Background Current treatments of chemotherapy-induced cardiomyopathy (CCM) are of limited efficacy. We assessed whether repeated intravenous injections of human extracellular vesicles from cardiac progenitor cells (EV-CPC) could represent a new therapeutic option and whether EV manufacturing according to a Good Manufacturing Practices (GMP)-compatible process did not impair their bioactivity. Methods Immuno-competent mice received intra-peritoneal injections (IP) of doxorubicin (DOX) (4 mg/kg each; cumulative dose: 12 mg/kg) and were then intravenously (IV) injected three times with EV-CPC (total dose: 30 billion). Cardiac function was assessed 9–11 weeks later by cardiac magnetic resonance imaging (CMR) using strain as the primary end point. Then, immuno-competent rats received 5 IP injections of DOX (3 mg/kg each; cumulative dose 15 mg/kg) followed by 3 equal IV injections of GMP-EV (total dose: 100 billion). Cardiac function was assessed by two dimensional-echocardiography. Results In the chronic mouse model of CCM, DOX + placebo-injected hearts incurred a significant decline in basal (global, epi- and endocardial) circumferential strain compared with sham DOX-untreated mice (p = 0.043, p = 0.042, p = 0.048 respectively) while EV-CPC preserved these indices. Global longitudinal strain followed a similar pattern. In the rat model, IV injections of GMP-EV also preserved left ventricular end-systolic and end-diastolic volumes compared with untreated controls. Conclusions Intravenously-injected extracellular vesicles derived from CPC have cardio-protective effects which may make them an attractive user-friendly option for the treatment of CCM.

Introduction: Current treatments of chemotherapy-induced cardiomyopathy (CCM) are of limited efficacy. Hypothesis: Extracellular vesicles (EV) might represent a new therapeutic option. Methods: Male immunocompetent mice were subjected to 3 intraperitoneal (IP) injections of doxorubicin (DOX, 4mg/kg) followed by 3 sequential intravenous injections of EV (10x10 9 /injection) collected from human induced pluripotent stem cell (hiPSC)-differentiated cardiovascular progenitor cells. Outcomes were assessed 8-11 weeks after the onset of DOX treatment on MRI and compared with those of mice injected with saline (controls) or DOX-untreated (sham). Explanted hearts were subjected to transcriptomics. Next, female rats received 5 IP injections of DOX (3mg/kg) followed by 3 IV injections of EV (33x10 9 /injection) similar to those of the mouse study except that they had been GMP-manufactured to be of clinical grade. Control rats were saline-injected or DOX-untreated (sham). Outcomes were assessed 1 month after the onset of DOX treatment on echocardiography. In additional in vitro experiments, hiPSC-derived cardiomyocytes were stressed by DOX w/wo EV and assessed for ATP content. Results: In mice, DOX+saline-injected hearts (n=14) incurred a significant decline in basal (global, epi- and endocardial) circumferential strain compared with the 14 sham mice (p=0.04 for each) while EV (n=11) preserved these indices which did not significantly differ from those of sham mice. Global longitudinal strain followed a similar pattern. EV also upregulated genes involved in the limitation of cardiac remodeling and preservation of contractile function. In rats, LV endsystolic volumes were significantly increased in saline-injected hearts (n=11) compared with the 6 sham (p=0.03) but were preserved after EV injections (n=12). Likewise, the percentages of "responder" rats which did not increase their enddiastolic LV volumes by more than 5% from their post-DOX pre-treatment values were 58% vs . 28% in EV- and saline-injected hearts, respectively. In vitro , EV increased ATP levels of DOX-stressed cardiomyocytes by 30% compared to placebo. Conclusions: The cardioprotective effects of IV-injected EV make them an attractive user-friendly option for the treatment of CCM.

