First-in-man use of a cardiovascular cell-derived secretome in heart failure. Case report.

BACKGROUND: There is increased evidence that the effects of stem cells can mostly be duplicated by administration of their secretome which might streamline the translation towards the clinics. METHODS: The 12-patient SECRET-HF phase 1 trial has thus been designed to determine the feasibility and safety of repeated intravenous injections of the extracellular vesicle (EV)-enriched secretome of cardiovascular progenitor cells differentiated from pluripotent stem cells in severely symptomatic patients with drug-refractory left ventricular (LV) dysfunction secondary to non-ischemic dilated cardiomyopathy. Here we report the case of the first treated patient (baseline NYHA class III; LV Ejection Fraction:25%) in whom a dose of 20 × 109 particles/kg was intravenously infused three times three weeks apart. FINDINGS: In addition to demonstrating the feasibility of producing a cardiac cell secretome compliant with Good Manufacturing Practice standards, this case documents the excellent tolerance of its repeated delivery, without any adverse events during or after infusions. Six months after the procedure, the patient is in NYHA Class II with improved echo parameters, a reduced daily need for diuretics (from 240 mg to 160 mg), no firing from the previously implanted automatic internal defibrillator and no alloimmunization against the drug product, thereby supporting its lack of immunogenicity. INTERPRETATION: The rationale underlying the intravenous route is that the infused EV-enriched secretome may act by rewiring endogenous immune cells, both circulating and in peripheral organs, to take on a reparative phenotype. These EV-modified immune cells could then traffic to the heart to effect tissue repair, including mitigation of inflammation which is a hallmark of cardiac failure. FUNDING: This trial is funded by the French Ministry of Health (Programme Hospitalier de Recherche CliniqueAOM19330) and the "France 2030" National Strategy Program (ANR-20-F2II-0003). It is sponsored by Assistance Publique-Hôpitaux de Paris.

Treatment of COVID-19-associated ARDS with umbilical cord-derived mesenchymal stromal cells in the STROMA-CoV-2 multicenter randomized double-blind trial: long-term safety, respiratory function, and quality of life.

BACKGROUND: The STROMA-CoV-2 study was a French phase 2b, multicenter, double-blind, randomized, placebo-controlled clinical trial that did not identify a significant efficacy of umbilical cord-derived mesenchymal stromal cells in patients with SARS-CoV-2-induced acute respiratory distress syndrome. Safety on day 28 was found to be good. The aim of our extended study was to assess the 6- and 12-month safety of UC-MSCs administration in the STROMA-CoV-2 cohort. METHODS: A detailed multi-domain assessment was conducted at 6 and 12 months following hospital discharge focusing on adverse events, lung computed tomography-scan, pulmonary and muscular functional status, and quality of life in the STROMA-CoV-2 cohort including SARS-CoV-2-related early (< 96 h) mild-to-severe acute respiratory distress syndrome. RESULTS: Between April 2020 and October 2020, 47 patients were enrolled, of whom 19 completed a 1-year follow-up. There were no significant differences in any endpoints or adverse effects between the UC-MSCs and placebo groups at the 6- and 12-month assessments. Ground-glass opacities persisted at 1 year in 5 patients (26.3%). Furthermore, diffusing capacity for carbon monoxide remained altered over 1 year, although no patient required oxygen or non-invasive ventilatory support. Quality of life revealed declines in mental, emotional and physical health throughout the follow-up period, and the six-minute walking distance remained slightly impaired at the 1-year patient assessment. CONCLUSIONS: This study suggests a favorable safety profile for the use of intravenous UC-MSCs in the context of the first French wave of SARS-CoV-2-related moderate-to-severe acute respiratory distress syndrome, with no adverse effects observed at 1 year.

Human PSC-derived cardiac cells and their products: therapies for cardiac repair.

Despite the dramatic improvements in the management of patients with chronic heart failure which have occurred over the last decades, some of them still exhaust conventional drug-based therapies without being eligible for more aggressive options like heart transplantation or implantation of a left ventricular assist device. Cell therapy has thus emerged as a possible means of filling this niche. Multiple cell types have now been tested both in the laboratory but also in the clinics and it is fair to acknowledge that none of the clinical trials have yet conclusively proven the efficacy of cell-based approaches. These clinical studies, however, have entailed the use of cells from various sources but of non-cardiac lineage origins. Although this might not be the main reason for their failures, the discovery of pluripotent stem cells capable of generating cardiomyocytes now raises the hope that such cardiac-committed cells could be therapeutically more effective. In this review, we will first describe where we currently are with regard to the clinical trials using PSC-differentiated cells and discuss the main issues which remain to be addressed. In parallel, because the capacity of cells to stably engraft in the recipient heart has increasingly been questioned, it has been hypothesized that a major mechanism of action could be the cell-triggered release of biomolecules that foster host-associated reparative pathways. Thus, in the second part of this review, we will discuss the rationale, clinically relevant advantages and pitfalls associated with the use of these PSC "products".

