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.

Thrombin receptor PAR1 silencing in endothelial colony-forming cells modifies stemness and vasculogenic properties.

BACKGROUND: The involvement of thrombin receptor PAR1 in blood vessel development has been largely demonstrated in knockout mice; however, its implication in adult mouse angiogenesis seems very moderate. OBJECTIVES: We aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony-forming cells (ECFCs). METHODS AND RESULTS: PAR1 activation on ECFCs using the selective PAR1-activating peptide induced a significant decrease in CD133 expression (RTQ-PCR analysis). In line, silencing of PAR1 gene expression with siRNA increased CD133 mRNA as well as intracellular CD133 protein expression. To confirm the link between CD133 and PAR1, we explored the association between PAR1 and CD133 levels in fast and slow fibroblasts prone to reprogramming. An imbalance between PAR1 and CD133 levels was evidenced, with a decreased expression of PAR1 in fast reprogramming fibroblasts expressing a high CD133 level. Regarding in vitro ECFC angiogenic properties, PAR1 silencing with specific siRNA induced cell proliferation evidenced by the overexpression of Ki67. However, it did not impact migration properties nor ECFC adhesion on smooth muscle cells or human arterial endothelial cells. In a mouse model of hind-limb ischemia, PAR1 silencing in ECFCs significantly increased postischemic revascularization compared to siCtrl-ECFCs along with a significant increase in cutaneous blood flows (P < .0001), microvessel density (P = .02), myofiber regeneration (P < .0001), and human endothelial cell incorporation in muscle (P < .0001). CONCLUSION: In conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs.

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.

Improving Autologous Fat Grafting in Regenerative Surgery through Stem Cell-Assisted Lipotransfer.

Autologous fat transplantation -i.e., lipofilling- has become a promising and popular technique in aesthetic and reconstructive surgery with several application such as breast reconstruction, facial and hand rejuvenation. However, the use of this technology is still limited due to an unpredictable and low graft survival rate (which ranges from 25%-80%). A systematic literature review was performed by thoroughly searching 12 terms using the PubMed database. The objective of this study is to present the current evidence for the efficacy of adjuvant regenerative strategies and cellular factors, which have been tested to improve fat graft retention. We present the main results (fat retention rate, histological analysis for pre-clinical studies and satisfaction/ complication for clinical studies) obtained from the studies of the three main fat grafting enrichment techniques: platelet-rich plasma (PRP), the stromal vascular fraction (SVF) and adipose-derived stem cells (ADSCs) and discuss the promising role of recent angiogenic cell enrichment that could induce early vascularization of fat graft. All in all, adding stem or progenitor cells to autologous fat transplantation might become a new concept in lipofilling. New preclinical models should be used to find mechanisms able to increase fat retention, assure safety and transfer these technologies to a good manufacturing practice (GMP) compliant facility, to manufacture an advanced therapy medicinal product (ATMP).

Regulatory B Cells Contribute to the Clinical Response After Bone Marrow-Derived Mesenchymal Stromal Cell Infusion in Patients With Systemic Sclerosis.

Mesenchymal stromal cells (MSCs) have recently emerged as an interesting therapeutic approach for patients with progressive systemic sclerosis (SSc), a rare and life-threatening orphan autoimmune disease. Whereas MSC immunomodulatory potential is considered as a central mechanism for their clinical benefit, very few data are available on the impact of MSCs on immune cell subsets in vivo. In the current extended study of a phase I/II clinical trial exploring the injection of a single dose of allogeneic bone marrow-MSCs (alloBM-MSCs) in patients with severe SSc (NCT02213705), we performed a longitudinal in-depth characterization of circulating immune cells in 19 MSC-treated patients, including 14 responders and 5 non-responders. By a combination of flow cytometry and transcriptomic analyses, we highlighted an increase in circulating CD24hiCD27posCD38lo/neg memory B cells, the main IL-10-producing regulatory B cell (Breg) subset, and an upregulation of IL10 expression in ex-vivo purified B cells, specifically in responder patients, early after the alloBM-MSC infusion. In addition, a deeper alteration of the B-cell compartment before alloBM-MSC treatment, including a higher expression of profibrotic cytokines IL6 and TGFß by sorted B cells was associated with a non-responder clinical status. Finally, BM-MSCs were able to directly upregulate IL-10 production in activated B cells in vitro. These data suggest that cytokine-producing B cells, in particular Breg, are pivotal effectors of BM-MSC therapeutic activity in SSc. Their quantification as activity biomarkers in MSC potency assays and patient selection criteria may be considered to reach optimal clinical benefit when designing MSC-based clinical trials.

