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.

Understanding the Angiogenic Characteristics of Clinical-Grade Mesenchymal Stromal Cells Isolated from Human Umbilical Cord.

Addressing the challenges in managing ischemic tissue repair and remodelling remains a prominent clinical concern. Current research is heavily concentrated on identifying innovative cell-based therapies with the potential to enhance revascularization in patients affected by these diseases. We have previously developed and validated a manufacturing process for human umbilical cord mesenchymal stromal cells (UC-MSCs)-based cell therapy medicinal product, according to Good Manufacturing Practices. In this study, we demonstrate that these UC-MSCs enhance the proliferation and migration of endothelial cells and the formation of capillary structures. Moreover, UC-MSCs and endothelial cells interact, allowing UC-MSCs to acquire a perivascular cell phenotype and consequently provide direct support to the newly formed vascular network. This characterization of the proangiogenic properties of this UC-MSCs based-cell therapy medicinal product is an essential step for its therapeutic assessment in the clinical context of vascular regeneration.

Self-Organization of Long-Lasting Human Endothelial Capillary-Like Networks Guided by DLP Bioprinting.

Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.

Acoustic levitation as a tool for cell-driven self-organization of human cell spheroids during long-term 3D culture.

Acoustic levitation, which allows contactless manipulation of micro-objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell-cell interactions, away from the walls of the chip, which leads to the spontaneous self-organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long-term culture of cells in acoustic levitation is feasible. We also demonstrated that this self-organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium-mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix.

Allogenic umbilical cord-derived mesenchymal stromal cells improve motor function in prenatal surgical repair of myelomeningocele: An ovine model study.

OBJECTIVE: To investigate the effects of an adjuvant allogenic umbilical cord mesenchymal stromal cell (UC-MSC) patch applied during fetal surgery on motor and sphincter function in the ovine MMC model. DESIGN: MMC defects were surgically created at 75 days of gestation and repaired 14 days later. POPULATION: Ovine MMC model: fetal lambs. METHODS: We compared lambs that received a UC-MSC patch with a control group of lambs that received an acellular patch. MAIN OUTCOME MEASURES: Clinical neurological assessment was performed at 2 and 24 hours of life and included determination of the Sheep Locomotor Rating scale (SLR), which has been validated in the ovine MMC model. Electrophysical examinations, spine scans and histological analyses were also performed. RESULTS: Of the 13 operated lambs, nine were born alive: five had of these had received a UC-MSC patch and four an acellular patch. At 24 hours of life, lambs in the UC-MSC group had a significantly higher score (14 versus 5, P = 0.04). Amyotrophy was significantly more common in the control group (75% versus 0%, P = 0.02). All the lambs in the control group and none of those in the UC-MSC group were incontinent. No significant differences were observed between the UC-MSC and control groups in terms of the presence of spontaneous EMG activity, nerve conduction or spinal evoked potentials. In the microscopic examination, lambs in the UC-MSC group had less fibrosis between the spinal cord and the dermis (mean thickness, 453 versus 3921 µm, P = 0.03) and around the spinal cord (mean thickness, 47 versus 158 µm, P < 0.001). Examination of the spinal cord in the area of the MMC defect showed a higher large neuron density in the UC-MSC group (14.5 versus 5.6 neurons/mm2, P < 0.001). No tumours were observed. CONCLUSIONS: Fetal repair of MMC using UC-MSC patches improves motor and sphincter function as well as spinal preservation and reduction of fibrosis.

Early predictive factors of failure in autologous CAR T-cell manufacturing and/or efficacy in hematologic malignancies.

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapies have shown significant benefits in the treatment of hematologic malignancies, such as B-cell acute lymphoblastic leukemia (B-ALL) and B-cell lymphoma. Despite the therapeutic advances offered by these innovative treatments, failures are still observed in 15% to 40% of patients with B-ALL and >50% of patients with B-cell lymphoma. Several hypotheses have emerged including CD19-negative or -positive relapses, low CAR T-cell activation and/or expansion in vivo, or T-cell exhaustion. To date, in the European Union, CAR T cells granted with marketing authorization are autologous and thus associated with a strong heterogeneity between products. Indeed, the manufacturing of a single batch requires cellular starting material collection by apheresis for each patient, with variable cellular composition, and then challenging pharmaceutical companies to standardize as much as possible the production process. In addition, these cost and time-consuming therapies are associated with a risk of manufacturing failure reaching 25%. Thus, there is a growing need to identify early risk factors of unsuccessful production and/or therapeutic escape. Quality of the apheresis product, pathology progression, as well as previous treatments have been reported as predictive factors of the variability in clinical response. The aim of this review is to report and discuss predictive factors that could help to anticipate the manufacturing success and clinical response.

Development and qualification of clinical grade decellularized and cryopreserved human esophagi.