Therapeutic potential of extracellular vesicles derived from cardiac progenitor cells in rodent models of chemotherapy-induced cardiomyopathy.

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.

When gene editing turns a nasty goddess into a friendly MEDUSA.

Marchiano and colleagues interrogate the underlying causes of ventricular arrhythmias occurring after human pluripotent stem cell-cardiomyocyte transplantation. Through stepwise analysis and gene editing of ion channel expression, they mitigate pace-maker-like activity, providing evidence that the automaticity responsible for these rhythmic events can be successfully controlled by appropriate gene edits.

Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are released from cells of the cardiovascular system, and are considered important mediators of intercellular and extracellular communications. Two types of EVs of particular interest are exosomes and microvesicles, which have been identified in all tissue and body fluids and carry a variety of molecules including RNAs, proteins, and lipids. EVs have potential for use in the diagnosis and prognosis of cardiovascular diseases and as new therapeutic agents, particularly in the setting of myocardial infarction and heart failure. Despite their promise, technical challenges related to their small size make it challenging to accurately identify and characterize them, and to study EV-mediated processes. Here, we aim to provide the reader with an overview of the techniques and technologies available for the separation and characterization of EVs from different sources. Methods for determining the protein, RNA, and lipid content of EVs are discussed. The aim of this document is to provide guidance on critical methodological issues and highlight key points for consideration for the investigation of EVs in cardiovascular studies.

Biomaterial-embedded extracellular vesicles improve recovery of the dysfunctional myocardium.

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.

Treatment of COVID-19-associated ARDS with mesenchymal stromal cells: a multicenter randomized double-blind trial.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-induced acute respiratory distress syndrome (ARDS) causes high mortality. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) have potentially relevant immune-modulatory properties, whose place in ARDS treatment is not established. This phase 2b trial was undertaken to assess the efficacy of UC-MSCs in patients with SARS-CoV-2-induced ARDS. METHODS: This multicentre, double-blind, randomized, placebo-controlled trial (STROMA-CoV-2) recruited adults (≥ 18 years) with SARS-CoV-2-induced early (< 96 h) mild-to-severe ARDS in 10 French centres. Patients were randomly assigned to receive three intravenous infusions of 106 UC-MSCs/kg or placebo (0.9% NaCl) over 5 days after recruitment. For the modified intention-to-treat population, the primary endpoint was the partial pressure of oxygen to fractional inspired oxygen (PaO2/FiO2)-ratio change between baseline (day (D) 0) and D7. RESULTS: Among the 107 patients screened for eligibility from April 6, 2020, to October 29, 2020, 45 were enrolled, randomized and analyzed. PaO2/FiO2 changes between D0 and D7 did not differ significantly between the UC-MSCs and placebo groups (medians [IQR] 54.3 [- 15.5 to 93.3] vs 25.3 [- 33.3 to 104.6], respectively; ANCOVA estimated treatment effect 7.4, 95% CI - 44.7 to 59.7; P = 0.77). Six (28.6%) of the 21 UC-MSCs recipients and six of 24 (25%) placebo-group patients experienced serious adverse events, none of which were related to UC-MSCs treatment. CONCLUSIONS: D0-to-D7 PaO2/FiO2 changes for intravenous UC-MSCs-versus placebo-treated adults with SARS-CoV-2-induced ARDS did not differ significantly. Repeated UC-MSCs infusions were not associated with any serious adverse events during treatment or thereafter (until D28). Larger trials enrolling patients earlier during the course of their ARDS are needed to further assess UC-MSCs efficacy in this context. TRIAL REGISTRATION: NCT04333368. Registered 01 April 2020, https://clinicaltrials.gov/ct2/history/NCT04333368 .

Dynamic contrast enhanced - MRI efficiency in detecting embolization-induced perfusion defects in a rabbit model of critical-limb-ischemia.

