Isolation, Expansion, and Endoscopic Delivery of Autologous Enteric Neuronal Stem Cells in Swine.

The enteric nervous system (ENS) is an extensive network of neurons and glia within the wall of the gastrointestinal (GI) tract that regulates many essential GI functions. Consequently, disorders of the ENS due to developmental defects, inflammation, infection, or age-associated neurodegeneration lead to serious neurointestinal diseases. Despite the prevalence and severity of these diseases, effective treatments are lacking as they fail to directly address the underlying pathology. Neuronal stem cell therapy represents a promising approach to treating diseases of the ENS by replacing the absent or injured neurons, and an autologous source of stem cells would be optimal by obviating the need for immunosuppression. We utilized the swine model to address key questions concerning cell isolation, delivery, engraftment, and fate in a large animal relevant to human therapy. We successfully isolated neural stem cells from a segment of small intestine resected from 1-month-old swine. Enteric neuronal stem cells (ENSCs) were expanded as neurospheres that grew optimally in low-oxygen (5%) culture conditions. Enteric neuronal stem cells were labeled by lentiviral green fluorescent protein (GFP) transduction, then transplanted into the same swine from which they had been harvested. Endoscopic ultrasound was then utilized to deliver the ENSCs (10,000-30,000 neurospheres per animal) into the rectal wall. At 10 and 28 days following injection, autologously derived ENSCs were found to have engrafted within rectal wall, with neuroglial differentiation and no evidence of ectopic spreading. These findings strongly support the feasibility of autologous cell isolation and delivery using a clinically useful and minimally invasive technique, bringing us closer to first-in-human ENSC therapy for neurointestinal diseases.

Transplanted ENSCs form functional connections with intestinal smooth muscle and restore colonic motility in nNOS-deficient mice.

BACKGROUND: Enteric neuropathies, which result from abnormalities of the enteric nervous system, are associated with significant morbidity and high health-care costs, but current treatments are unsatisfactory. Cell-based therapy offers an innovative approach to replace the absent or abnormal enteric neurons and thereby restore gut function. METHODS: Enteric neuronal stem cells (ENSCs) were isolated from the gastrointestinal tract of Wnt1-Cre;R26tdTomato mice and generated neurospheres (NS). NS transplants were performed via injection into the mid-colon mesenchyme of nNOS-/- mouse, a model of colonic dysmotility, using either 1 (n = 12) or 3 (n = 12) injections (30 NS per injection) targeted longitudinally 1-2 mm apart. Functional outcomes were assessed up to 6 weeks later using electromyography (EMG), electrical field stimulation (EFS), optogenetics, and by measuring colorectal motility. RESULTS: Transplanted ENSCs formed nitrergic neurons in the nNOS-/- recipient colon. Multiple injections of ENSCs resulted in a significantly larger area of coverage compared to single injection alone and were associated with a marked improvement in colonic function, demonstrated by (1) increased colonic muscle activity by EMG recording, (2) faster rectal bead expulsion, and (3) increased fecal pellet output in vivo. Organ bath studies revealed direct neuromuscular communication by optogenetic stimulation of channelrhodopsin-expressing ENSCs and restoration of smooth muscle relaxation in response to EFS. CONCLUSIONS: These results demonstrate that transplanted ENSCs can form effective neuromuscular connections and improve colonic motor function in a model of colonic dysmotility, and additionally reveal that multiple sites of cell delivery led to an improved response, paving the way for optimized clinical trial design.