Distinguishing Plasmin-Generating Microvesicles: Tiny Messengers Involved in Fibrinolysis and Proteolysis.

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency.

Grafted dinuclear zinc complexes for selective recognition of phosphatidylserine: Application to the capture of extracellular membrane microvesicles.

Microvesicles (MVs) are key markers in human body fluids that reflect cellular activation related to diseases as thrombosis. These MVs display phosphatidylserine at the outer leaflet of their plasma membrane as specific recognition moieties. The work reported in this manuscript focuses on the development of an original method where MVs are captured by bimetallic zinc complexes. A set of ligands have been synthetized based on a phenol spacer bearing in para position an amine group appended to a short or a longer alkyl chain (for grafting on surface) and bis(dipicolylamine) arms in ortho position (for zinc coordination). The corresponding dibridged zinc phenoxido and hydroxido complexes have been prepared in acetronitrile in presence of triethylamine and characterized by several spectroscopic techniques. The pH-driven interconversion studies for both complexes in H2O:DMSO (70:30) evidence that at physiologic pH the main species are mono-bridged by the phenoxido spacer. An X-Ray structure obtained from complex 2 (based on the ligand with the amine group on the short chain) in aqueous medium confirms the presence of a mono-bridged complex. Then, the complexes have been used for interaction studies with short-chain phospholipids. Both have established the selective recognition of the anionic phosphatidylserine model versus zwitterionic phospholipids (in solution by 31P NMR and after immobilization on solid support by surface plasmon resonance (SPR)). Moreover, both complexes have also demonstrated their ability to capture MVs isolated from human plasma. These complexes are thus promising candidates for MVs probing by a new approach based on coordination chemistry.

Combined Transplantation of Human MSCs and ECFCs Improves Cardiac Function and Decrease Cardiomyocyte Apoptosis After Acute Myocardial Infarction.

BACKGROUND: Ischemic heart disease, often caused by an acute myocardial infarction (AMI) is one of the leading causes of morbidity and mortality worldwide. Despite significant advances in medical and procedural therapies, millions of AMI patients progress to develop heart failure every year. METHODS: Here, we examine the combination therapy of human mesenchymal stromal cells (MSCs) and endothelial colony-forming cells (ECFCs) to reduce the early ischemic damage (MSCs) and enhance angiogenesis (ECFCs) in a pre-clinical model of acute myocardial infarction. NOD/SCID mice were subjected to AMI followed by transplantation of MSCs and ECFCs either alone or in combination. Cardiomyocyte apoptosis and cardiac functional recovery were assessed in short- and long-term follow-up studies. RESULTS: At 1 day after AMI, MSC- and ECFC-treated animals demonstrated significantly lower cardiomyocyte apoptosis compared to vehicle-treated animals. This phenomenon was associated with a significant reduction in infarct size, cardiac fibrosis, and improvement in functional cardiac recovery 4 weeks after AMI. CONCLUSIONS: The use of ECFCs, MSCs, and the combination of both cell types reduce cardiomyocyte apoptosis, scar size, and adverse cardiac remodeling, compared to vehicle, in a pre-clinical model of AMI. These results support the use of this combined cell therapy approach in future human studies during the acute phase of ischemic cardiac injury.

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.

Non-Classical HLA Determinants of the Clinical Response after Autologous Stem Cell Transplantation for Systemic Sclerosis.