Extracellular vesicles (EV) are increasingly recognized as a therapeutic option in heart failure. They are usually administered by direct intramyocardial injections with the caveat of a rapid wash-out from the myocardium which might weaken their therapeutic efficacy. To improve their delivery in the failing myocardium, we designed a system consisting of loading EV into a clinical-grade hyaluronic acid (HA) biomaterial. EV were isolated from umbilical cord-derived mesenchymal stromal cells. The suitability of HA as a delivery platform was then assessed in vitro. Rheology studies demonstrated the viscoelastic and shear thinning behaviors of the selected HA allowing its easy injection. Moreover, the release of HA-embedded EV was sustained over more than 10 days, and EV bioactivity was not altered by the biomaterial. In a rat model of myocardial ischemia reperfusion, we showed that HA-embedded EV preserved cardiac function (echocardiography), improved angiogenesis and decreased both apoptosis and fibrosis (histology and transcriptomics) when compared to intramyocardial administration of EV alone. These data thus strengthen the concept that inclusion of EV into a clinically useable biomaterial might optimize their beneficial effects on post-ischemic cardiac repair.

Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.

Background: Extracellular vesicles (EV) mediate the therapeutic effects of stem cells but it is unclear whether this involves cardiac regeneration mediated by endogenous cardiomyocyte proliferation. Methods: Bi-transgenic MerCreMer/ZEG (n = 15/group) and Mosaic Analysis With Double Markers (MADM; n = 6/group) mouse models underwent permanent coronary artery ligation and received, 3 weeks later, 10 billion EV (from human iPS-derived cardiovascular progenitor cells [CPC]), or saline, injected percutaneously under echo guidance in the peri-infarcted myocardium. Endogenous cardiomyocyte proliferation was tracked by EdU labeling and biphoton microscopy. Other end points, including cardiac function (echocardiography and MRI), histology and transcriptomics were blindly assessed 4-6 weeks after injections. Results: There was no proliferation of cardiomyocytes in either transgenic mouse strains. Nevertheless, EV improved cardiac function in both models. In MerCreMer/ZEG mice, LVEF increased by 18.3 ± 0.2% between baseline and the end-study time point in EV-treated hearts which contrasted with a decrease by 2.3 ± 0.2% in the PBS group; MADM mice featured a similar pattern as intra-myocardial administration of EV improved LVEF by 13.3 ± 0.16% from baseline whereas it decreased by 14.4 ± 0.16% in the control PBS-injected group. This functional improvement was confirmed by MRI and associated with a reduction in infarct size, the decreased expression of several pro-fibrotic genes and an overexpression of the anti-fibrotic miRNA 133-a1 compared to controls. Experiments with an anti-miR133-a demonstrated that the cardio-reparative effects of EV were partly abrogated. Conclusions: EV-CPC do not trigger cardiomyocyte proliferation but still improve cardiac function by other mechanisms which may include the regulation of fibrosis.

AIMS:The cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation. METHODS AND RESULTS:Flow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages. CONCLUSIONS:EV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.

Introduction: Extracellular Vesicles (EV) are increasingly recognized as key mediators of the effects of cellular therapy but repeated dosing is likely required for optimizing their therapeutic benefits. This, in turn, requires a noninvasive delivery route. Hypothesis: Repeated intravenous (IV) EV infusions might improve cardiac function in chemotherapy-induced cardiomyopathy. Methods: All EV were collected from human iPSC-derived cardiovascular progenitor cells. In vitro experiments : iPSC-derived cardiomyocytes were stressed 3 times by doxorubicin (Dox, 0.2 μM), once every 48 hours (D0, D2, D4), and then exposed to EV once (D6) or twice (D6 and D8). Outcomes were assessed on D8 and D10 on mitochondrial function and intracellular ATP levels assessed by the Seahorse XF Cell Mito Stress Test and ATPlite Luminescence Assay System, respectively. In vivo experiments : Male BALB/c mice were subjected to 3 weekly injections of Dox (total cumulated dose: 12 mg/kg) followed by 3 IV infusions of EV (10 9 per dose, n=11) or an equivalent volume of saline (n=14) over 1-2 weeks. The main outcome measure was Global Longitudinal Strain (GLS), a robust and early marker of LV dysfunction, blindly assessed by MRI at baseline and 8-11 weeks following the onset of Dox treatment. Explanted hearts were then processed for histology and transcriptomics. Results: Compared with Dox-stressed controls, EV increased ATP levels by 30%±8% (n=7, each in triplicate, p=0.047; m±SEM) and enhanced both mitochondrial respiration and anaerobic glycolysis. In vivo , GLS at baseline averaged -14.98±0.24. It was well preserved after EV treatment with a decrease at end-study of only 2.96%±7.18% from baseline while it fell by 11.90%±3.51% in PBS-injected controls. Similar patterns of changes were seen for longitudinal endocardial strain which declined by 0.22%±7.29% and 8.39%±3.42% from baseline in EV- and PBS-injected hearts, respectively, while the corresponding declines for epicardial strains were 3.92%±7.20% and 13.44%±3.28%. Basal global circumferential strain was also better preserved in EV-treated hearts. Conclusions: Repeated IV infusions of EV decrease chemo-triggered cardiomyocyte toxicities and help preserving cardiac function in chemotherapy-induced cardiomyopathy.