Cell therapy attenuates endothelial dysfunction in hypertensive rats with heart failure and preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is defined by increased left ventricular (LV) stiffness, impaired vascular compliance, and fibrosis. Although systemic inflammation, driven by comorbidities, has been proposed to play a key role, the precise pathogenesis remains elusive. To test the hypothesis that inflammation drives endothelial dysfunction in HFpEF, we used cardiosphere-derived cells (CDCs), which reduce inflammation and fibrosis, improving function, structure, and survival in HFpEF rats. Dahl salt-sensitive rats fed a high-salt diet developed HFpEF, as manifested by diastolic dysfunction, systemic inflammation, and accelerated mortality. Rats were randomly allocated to receive intracoronary infusion of CDCs or vehicle. Two weeks later, inflammation, oxidative stress, and endothelial function were analyzed. Single-cell RNA sequencing of heart tissue was used to assay transcriptomic changes. CDCs improved endothelial-dependent vasodilation while reducing oxidative stress and restoring endothelial nitric oxide synthase (eNOS) expression. RNA sequencing revealed CDC-induced attenuation of pathways underlying endothelial cell leukocyte binding and innate immunity. Exposure of endothelial cells to CDC-secreted extracellular vesicles in vitro reduced VCAM-1 protein expression and attenuated monocyte adhesion and transmigration. Cell therapy with CDCs corrects diastolic dysfunction, reduces oxidative stress, and restores vascular reactivity. These findings lend credence to the hypothesis that inflammatory changes of the vascular endothelium are important, if not central, to HFpEF pathogenesis.NEW & NOTEWORTHY We tested the concept that inflammation of endothelial cells is a major pathogenic factor in HFpEF. CDCs are heart-derived cell products with verified anti-inflammatory therapeutic properties. Infusion of CDCs reduced oxidative stress, restored eNOS abundance, lowered monocyte levels, and rescued the expression of multiple disease-associated genes, thereby restoring vascular reactivity. The salutary effects of CDCs support the hypothesis that inflammation of endothelial cells is a proximate driver of HFpEF.

Regulatory T cell activation, proliferation, and reprogramming induced by extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) from heart stromal/progenitor cells modulate innate immunity, with salutary effects in a variety of cardiac disease models. Little is known, however, about the effects of these EVs on adaptive immunity. METHODS: Ex vivo differentiation of naïve CD4+ T cells was conducted to assess the effect of EVs on cytokine production and proliferation of Th1, Th2, Th17, and regulatory T (Treg) cells. These effects were further tested in vivo using the experimental autoimmune myocarditis (EAM) model. RESULTS: Using differentiated CD4+ T cells, we show that EVs secreted by human-derived heart stromal/progenitor cells selectively influence the phenotype, activity, and proliferation of regulatory T (Treg) cells. Exposure of Treg cells to EVs results in faster proliferation, augmented production of IL-10, and polarization toward an intermediate FOXP3+RORγt+ phenotype. In experimental autoimmune myocarditis, EVs attenuate cardiac inflammation and functional decline, in association with increased numbers of splenic IL10+-Treg cells. CONCLUSIONS: T cell modulation by EVs represents a novel therapeutic approach to inflammation, harnessing endogenous immunosuppressive mechanisms that may be applied in solid organ transplantation, graft-versus-host disease, and autoimmune disorders.

Exosomally derived Y RNA fragment alleviates hypertrophic cardiomyopathy in transgenic mice.

Cardiosphere-derived cell exosomes (CDCexo) and YF1, a CDCexo-derived non-coding RNA, elicit therapeutic bioactivity in models of myocardial infarction and hypertensive hypertrophy. Here we tested the hypothesis that YF1, a 56-nucleotide Y RNA fragment, could alleviate cardiomyocyte hypertrophy, inflammation, and fibrosis associated with hypertrophic cardiomyopathy (HCM) in transgenic mice harboring a clinically relevant mutation in cardiac troponin I (cTnIGly146). By quantitative PCR, YF1 was detectable in bone marrow, spleen, liver, and heart 30 min after intravenous (i.v.) infusion. For efficacy studies, mice were randomly allocated to receive i.v. YF1 or vehicle, monitored for ambulatory and cardiac function, and sacrificed at 4 weeks. YF1 (but not vehicle) improved ambulation and reduced cardiac hypertrophy and fibrosis. In parallel, peripheral mobilization of neutrophils and proinflammatory monocytes was decreased, and fewer macrophages infiltrated the heart. RNA-sequencing of macrophages revealed that YF1 confers substantive and broad changes in gene expression, modulating pathways associated with immunological disease and inflammatory responses. Together, these data demonstrate that YF1 can reverse hypertrophic and fibrotic signaling pathways associated with HCM, while improving function, raising the prospect that YF1 may be a viable novel therapeutic candidate for HCM.