Systemic Sclerosis (SSc) is a chronic autoimmune disease with high morbidity and mortality. Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is the best therapeutic option for rapidly progressive SSc, allowing increased survival with regression of skin and lung fibrosis. The immune determinants of the clinical response after AHSCT have yet to be well characterized. In particular, the pivotal role of the Human Leukocyte Antigen (HLA) system is not well understood, including the role of non-classical immuno-modulatory HLA-E and HLA-G molecules in developing tolerance and the role of Natural Killer cells (NK) in the immunomodulation processes. We retrospectively tested whether the genetic and/or circulating expression of the non-classical HLA-E and HLA-G loci, as well as the imputed classical HLA determinants of HLA-E expression, influence the observed clinical response to AHSCT at 12- and 24-month follow-up. In a phenotypically well-defined sample of 46 SSc patients classified as clinical responders or non-responders, we performed HLA genotyping using next-generation sequencing and circulating levels of HLA-G and quantified HLA-E soluble isoforms by ELISA. The -21HLA-B leader peptide dimorphism and the differential expression level of HLA-A and HLA-C alleles were imputed. We observed a strong trend towards better clinical response in HLA-E*01:03 or HLA-G 14bp Del allele carriers, which are known to be associated with high expression of the corresponding molecules. At 12-month post-AHSCT follow-up, higher circulating levels of soluble HLA-E were associated with higher values of modified Rodnan Skin Score (mRSS) (p = 0.0275), a proxy of disease severity. In the non-responder group, the majority of patients carried a double dose of the HLA-B Threonine leader peptide, suggesting a non-efficient inhibitory effect of the HLA-E molecules. We did not find any correlation between the soluble HLA-G levels and the observed clinical response after AHSCT. High imputed expression levels of HLA-C alleles, reflecting more efficient NK cell inhibition, correlated with low values of the mRSS 3 months after AHSCT (p = 0.0087). This first pilot analysis of HLA-E and HLA-G immuno-modulatory molecules suggests that efficient inhibition of NK cells contributes to clinical response after AHSCT for SSc. Further studies are warranted in larger patient cohorts to confirm our results.

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 .

Adipose tissue-derived stromal vascular fraction for treating hands of patients with systemic sclerosis: a multicentre randomized trial Autologous AD-SVF versus placebo in systemic sclerosis.

OBJECTIVE: To assess the superiority of adipose tissue-derived stromal vascular fraction (AD-SVF) injection into the fingers vs placebo in reducing hand disability in systemic sclerosis (SSc) patients. METHODS: We performed a double-blind, multicentre, phase II trial from October 2015 to January 2018 in France. SSc patients with a Cochin Hand Function Scale (CHFS) ≥20/90 were randomized 1:1 to receive injection of AD-SVF or placebo. AD-SVF was obtained using the automated processing Celution 800/CRS system. The placebo was lactated Ringer's solution. The primary efficacy end point was the change of the CHFS score from baseline to 3 months. Secondary efficacy endpoints included the CHFS score at 6 months, hand function, vasculopathy, hand pain, skin fibrosis, sensitivity of the finger pulps, Scleroderma Health Assessment Questionnaire, patients and physician satisfaction, and safety. RESULTS: Forty patients were randomized. The AD-SVF and placebo groups were comparable for age, sex ratio, disease duration, skin fibrosis of the hands and main cause of hand disability. After 3 months' follow-up, hand function significantly improved in both groups with no between-group difference of CHFS (mean change of -9.2 [12.2] in the AD-SVF group vs -7.6 [13.2] in the placebo group). At 6 months, hand function improved in both groups. CONCLUSION: This study showed an improvement of hand function in both groups over time, with no superiority of the AD-SVF. Considering the limits of this trial, studies on a larger population of patients with homogeneous phenotype and hand handicap should be encouraged to accurately assess the benefit of AD-SVF therapy. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT02558543. Registered on September 24, 2015.

In utero treatment of myelomeningocele with allogenic umbilical cord-derived mesenchymal stromal cells in an ovine model.

PURPOSE OF THE STUDY: The purpose of our study was to investigate the effects of ovine umbilical cord-derived mesenchymal stromal cells (UC-MSCs) seeded in a fibrin patch as an adjuvant therapy for fetal myelomeningocele repair in the ovine model. MATERIALS AND METHODS: MMC defects were surgically created at 75 days of gestation and repaired 15 days later with UC-MSCs patch or an acellular patch. At birth, motor function, tail movements, and voiding abilities were recorded. Histological and immunohistochemical analysis included study of MMC defect's healing, spinal cord, UC-MSCs survival, and screening for tumors. RESULTS: Six lambs were born alive in each group. There was no difference between the two groups on the median sheep locomotor rating score but all lambs in the control group had a score between lower than 3 compared to 50% in UC-MSCs group. There were more lambs with tail movements and voiding ability in UC-MSCs group (83% vs 0% and 50% vs 0%, respectively). gray matter area and large neurons density were higher in UC-MSCs group (2.5 vs 0.8 mm2 and 19.3 vs 1.6 neurons/mm2 of gray matter, respectively). Fibrosis thickness at the myelomeningocele scar level was reduced in UC-MSCs group (1269 µm vs 2624 µm). No tumors were observed. CONCLUSION: Fetal repair of myelomeningocele using allogenic UC-MSCs patch provides a moderate improvement in neurological functions, gray matter and neuronal preservation and prevented from fibrosis development at the myelomeningocele scar level.