Tissue engineering is a promising alternative to current full thickness circumferential esophageal replacement methods. The aim of our study was to develop a clinical grade Decellularized Human Esophagus (DHE) for future clinical applications. After decontamination, human esophagi from deceased donors were placed in a bioreactor and decellularized with sodium dodecyl sulfate (SDS) and ethylendiaminetetraacetic acid (EDTA) for 3 days. The esophagi were then rinsed in sterile water and SDS was eliminated by filtration on an activated charcoal cartridge for 3 days. DNA was removed by a 3-hour incubation with DNase. A cryopreservation protocol was evaluated at the end of the process to create a DHE cryobank. The decellularization was efficient as no cells and nuclei were observed in the DHE. Sterility of the esophagi was obtained at the end of the process. The general structure of the DHE was preserved according to immunohistochemical and scanning electron microscopy images. SDS was efficiently removed, confirmed by a colorimetric dosage, lack of cytotoxicity on Balb/3T3 cells and mesenchymal stromal cell long term culture. Furthermore, DHE did not induce lymphocyte proliferation in-vitro. The cryopreservation protocol was safe and did not affect the tissue, preserving the biomechanical properties of the DHE. Our decellularization protocol allowed to develop the first clinical grade human decellularized and cryopreserved esophagus.

Endothelial CD34 expression and regulation of immune cell response in-vitro.

Endothelial cells cover the lining of different blood vessels and lymph nodes, and have major functions including the transport of blood, vessel homeostasis, inflammatory responses, control of transendothelial migration of circulating cells into the tissues, and formation of new blood vessels. Therefore, understanding these cells is of major interest. The morphological features, phenotype and function of endothelial cells varies according to the vascular bed examined. The sialomucin, CD34, is widely used as an endothelial marker. However, CD34 is differentially expressed on endothelial cells in different organs and in pathological conditions. Little is known about regulation of endothelial CD34 expression or function. Expression of CD34 is also strongly regulated in-vitro in endothelial cell models, including human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC). We have therefore analysed the expression and function of CD34 by comparing CD34high and CD34low endothelial cell subpopulations. Transcriptomic analysis showed that CD34 gene and protein expressions are highly correlated, that CD34high cells proliferate less but express higher levels of IL-33 and Angiopoietin 2, compared with CD34low cells. Higher secretion levels of IL-33 and Angiopoietin 2 by CD34high HUVECs was confirmed by ELISA. Finally, when endothelial cells were allowed to interact with peripheral blood mononuclear cells, CD34high endothelial cells activated stronger proliferation of regulatory T lymphocytes (Tregs) compared to CD34low cells whereas expansion of other CD4+-T cell subsets was equivalent. These results suggest that CD34 expression by endothelial cells in-vitro associates with their ability to proliferate and with an immunogenic ability that favours the tolerogenic response.

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.

Nestin is a New Partner in Endothelial Colony Forming Cell Angiogenic Potential.

Nestin, an intermediate filament protein expressed by progenitor cells, is associated with tissue regeneration. Although nestin expression has been reported in poorly differentiated and newly formed blood vessels, its role in endothelial cells remains unclear. In this study, we investigated the involvement of nestin in the angiogenic properties of endothelial colony-forming cells (ECFCs) derived from human umbilical cord blood. Our results demonstrate that ECFCs express high levels of nestin, and that its inhibition by small interfering RNAs decreased ECFC proliferation, migration in response to SDF-1 and VEGF-A, tubulogenesis, and adhesion on collagen. These effects are associated with modulation of focal adhesion kinase phosphorylation. Furthermore, nestin silencing resulted in reduced revascularization in a mouse hindlimb ischemia model. In conclusion, these findings provide evidence that nestin more than being a structural protein, is an active player in ECFC angiogenic properties.

Feasibility of an acoustophoresis-based system for a high-throughput cell washing: application to bioproduction.

BACKGROUND AIMS: These last decades have seen the emergence and development of cell-based therapies, notably those based on mesenchymal stromal cells (MSCs). The advancement of these promising treatments requires increasing the throughput of processed cell for industrialization in order to reduce production costs. Among the various bioproduction challenges, downstream processing, including medium exchange, cell washing, cell harvesting and volume reduction, remains a critical step for which improvements are needed. Typically, these processes are performed by centrifugation. However, this approach limits the automation, especially in small batch productions where it is performed manually in open system. METHODS: An acoustophoresis-based system was developed for cell washing. The cells were transferred from one stream to another via the acoustic forces and were collected in a different medium. The optimal flow rates of the different streams were assessed using red blood cells suspended in an albumin solution. Finally, the impact of acoustic washing on adipose tissue-derived MSCs (AD-MSCs) transcriptome was investigated by RNA-sequencing. RESULTS: With a single passage through the acoustic device at input flow rate of 45 mL/h, the albumin removal was up to 90% while recovering 99% of RBCs. To further increase the protein removal, a loop washing in two steps was performed and has allowed an albumin removal ≥99% and a red blood cell/AD-MSCs recovery of 99%. After loop washing of AD-MSCs, only two genes, HES4 and MIR-3648-1, were differently expressed compared with the input. CONCLUSIONS: In this study, we developed a continuous cell-washing system based on acoustophoresis. The process allows a theoretically high cell throughput while inducing little gene expression changes. These results indicate that cell washing based on acoustophoresis is a relevant and promising solution for numerous applications in cell manufacturing.