Mechanistic and therapeutic distinctions between cardiosphere-derived cell and mesenchymal stem cell extracellular vesicle non-coding RNA.

Cell therapy limits ischemic injury following myocardial infarction (MI) by preventing cell death, modulating the immune response, and promoting tissue regeneration. The therapeutic efficacy of cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs) is associated with extracellular vesicle (EV) release. Prior head-to-head comparisons have shown CDCs to be more effective than MSCs in MI models. Despite differences in cell origin, it is unclear why EVs from different adult stem cell populations elicit differences in therapeutic efficacy. Here, we compare EVs derived from multiple human MSC and CDC donors using diverse in vitro and in vivo assays. EV membrane protein and non-coding RNA composition are highly specific to the parent cell type; for example, miR-10b is enriched in MSC-EVs relative to CDC-EVs, while Y RNA fragments follow the opposite pattern. CDC-EVs enhance the Arg1/Nos2 ratio in macrophages in vitro and reduce MI size more than MSC-EVs and suppress inflammation during acute peritonitis in vivo. Thus, CDC-EVs are distinct from MSC-EVs, confer immunomodulation, and protect the host against ischemic myocardial injury and acute inflammation.

Mechanisms of atrial fibrillation in aged rats with heart failure with preserved ejection fraction.

BACKGROUND: Although ∼20% of the elderly population develops atrial fibrillation (AF), little is known about the mechanisms. Heart failure with preserved ejection fraction (HFpEF), which is associated with AF, is more common in aged women than in men. OBJECTIVE: The purpose of this study was to identify potential mechanisms of AF in an age-related HFpEF model. METHODS: In aged female Fischer F344 rats (21- to 24-month-old), which are prone to HFpEF, we induced AF by atrial pacing. Young Fischer F344 female rats (3- to 4-month-old) and age-matched Sprague Dawley female rats (27-month-old) served as controls. Phenotyping included echocardiography to assess left ventricular structure/function; in vivo electrophysiology and ex vivo high-resolution optical mapping to assess AF vulnerability; systemic and atrial inflammatory profiling; atrial histology; and expression of inflammasome signaling proteins. RESULTS: Aged rats developed left ventricular hypertrophy, left atrial enlargement, diastolic dysfunction, and pulmonary congestion, without ejection fraction impairment, thus meeting the criteria for HFpEF. Increased serum inflammatory markers, hypertension, and obesity further characterize aged females. Sinoatrial and atrioventricular node dysfunction was associated with the high inducibility of AF in aged rats. Ex vivo electrical activation mapping revealed abnormal ß-adrenergic responsiveness and slowed conduction velocity. Atrial inflammasome signaling was enhanced in aged rats, which may contribute to fibrotic remodeling and high AF susceptibility. CONCLUSION: Together, our data demonstrate that aging-related atrial remodeling and HFpEF are associated with atrial enlargement, fibrosis, conduction abnormalities, and nodal dysfunction, favoring a substrate conducive to AF.

Macrophages in cardiac repair: Environmental cues and therapeutic strategies.

Mammals, in contrast to urodeles and teleost fish, lose the ability to regenerate their hearts soon after birth. Central to this regenerative response are cardiac macrophages, which comprise a heterogeneous population of cells with origins from the yolk sac, fetal liver, and bone marrow. These cardiac macrophages maintain residency in the myocardium through local proliferation and partial replacement over time by circulating monocytes. The intrinsic plasticity of cardiac macrophages in the adult heart promotes dynamic phenotypic changes in response to environmental cues, which may either protect against injury or promote maladaptive remodeling. Thus, therapeutic strategies promoting myocardial repair are warranted. Adult stromal cell-derived exosomes have shown therapeutic promise by skewing macrophages toward a cardioprotective phenotype. While several key exosomal non-coding RNA have been identified, additional factors responsible for cardiomyocyte proliferation remain to be elucidated. Here I review cardiac macrophages in development and following injury, unravel environmental cues modulating macrophage activation, and assess novel approaches for targeted delivery.

Mechanism of Enhanced MerTK-Dependent Macrophage Efferocytosis by Extracellular Vesicles.