Safety and preliminary efficacy of allogeneic bone marrow-derived multipotent mesenchymal stromal cells for systemic sclerosis: a single-centre, open-label, dose-escalation, proof-of-concept, phase 1/2 study.

BACKGROUND: Systemic sclerosis remains an orphan life-threatening autoimmune disease. The unique immunomodulatory, proangiogenic, and antifibrotic properties of mesenchymal stromal cells provide a strong rationale for mesenchymal stromal cell-based therapy for systemic sclerosis, and treatment with mesenchymal stromal cells has shown benefits in preclinical models of this disease. The safety of allogeneic bone marrow-derived mesenchymal stromal cell administration in patients with severe systemic sclerosis has not yet been established. We aimed to test the safety and feasibility of a single intravenous injection of intrafamilial allogeneic bone marrow-derived mesenchymal stromal cells to treat severe diffuse systemic sclerosis. METHODS: We did an open-label, dose-escalation, proof-of-concept, phase 1/2 study at Saint-Louis-Hospital, Paris, France. Eligible patients were aged 18-70 years with severe diffuse systemic sclerosis, who fulfilled the 2013 American College of Rheumatology and European League Against Rheumatism systemic sclerosis criteria, had a minimum modified Rodnan skin score of 15 (range 0-51), had severe lung, heart, or kidney involvement, and had inadequate response or contraindications to conventional immunosuppressive therapy or autologous haematopoietic stem cell transplantation. Patients with severe comorbidities were excluded. The first ten recipients were to receive a single intravenous infusion of 1 × 106 bone marrow-derived mesenchymal stromal cells per kg bodyweight, and the subsequent ten recipients were to be infused with a single dose of 3 × 106 bone marrow-derived mesenchymal stromal cells per kg bodyweight. The primary endpoint was immediate tolerance during infusion and within the first 10 days after infusion, measured as the occurrence of serious adverse events (grade 3 or higher) in all infused patients. Safety was assessed in all participants during the 24-month follow-up period. This study is registered with ClinicalTrials.gov, NCT02213705. FINDINGS: Between March 24, 2014, and Jan 6, 2020, 20 cisgender individuals (13 women and seven men) with severe diffuse systemic sclerosis were enrolled. All 20 patients were included in the primary outcome analysis. No infusion-related severe adverse events and three infusion-related adverse events occurred in the first 10 days after treatment; one patient had grade 1 flushing and another patient had grade 1 nausea and grade 2 asthenia. After ten days and up to a median follow-up of 24·1 months (IQR 20·8-24·5), 36 non-treatment-related severe adverse events in 14 (70%) patients and no treatment-related adverse event were reported. INTERPRETATION: A single infusion of allogeneic bone marrow-derived mesenchymal stromal cells was safe in patients with severe diffuse systemic sclerosis. Future placebo-controlled trials will help to definitively ascertain the efficacy of mesenchymal stromal cell-based cell therapy from various tissue sources in larger number of patients with systemic sclerosis. FUNDING: French Ministry of Health, Capucine Association, Fonds de Dotation de l'AFER pour la Recherche Médicale, and Agence Nationale de la Recherche (Infrastructure Program Ecell), France.

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.

Inter-center comparison of good manufacturing practices-compliant stromal vascular fraction and proposal for release acceptance criteria: a review of 364 productions.