Critical limb ischemia (CLI) is a severe disease which affects about 2 million people in the US. Its prevalence is assessed at 800/100,000 population. However, no reliable tools are currently available to assess perfusion defects at the muscle tissue level. DCE-MRI is a technique that holds the potential to be effective in achieving this goal. However, preclinical studies performed with DCE-MRI have indicated low sensitivity assessing perfusion at resting state. To improve these previous results, in this work we propose new methodologies for data acquisition and analysis and we also revisit the biological model used for evaluation. Eleven rabbits underwent embolization of a lower limb. They were imaged at day 7 after embolization using DCE-MRI, performed on a 4.7 T small imaging device. Among them, n = 4 rabbits were used for MRI sequence optimization and n = 6 for data analysis after one exclusion. Normalized Areas under the curve (AUCn), and kinetic parameters such as Ktrans and Vd resulting from the Tofts-Kety modeling (KTM) were calculated on the embolized and contralateral limbs. Average and heterogeneity features, consisting on standard-deviation and quantiles, were calculated on muscle groups and whole limbs. The Wilcoxon and Fisher-tests were performed to compare embolized and contralateral regions of interests. The Wilcoxon test was also used to compare features of parametric maps. Quantiles of 5 and 95% in the contralateral side were used to define low and high outliers. A P-value <0.05 was considered statistically significant. Average features were inefficient to identify injured muscles, in agreement with the low sensitivity of the technique previously reported by the literature. However, these findings were dramatically improved by the use of additional heterogeneity features (97% of total accuracy for group muscles, P < 0.01 and 100% of total accuracy for the total limbs). The mapping analysis and automatic outlier detection quantification improvement was explained by the presence of local hyperemia that impair the average calculations. The analysis with KTM did not provide any additional information compared to AUCn. The DCE technique can be effective in detecting embolization-induced disorders of limb muscles in a CLI model when heterogeneity is taken into account in the data processing, even without vascular stimulation. The simultaneous presence of areas of ischemia and hyperemia appeared as a signature of the injured limbs. These areas seem to reflect the simultaneous presence of infarcted areas and viable peripheral areas, characterized by a vascular response that is visible in DCE.

Basic and Translational Research in Cardiac Repair and Regeneration: JACC State-of-the-Art Review.

This paper aims to provide an important update on the recent preclinical and clinical trials using cell therapy strategies and engineered heart tissues for the treatment of postinfarction left ventricular remodeling and heart failure. In addition to the authors' own works and opinions on the roadblocks of the field, they discuss novel approaches for cardiac remuscularization via the activation of proliferative mechanisms in resident cardiomyocytes or direct reprogramming of somatic cells into cardiomyocytes. This paper's main mindset is to present current and future strategies in light of their implications for the design of future patient trials with the ultimate objective of facilitating the translation of discoveries in regenerative myocardial therapies to the clinic.

Extracellular vesicles fail to trigger the generation of new cardiomyocytes in chronically infarcted hearts.

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.

Development of a human umbilical cord-derived mesenchymal stromal cell-based advanced therapy medicinal product to treat immune and/or inflammatory diseases.

BACKGROUND: Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) revealed their key role in immune regulation, offering promising therapeutic perspectives for immune and inflammatory diseases. We aimed to develop a production process of an UC-MSC-based product and then to characterize UC-MSC properties and immunomodulatory activities in vitro, related to their clinical use and finally, to transfer this technology to a good manufacturing practice (GMP) compliant facility, to manufacture an advanced therapy medicinal product (ATMP). METHODS: Fifteen human umbilical cords (UCs) were collected to develop the production process. Three batches of UC-MSCs from a single donor were characterized at basal state and after in vitro pro-inflammatory stimulation by interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Proliferation, immunophenotype, activation markers' expression and the inhibition of T cell proliferation were assessed. Finally, this technology was transferred to a GMP-compliant facility to manufacture an UC-MSC-based ATMP, from a single donor, using the explant method followed by the establishment of master and work cell stocks. RESULTS: Twelve UCs were processed successfully allowing to isolate UC-MSCs with doubling time and population doubling remaining stable until passage 4. CD90, CD105, CD73, CD44, CD29, CD166 expression was positive; CD14, CD45, CD31, HLA-DR, CD40, CD80 and CD86 expression was negative, while CD146 and HLA-ABC expression was heterogeneous. Cell morphology, proliferation and immunophenotype were not modified by inflammatory treatment. Indoleamine 2,3-dioxygenase (IDO) expression was significantly induced by IFNγ and IFNγ + TNFα versus non-treated cells. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression was induced significantly after priming. T cell proliferation was significantly decreased in the presence of UC-MSCs in a dose-dependent manner. This inhibitory effect was improved by IFNγ or IFNγ + TNFα, at UC-MSCs:PBMC ratio 1:10 and 1:30, whereas only IFNγ allowed to decrease significantly T cell proliferation at ratio 1:100. The manufacturing process of the UC-MSC-based ATMP was qualified and authorized by the French regulatory agency for clinical use (NCT04333368). CONCLUSION: This work allowed to develop an investigational UC-MSC-based ATMP authorized for clinical use. Our results showed that an inflammatory environment preserves the biological properties of UC-MSCs with an improvement of their immunomodulatory functions.

Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France".

Extracellular vesicles (EV) are emergent therapeutic effectors that have reached clinical trial investigation. To translate EV-based therapeutic to clinic, the challenge is to demonstrate quality, safety, and efficacy, as required for any medicinal product. EV research translation into medicinal products is an exciting and challenging perspective. Recent papers, provide important guidance on regulatory aspects of pharmaceutical development, defining EVs for therapeutic applications and critical considerations for the development of potency tests. In addition, the ISEV Task Force on Regulatory Affairs and Clinical Use of EV-based Therapeutics as well as the Exosomes Committee from the ISCT are expected to contribute in an active way to the development of EV-based medicinal products by providing update on the scientific progress in EVs field, information to patients and expert resource network for regulatory bodies. The contribution of our work group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France", created in 2020, can be positioned in complement to all these important initiatives. Based on complementary scientific, technical, and medical expertise, we provide EV-specific recommendations for manufacturing, quality control, analytics, non-clinical development, and clinical trials, according to current European legislation. We especially focus on early phase clinical trials concerning immediate needs in the field. The main contents of the investigational medicinal product dossier, marketing authorization applications, and critical guideline information are outlined for the transition from research to clinical development and ultimate market authorization.

Reparative cell therapy for the heart: critical internal appraisal of the field in response to recent controversies.

The concept that cell-based repair of myocardial injury might be possible was introduced almost two decades ago; however, the field of cardiovascular reparative medicine has been criticized as translation to clinically effective approaches has been slow. The recent retraction of a series of papers has further impacted perception of this area of research. As researchers, clinicians, and teachers in this field, we felt it incumbent to critically appraise the current state of cardiac cell repair, determine what can be learned from past mistakes, and formulate best practices for future work. This special communication summarizes an introspective assessment of what has fallen short, how to prevent similar issues, and how the field might best move forward in the service of science and patients.

Extracellular vesicles from human cardiovascular progenitors trigger a reparative immune response in infarcted hearts.

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.

Insights into therapeutic products, preclinical research models, and clinical trials in cardiac regenerative and reparative medicine: where are we now and the way ahead. Current opinion paper of the ESC Working Group on Cardiovascular Regenerative and Reparative Medicine.

Great expectations have been set around the clinical potential of regenerative and reparative medicine in the treatment of cardiovascular diseases [i.e. in particular, heart failure (HF)]. Initial excitement, spurred by encouraging preclinical data, resulted in a rapid translation into clinical research. The sobering outcome of the resulting clinical trials suggests that preclinical testing may have been insufficient to predict clinical outcome. A number of barriers for clinical translation include the inherent variability of the biological products and difficulties to develop potency and quality assays, insufficient rigour of the preclinical research and reproducibility of the results, manufacturing challenges, and scientific irregularities reported in the last years. The failure to achieve clinical success led to an increased scrutiny and scepticism as to the clinical readiness of stem cells and gene therapy products among clinicians, industry stakeholders, and funding bodies. The present impasse has attracted the attention of some of the most active research groups in the field, which were then summoned to analyse the position of the field and tasked to develop a strategy, to re-visit the undoubtedly promising future of cardiovascular regenerative and reparative medicine, based on lessons learned over the past two decades. During the scientific retreat of the ESC Working Group on Cardiovascular Regenerative and Reparative Medicine (CARE) in November 2018, the most relevant and timely research aspects in regenerative and/or reparative medicine were presented and critically discussed, with the aim to lay out a strategy for the future development of the field. We report herein the main ideas and conclusions of that meeting.