OBJECTIVE: Extracellular vesicles secreted by cardiosphere-derived cells (CDCev) polarize macrophages toward a distinctive phenotype with enhanced phagocytic capacity (MCDCev). These changes underlie cardioprotection by CDCev and by the parent CDCs, notably attenuating the no-reflow phenomenon following myocardial infarction, but the mechanisms are unclear. Here, we tested the hypothesis that MCDCev are especially effective at scavenging debris from dying cells (ie, efferocytosis) to attenuate irreversible damage post-myocardial infarction. Approach and Results: In vitro efferocytosis assays with bone marrow-derived macrophages, and in vivo transgenic rodent models of myocardial infarction, demonstrate enhanced apoptotic cell clearance with MCDCev. CDCev exposure induces sustained MerTK expression in MCDCev through extracellular vesicle transfer of microRNA-26a (via suppression of Adam17); the cardioprotective response is lost in animals deficient in MerTK. Single-cell RNA-sequencing revealed phagocytic pathway activation in MCDCev, with increased expression of complement factor C1qa, a phagocytosis facilitator. CONCLUSIONS: Together, these data demonstrate that extracellular vesicle modulation of MerTK and C1qa expression leads to enhanced macrophage efferocytosis and cardioprotection.

Repeated cell transplantation and adjunct renal denervation in ischemic heart failure: exploring modalities for improving cell therapy efficacy.

Enthusiasm for cell therapy for myocardial injury has waned due to equivocal benefits in clinical trials. In an attempt to improve efficacy, we investigated repeated cell therapy and adjunct renal denervation (RDN) as strategies for augmenting cardioprotection with cardiosphere-derived cells (CDCs). We hypothesized that combining CDC post-conditioning with repeated CDC doses or delayed RDN therapy would result in superior function and remodeling. Wistar-Kyoto (WKY) rats or spontaneously hypertensive rats (SHR) were subjected to 45 min of coronary artery ligation followed by reperfusion for 12-14 weeks. In the first study arm, SHR were treated with CDCs (0.5 × 106 i.c.) or PBS 20 min following reperfusion, or additionally treated with CDCs (1.0 × 106 i.v.) at 2, 4, and 8 weeks. In the second arm, at 4 weeks following myocardial infarction (MI), SHR received CDCs (0.5 × 106 i.c.) or CDCs + RDN. In the third arm, WKY rats were treated with i.c. CDCs administered 20 min following reperfusion and RDN or a sham at 4 weeks. Early i.c. + multiple i.v. dosing, but not single i.c. dosing, of CDCs improved long-term left ventricular (LV) function, but not remodeling. Delayed CDC + RDN therapy was not superior to single-dose delayed CDC therapy. Early CDC + delayed RDN therapy improved LV ejection fraction and remodeling compared to both CDCs alone and RDN alone. Given that both RDN and CDCs are currently in the clinic, our findings motivate further translation targeting a heart failure indication with combined approaches.

Reverse electrical remodeling in rats with heart failure and preserved ejection fraction.

Sudden death is the most common mode of exodus in patients with heart failure and preserved ejection fraction (HFpEF). Cardiosphere-derived cells (CDCs) reduce inflammation and fibrosis in a rat model of HFpEF, improving diastolic function and prolonging survival. We tested the hypothesis that CDCs decrease ventricular arrhythmias (VAs) and thereby possibly contribute to prolonged survival. Dahl salt-sensitive rats were fed a high-salt diet to induce HFpEF. Allogeneic rat CDCs (or phosphate-buffered saline as placebo) were injected in rats with echo-verified HFpEF. CDC-injected HFpEF rats were less prone to VA induction by programmed electrical stimulation. Action potential duration (APD) was shortened, and APD homogeneity was increased by CDC injection. Transient outward potassium current density was upregulated in cardiomyocytes from CDC rats relative to placebo, as were the underlying transcript (Kcnd3) and protein (Kv4.3) levels. Fibrosis was attenuated in CDC-treated hearts, and survival was increased. Sudden death risk also trended down, albeit nonsignificantly. CDC therapy decreased VA in HFpEF rats by shortening APD, improving APD homogeneity, and decreasing fibrosis. Unlike other stem/progenitor cells, which often exacerbate arrhythmias, CDCs reverse electrical remodeling and suppress arrhythmogenesis in HFpEF.