BACKGROUND: Even though the manufacturing processes of the stromal vascular fraction for clinical use are performed in compliance with the good manufacturing practices applying to advanced therapy medicinal products, specifications related to stromal vascular fraction quality remain poorly defined. We analyzed stromal vascular fraction clinical batches from two independent good manufacturing practices-compliant manufacturing facilities, the Swiss Stem Cell Foundation (SSCF) and Marseille University Hospitals (AP-HM), with the goal of defining appropriate and harmonized release acceptance criteria. METHODS: This retrospective analysis reviewed the biological characteristics of 364 batches of clinical-grade stromal vascular fraction. Collected data included cell viability, recovery yield, cell subset distribution of stromal vascular fraction, and microbiological quality. RESULTS: Stromal vascular fraction from SSCF cohort demonstrated a higher viability (89.33% ± 4.30%) and recovery yield (2.54 × 105 ± 1.22 × 105 viable nucleated cells (VNCs) per mL of adipose tissue) than stromal vascular fraction from AP-HM (84.20% ± 5.96% and 2.25 × 105 ± 1.11 × 105 VNCs per mL). AP-HM batches were significantly less contaminated (95.71% of sterile batches versus 74.15% for SSCF batches). The cell subset distribution was significantly different (higher proportion of endothelial cells and lower proportion of leukocytes and pericytes in SSCF cohort). CONCLUSIONS: Both centers agreed that a good manufacturing practices-compliant stromal vascular fraction batch should exert a viability equal or superior to 80%, a minimum recovery yield of 1.50 × 105 VNCs per mL of adipose tissue, a proportion of adipose-derived stromal cells at least equal to 20%, and a proportion of leukocytes under 50%. In addition, a multiparameter gating strategy for stromal vascular fraction analysis is proposed.

Umbilical cord blood transplants facilitated by the French cord blood banks network. On behalf of the Agency of Biomedicine, Eurocord and the French society of bone marrow transplant and cell therapy (SFGM-TC).

The public French Cord Blood Banks Network was established in 1999 with the objective of standardizing the practices governing umbilical cord blood (UCB) banking in France. The Network adopted a strategy to optimize its inventory and improve the quality of its banked units based on a quality improvement process using outcome data regularly provided by Eurocord. This study aimed to describe the results, over 10 years, of UCBT facilitated by a national network that used the same criteria of UCB collection and banking and to assess how modifications of banking criteria and unit selection might influence transplant outcomes. Nine hundred and ninety-nine units (593 single-unit and 203 double-unit grafts) were released by the Network to transplant 796 patients with malignant (83%) and non-malignant (17%) diseases. Median cell dose exceeded 3.5 × 107 TNC/kg in 86%. There was a trend to select units more recently collected and with higher cell dose. Neutrophil engraftment was 88.2% (85.7-90.7) and 79.3% (72.6-86.5) respectively for malignant and non-malignant diseases with a trend to faster recovery with higher cell doses. The respective 3-year transplant-related mortality were 31.1% (27.5-35.1) and 34.3% (27.0-43.5). OS was 49% ± 4 in malignant and 62% ± 4 in non-malignant disorders. In multivariate analysis, cell dose was the only unit-related factor associated with outcomes. Our results reflect the benefit on clinical outcomes of the strategy adopted by the Network to bank units with higher cell counts.

Human umbilical cord-derived mesenchymal stem/stromal cells: a promising candidate for the development of advanced therapy medicinal products.

Umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs) emerge as a perspective for therapeutic use in immune and inflammatory diseases. Indeed, immunomodulatory and anti-inflammatory properties, associated to fewer ethical, availability, and safety issues, position UC-MSCs as a promising active substance to develop medicinal products. Since 2007, UC-MSC-based products are classified as advanced therapy medicinal products (ATMP) according to the European Regulation 1394/2007/EC. This new regulatory status required a total adaptation of stakeholders wishing to develop UC-MSC-based ATMPs. Cell production in tissue and cell banks has been replaced by the manufacturing of a medicine, in authorized establishments, according to the good manufacturing practices (GMP) specific to ATMPs. After a brief description of UC-MSCs, we described in this review their recent use in a large panel of immune and inflammatory pathologies, including early and late phase clinical trials. Despite the use of the same product, we noticed an important heterogeneity in terms of indication, posology and study design. Then, we discussed regulatory and manufacturing challenges for stakeholders, especially in terms of process harmonization and cells characterization. Our aim was to point that despite MSCs use for several decades, the development of an UC-MSC-based ATMP remains at this day a real challenge for both academic institutions and pharmaceutical companies.