Distinct subsets of multi-lymphoid progenitors support ontogeny-related changes in human lymphopoiesis.

Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.

Cancer Cell De-Differentiation: Plasticity-Driven Stratagem For Tumor Metastasis and Recurrence.

Tumor recurrence is a colossal challenge in clinical oncology. This multifactorial problem is attributed to the emergence of additional genetic mutations and the presence of dormant cancer cells. However, the plasticity of non-stem cancer cells and the acquisition of cancer stem cell (CSC) functionality is another contributing factor to tumor recurrence. Herein, I focus attention on the mechanisms that fuel cancer cell de-differentiation and the interplay between intra-cellular regulators and tumor microenvironment (TME) landscape that promotes cancer cell stemness. Our understanding of the mechanisms underlying tumor cell de-differentiation is crucial for developing innovative therapeutic strategies that prevent cancer from ever recurring.

The use of ICU resources in CAR-T cell recipients: a hospital-wide study.

BACKGROUND: CAR-T cell (chimeric antigen receptor T) therapy has emerged as an effective treatment of refractory hematological malignancies. Intensive care management is intrinsic to CAR-T cell therapy. We aim to describe and to assess outcomes in critically ill CAR-T cell recipients. STUDY DESIGN AND METHODS: Hospital-wide retrospective study. Consecutive CAR-T cell recipients requiring ICU admission from July 2017 and December 2020 were included. RESULTS: 71 patients (median age 60 years [37-68]) were admitted to the ICU 6 days [4-7] after CAR-T cell infusion. Underlying malignancies included diffuse large B cell lymphoma (n = 53, 75%), acute lymphoblastic leukemia (17 patients, 24%) and multiple myeloma (n = 1, 1.45%). Performance status (PS) was 1 [1-2]. Shock was the main reason for ICU admission (n = 40, 48%). Isolated cytokine release syndrome (CRS) was the most common complication (n = 33, 46%), while 21 patients (30%) had microbiologically documented bacterial infection (chiefly catheter-related infection). Immune effector cell-associated neurotoxicity syndrome was reported in 26 (37%) patients. At ICU admission, vasopressors were required in 18 patients (25%) and invasive mechanical ventilation in two. Overall, 49 (69%) and 40 patients (56%) received tocilizumab or steroids, respectively. Determinant of mortality were the reason for ICU admission (disease progression vs. sepsis or CRS (HR 4.02 [95%CI 1.10-14.65]), Performance status (HR 1.97/point [95%CI 1.14-3.41]) and SOFA score (HR 1.16/point [95%CI 1.01-1.33]). CONCLUSIONS: Meaningful survival could be achieved in up to half the CAR-T cell recipients. The severity of organ dysfunction is a major determinant of death, especially in patients with altered performance status or disease progression.

Biodistribution of allogenic umbilical cord-derived mesenchymal stromal cells after fetal repair of myelomeningocele in an ovine model.

BACKGROUND: Myelomeningocele (MMC) is a spinal cord congenital defect that leads to paraplegia, sphincter disorders and potential neurocognitive disabilities. Prenatal surgery of MMC provides a significant benefit compared to surgery at birth. Mesenchymal stromal cell (MSC) therapy as an adjuvant treatment for prenatal surgery showed promising results in animal experiments which could be considered for clinical use in human fetuses. Despite numerous reassuring studies on the safety of MSCs administration in humans, no study focused on MSCs biodistribution after a local MSCs graft on the fetal spinal cord. AIM: The purpose of our study was to assess the biodistribution of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) at birth in lambs who had a prenatal myelomeningocele repair using a fibrin patch seeded with allogenic UC-MSCs. METHODS: After isolation, UC-MSCs were tagged using a green fluorescent protein (GFP)-containing lentiviral vector. MMC defects were surgically created at 75 days of gestation and repaired 15 days later using UC-MSCs patch. Lambs were delivered at 142 days and sacrificed. DNA extraction was performed among biopsies of the different organs and q-PCR analysis was used to detect the expression of GFP (GFP DNA coding sequence). RESULTS: In our 6 surviving lambs grafted with UC-MSCs, GFP lentivirus genomic DNA was not detected in the organs. CONCLUSION: These reassuring data will support translational application in humans, especially since the first human clinical trial using mesenchymal stromal cells for in-utero treatment of MMC started recently in U.S.A.

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 .