Intravenous xenogeneic human cardiosphere-derived cell extracellular vesicles (exosomes) improves behavioral function in small-clot embolized rabbits.

Acute ischemic stroke is devastating to patients and their families because of few viable therapeutic options to promote recovery after reperfusion windows close. Recent breakthroughs in biotechnology have resulted in a reproducible patented process for the purification of extracellular vesicles (EVs) from human cardiosphere-derived cells (CDCs). Because CDC-EVs have many features potentially beneficial to treat acute ischemic stroke, CDC-EVs were evaluated in an established small-clot rabbit embolic stroke model, where clinically relevant end points were used to assess recovery in a more translational large animal model. Biodistribution studies with fluorescent DiD-labeled CDC-EVs showed intense uptake in the ischemic region of the brain. In this report, we show that intravenous (IV) CDC-EVs (0.75 mg/kg) administered 1-hour post-embolization significantly attenuate behavioral deficits following an embolic stroke in rabbits. In CDC-EV-treated rabbits, P50 (3.63 ±â€¯1.27 mg, n = 24) was increased by 245% over vehicle control (1.05 ±â€¯0.15 mg, n = 24); by comparison, rt-PA increased P50 by 91% (2.01 ±â€¯0.24 mg, n = 23). Importantly, the therapy was also without adverse effects on intracerebral hemorrhage or survival rate of embolized rabbits. Thus, as a first step toward widespread use, CDC-EVs, given adjunctively to routine reperfusion therapy, merit further investigation as a therapeutic candidate for stroke.

Newt cells secrete extracellular vesicles with therapeutic bioactivity in mammalian cardiomyocytes.

Newts can regenerate amputated limbs and cardiac tissue, unlike mammals which lack broad regenerative capacity. Several signaling pathways involved in cell proliferation, differentiation and survival during newt tissue regeneration have been elucidated, however the factors that coordinate signaling between cells, as well as the conservation of these factors in other animals, are not well defined. Here we report that media conditioned by newt limb explant cells (A1 cells) protect mammalian cardiomyocytes from oxidative stress-induced apoptosis. The cytoprotective effect of A1-conditioned media was negated by exposing A1 cells to GW4869, which suppresses the generation of extracellular vesicles (EVs). A1-EVs are similar in diameter (~100-150 nm), structure, and share several membrane surface and cargo proteins with mammalian exosomes. However, isolated A1-EVs contain significantly higher levels of both RNA and protein per particle than mammalian EVs. Additionally, numerous cargo RNAs and proteins are unique to A1-EVs. Of particular note, A1-EVs contain numerous mRNAs encoding nuclear receptors, membrane ligands, as well as transcription factors. Mammalian cardiomyocytes treated with A1-EVs showed increased expression of genes in the PI3K/AKT pathway, a pivotal player in survival signaling. We conclude that newt cells secrete EVs with diverse, distinctive RNA and protein contents. Despite ~300 million years of evolutionary divergence between newts and mammals, newt EVs confer cytoprotective effects on mammalian cardiomyocytes.

