in improved cell survival and retention, leading to more angiogenesis, smaller scar size, and better cardiac function in comparison with control MSCs. GDF11 enhanced viability and therapeutic efficiency of MSCs by promoting mitochondrial fusion through TGF‐β receptor/Smad2/3/YME1L‐OPA1 signaling pathway. This novel role of GDF11 may be used for a new approach of stem cell therapy for myocardial infarction.