Introduction: Extracellular Vesicles (EV) recapitulate the benefits of cell therapy for heart repair. Their mechanism of action remains unsettled. Hypothesis: EV may contribute to heart repair by de novo cardiogenesis. Methods: To answer this question, we used 2 bi-transgenic mouse models: the fate-mapping MerCreMer/ZEG and the Mosaic Analysis With Double Markers (MADM). Myocardial infarction was induced by permanent coronary artery ligation. Those with a LVEF ≤ 45% were treated 3 weeks later with EV (from human iPS-derived cardiovascular progenitor cells; 10x10 9 particles) or PBS, injected under echo guidance in the peri-infarcted area (MerCreMer/ZEG: n=15/group and MADM: n=6/group). To track endogenous cardiomyocyte (CM) proliferation, we used EdU labeling in MerCreMer/ZEG delivered by osmotic pumps implanted for 7-10 days post-injection and biphoton microscopy in MADM models. Cardiac function was assessed 4-6 weeks after injection by echocardiography and MRI, blinded to treatment group. Hearts were then subjected to histological and transcriptomic analyses (qPCR and genome-wide microarray). Results: In PBS controls, EF remained stable over time in MerCreMer/ZEG mice and decreased from 34.5% ± 6.0% to 30.7% ± 7.5% in MADM mice by the end of the study. Conversely, EV injections increased EF from 32.1% ± 9.5% to 36.1% ± 7.45 % in MerCreMer/ZEG and from 36.2 %± 8.7% to 40.5% ± 8.9% in MADM mice. A significant difference in the change from baseline was found between EV and controls: 20.7% ± 10.5 % (p=0.048) and 28.0% ± 11.0 %, (p=0.045) for MerCreMer/ZEG and MADM groups, respectively. This improvement was confirmed by MRI in MerCreMer/ZEG mice (p=0.05). Improvement in EF was unrelated to the appearance of new CM, as shown by the absence of difference in TnT+/EdU+/GFP+ cell numbers and the lack of activation of the YAP/TAZ pathway between control and EV groups. However, EV reduced infarct size by 11.9% ± 5.75% (p=0.04), which was accompanied by decreased expression of 4 pro-fibrotic genes (Col1a2, Col3a1, Lox, Col1a2 by qPCR) in heart tissue and a 2.13X overexpression of the anti-fibrotic miRNA 133a-1 compared to controls (n=3/group; p=0.001). Conclusions: EV likely improve cardiac function by modulation of fibrosis rather than by de novo cardiogenesis.

Although the first wave of cell therapy trials has not commonly yielded clinically meaningful improvements, some encouraging hints have emerged which suggest that stem cells or their secreted products could ultimately find a place within the armamentarium of therapies that can be offered to patients with heart failure. In this setting, pluripotent stem cells raise a particular interest because of their unique ability to generate lineage-specific cells which can be transplanted at the desired stage of differentiation. This review discusses the current status of research in this field, the persisting roadblocks that need to be overcome and the approaches which might hasten the clinical applications of this cell type.

Although the clinical outcomes of cell therapy trials have not met initial expectations, emerging evidence suggests that injury-mediated tissue damage might benefit from the delivery of cells or their secreted products. Pluripotent stem cells (PSCs) are promising cell sources primarily because of their capacity to generate stage- and lineage-specific differentiated derivatives. However, they carry inherent challenges for safe and efficacious clinical translation. This Review describes completed or ongoing trials of PSCs, discusses their potential mechanisms of action, and considers how to address the challenges required for them to become a major therapy, using heart repair as a case study.