Delayed Repolarization Underlies Ventricular Arrhythmias in Rats With Heart Failure and Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) represents approximately half of heart failure, and its incidence continues to increase. The leading cause of mortality in HFpEF is sudden death, but little is known about the underlying mechanisms. METHODS: Dahl salt-sensitive rats were fed a high-salt diet (8% NaCl) from 7 weeks of age to induce HFpEF (n=38). Rats fed a normal-salt diet (0.3% NaCl) served as controls (n=13). Echocardiograms were performed to assess systolic and diastolic function from 14 weeks of age. HFpEF-verified and control rats underwent programmed electrical stimulation. Corrected QT interval was measured by surface ECG. The mechanisms of ventricular arrhythmias (VA) were probed by optical mapping, whole-cell patch clamp to measure action potential duration and ionic currents, and quantitative polymerase chain reaction and Western blotting to investigate changes in ion channel expression. RESULTS: After 7 weeks of a high-salt diet, 31 of 38 rats showed diastolic dysfunction and preserved ejection fraction along with signs of heart failure and hence were diagnosed with HFpEF. Programmed electric stimulation demonstrated increased susceptibility to VA in HFpEF rats (P<0.001 versus controls). The arrhythmogenicity index was increased (P<0.001) and the corrected QT interval on ECG was prolonged (P<0.001) in HFpEF rats. Optical mapping of HFpEF hearts demonstrated prolonged action potentials (P<0.05) and multiple reentry circuits during induced VA. Single-cell recordings of cardiomyocytes isolated from HFpEF rats confirmed a delay of repolarization (P=0.001) and revealed downregulation of transient outward potassium current (Ito; P<0.05). The rapid components of the delayed rectifier potassium current (IKr) and the inward rectifier potassium current (IK1) were also downregulated (P<0.05), but the current densities were much lower than for Ito. In accordance with the reduction of Ito, both Kcnd3 transcript and Kv4.3 protein levels were decreased in HFpEF rat hearts. CONCLUSIONS: Susceptibility to VA was markedly increased in rats with HFpEF. Underlying abnormalities include QT prolongation, delayed repolarization from downregulation of potassium currents, and multiple reentry circuits during VA. Our findings are consistent with the hypothesis that potassium current downregulation leads to abnormal repolarization in HFpEF, which in turn predisposes to VA and sudden cardiac death.

Exosomal MicroRNA Transfer Into Macrophages Mediates Cellular Postconditioning.

BACKGROUND: Cardiosphere-derived cells (CDCs) confer cardioprotection in acute myocardial infarction by distinctive macrophage (Mϕ) polarization. Here we demonstrate that CDC-secreted exosomes (CDCexo) recapitulate the cardioprotective effects of CDC therapy known as cellular postconditioning. METHODS: Rats and pigs underwent myocardial infarction induced by ischemia/reperfusion before intracoronary infusion of CDCexo, inert fibroblast exosomes (Fbexo; control), or vehicle. Two days later, infarct size was quantified. Macrophages were isolated from cardiac tissue or bone marrow for downstream analyses. RNA sequencing was used to determine exosome content and alterations in gene expression profiles in Mϕ. RESULTS: Administration of CDCexo but not Fbexo after reperfusion reduces infarct size in rat and pig models of myocardial infarction. Furthermore, CDCexo reduce the number of CD68+ Mϕ within infarcted tissue and modify the polarization state of Mϕ so as to mimic that induced by CDCs. CDCexo are enriched in several miRNAs (including miR-146a, miR-181b, and miR-126) relative to Fbexo. Reverse pathway analysis of whole-transcriptome data from CDCexo-primed Mϕ implicated miR-181b as a significant (P=1.3x10-21) candidate mediator of CDC-induced Mϕ polarization, and PKCδ (protein kinase C δ) as a downstream target. Otherwise inert Fbexo loaded selectively with miR-181b alter Mϕ phenotype and confer cardioprotective efficacy in a rat model of myocardial infarction. Adoptive transfer of PKCδ-suppressed Mϕ recapitulates cardioprotection. CONCLUSIONS: Our data support the hypothesis that exosomal transfer of miR-181b from CDCs into Mϕ reduces PKCδ transcript levels and underlies the cardioprotective effects of CDCs administered after reperfusion.

Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction.

Aims: Naturally secreted nanovesicles known as exosomes are required for the regenerative effects of cardiosphere-derived cells (CDCs), and exosomes mimic the benefits of CDCs in rodents. Nevertheless, exosomes have not been studied in a translationally realistic large-animal model. We sought to optimize delivery and assess the efficacy of CDC-secreted exosomes in pig models of acute (AMI) and convalescent myocardial infarction (CMI). Methods and Results: In AMI, pigs received human CDC exosomes (or vehicle) by intracoronary (IC) or open-chest intramyocardial (IM) delivery 30 min after reperfusion. No-reflow area and infarct size (IS) were assessed histologically at 48 h. Intracoronary exosomes were ineffective, but IM exosomes decreased IS from 80 ± 5% to 61 ± 12% (P= 0.001) and preserved left ventricular ejection fraction (LVEF). In a randomized placebo-controlled study of CMI, pigs 4 weeks post-myocardial infarction (MI) underwent percutaneous IM delivery of vehicle (n = 6) or CDC exosomes (n = 6). Magnetic resonance imaging (MRI) performed before and 1 month after treatment revealed that exosomes (but not vehicle) preserved LV volumes and LVEF (−0.1 ± 2.2% vs. −5.4 ± 3.6%, P= 0.01) while decreasing scar size. Histologically, exosomes decreased LV collagen content and cardiomyocyte hypertrophy while increasing vessel density. Conclusion: Cardiosphere-derived cell exosomes delivered IM decrease scarring, halt adverse remodelling and improve LVEF in porcine AMI and CMI. While conceptually attractive as cell-free therapeutic agents for myocardial infarction, exosomes have the disadvantage that IM delivery is necessary.

Y RNA fragment in extracellular vesicles confers cardioprotection via modulation of IL-10 expression and secretion.

Cardiosphere-derived cells (CDCs) reduce myocardial infarct size via secreted extracellular vesicles (CDC-EVs), including exosomes, which alter macrophage polarization. We questioned whether short non-coding RNA species of unknown function within CDC-EVs contribute to cardioprotection. The most abundant RNA species in CDC-EVs is a Y RNA fragment (EV-YF1); its relative abundance in CDC-EVs correlates with CDC potency in vivo Fluorescently labeled EV-YF1 is actively transferred from CDCs to target macrophages via CDC-EVs. Direct transfection of macrophages with EV-YF1 induced transcription and secretion of IL-10. When cocultured with rat cardiomyocytes, EV-YF1-primed macrophages were potently cytoprotective toward oxidatively stressed cardiomyocytes through induction of IL-10. In vivo, intracoronary injection of EV-YF1 following ischemia/reperfusion reduced infarct size. A fragment of Y RNA, highly enriched in CDC-EVs, alters Il10 gene expression and enhances IL-10 protein secretion. The demonstration that EV-YF1 confers cardioprotection highlights the potential importance of diverse exosomal contents of unknown function, above and beyond the usual suspects (e.g., microRNAs and proteins).

Repeated transplantation of allogeneic cardiosphere-derived cells boosts therapeutic benefits without immune sensitization in a rat model of myocardial infarction.

BACKGROUND: A single dose of allogeneic cardiosphere-derived cells (CDCs) improves cardiac function and reduces scarring, and increases viable myocardium in the infarcted rat and pig heart without eliciting a detrimental immune response. Clinical trials using single doses of allogeneic human CDCs are underway. It is unknown whether repeat dosing confers additional benefit or if it elicits an immune response. METHODS: Wistar-Kyoto rats underwent coronary artery ligation and intramyocardial injection of CDCs, with a second thoracotomy and repeat CDC injection 3 weeks later. Treatment permutations included 2 doses of allogeneic Brown-Norway CDCs (n = 24), syngeneic Wistar-Kyoto CDCs (n = 24), xenogeneic human CDCs (n = 24) or saline (n = 8). Cardiac function was assessed by transthoracic echocardiography, infarct size and inflammatory infiltration by histology, and cellular and humoral immune responses by lymphocyte proliferation and alloantibody assays. RESULTS: Repeat dosing of allogeneic and syngeneic CDCs improved ejection fraction by 5.2% (95% CI 2.1 to 8.3) and 6.8% (95% CI 3.8 to 9.8) after the first dose, and by 3.4% (95% CI 0.1% to 6.8%) and 6.4% (95% CI 4.2% to 8.6%) after the second dose. Infarct size was equally reduced with repeat dosing of syngeneic and allogeneic CDCs relative to xenogeneic and control treatments (p < 0.0001). Significant rejection-like infiltrates were present only in the xenogeneic group; likewise, lymphocyte proliferation and antibody assays were positive in the xenogeneic and negative in syngeneic and allogeneic groups. CONCLUSIONS: Repeat dosing of allogeneic CDCs in immunocompetent rats is safe and effective, consistent with the known immunomodulatory and anti-inflammatory properties of CDCs. These findings motivate clinical testing of repeatedly dosed CDCs for chronic heart disease.