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Background: Many clinical trials have reported the use of mesenchymal stromal cells (MSCs) following the indication of severe SARS-CoV-2 infection. However, in the COVID19 pandemic context, academic laboratories had to adapt a production process to obtain MSCs in a very short time. Production processes, especially freezing/thawing cycles, or culture medium have impacts on MSC properties. We evaluated the impact of an intermediate cryopreservation state during MSC culture to increase production yields. Methods: Seven Wharton’s jelly (WJ)-MSC batches generated from seven different umbilical cords with only one cryopreservation step and 13 WJ-MSC batches produced with intermediate freezing were formed according to good manufacturing practices. The identity (phenotype and clonogenic capacities), safety (karyotype, telomerase activity, sterility, and donor qualification), and functionality (viability, mixed lymphocyte reaction) were analyzed. Results: No significant differences between MSC production processes were observed, except for the clonogenic capacity, which was decreased, although it always remained above our specifications. Conclusions: Intermediate cryopreservation allows an increase in the production yield and has little impact on the basic characteristics of MSCs.

BackgroundWe explored whether the disialoganglioside GD2 (GD2) is expressed in small cell lung cancer (SCLC) and non-SCLC (NSCLC) and can be targeted by GD2-specific chimeric antigen receptor (CAR) T cells.MethodsGD2 expression was evaluated in tumor cell lines and tumor biopsies by flow cytometry and immunohistochemistry. We used a GD2.CAR that coexpress the IL-15 to promote T-cell proliferation and persistence, and the inducible caspase 9 gene safety switch to ablate GD2.CAR-T cells in case of unforeseen toxicity. The antitumor activity of GD2.CAR-T cells was evaluated using in vitro cocultures and in xenograft models of orthotopic and metastatic tumors. The modulation of the GD2 expression in tumor cell lines in response to an epigenetic drug was also evaluated.ResultsGD2 was expressed on the cell surface of four of fifteen SCLC and NSCLC cell lines (26.7%) tested by flow cytometry, and in 39% of SCLC, 72% of lung adenocarcinoma and 56% of squamous cell carcinoma analyzed by immunohistochemistry. GD2 expression by flow cytometry was also found on the cell surface of tumor cells freshly isolated from tumor biopsies. GD2.CAR-T cells exhibited antigen-dependent cytotoxicity in vitro and in vivo in xenograft models of GD2-expressing lung tumors. Finally, to explore the applicability of this approach to antigen low expressing tumors, we showed that pretreatment of GD2low/neg lung cancer cell lines with the Enhancer of zeste homolog 2 inhibitor tazemetostat upregulated GD2 expression at sufficient levels to trigger GD2.CAR-T cell cytotoxic activity.ConclusionsGD2 is a promising target for CAR-T cell therapy in lung cancer. Tazemetostat treatment could be used to upregulate GD2 expression in tumor cells, enhancing their susceptibility to CAR-T cell targeting.

The development of Chimeric Antigen Receptor T cells therapy initiated by the United States and China is still currently led by these two countries with a high number of clinical trials, with Europe lagging in launching its first trials. In this systematic review, we wanted to establish an overview of the production of CAR-T cells in clinical trials around the world, and to understand the causes of this delay in Europe. We particularly focused on the academic centers that are at the heart of research and development of this therapy. We counted 1087 CAR-T cells clinical trials on ClinicalTrials.gov (Research registry ID: reviewregistry1542) on the date of 25 January 2023. We performed a global analysis, before analyzing the 58 European trials, 34 of which sponsored by academic centers. Collaboration between an academic and an industrial player seems to be necessary for the successful development and application for marketing authorization of a CAR-T cell, and this collaboration is still cruelly lacking in European trials, unlike in the leading countries. Europe, still far behind the two leading countries, is trying to establish measures to lighten the regulations surrounding ATMPs and to encourage, through the addition of fundings, clinical trials involving these treatments.

Background Mechanical ventilation for pneumonia may contribute to lung injury due to factors that include mitochondrial dysfunction, and mesenchymal stem cells may attenuate injury. This study hypothesized that mechanical ventilation induces immune and mitochondrial dysfunction, with or without pneumococcal pneumonia, that could be mitigated by mesenchymal stem cells alone or combined with antibiotics. Methods Male rabbits underwent protective mechanical ventilation (8 ml/kg tidal volume, 5 cm H2O end-expiratory pressure) or adverse mechanical ventilation (20 ml/kg tidal-volume, zero end-expiratory pressure) or were allowed to breathe spontaneously. The same settings were then repeated during pneumococcal pneumonia. Finally, infected animals during adverse mechanical ventilation received human umbilical cord–derived mesenchymal stem cells (3 × 106/kg, intravenous) and/or ceftaroline (20 mg/kg, intramuscular) or sodium chloride, 4 h after pneumococcal challenge. Twenty-four-hour survival (primary outcome), lung injury, bacterial burden, immune and mitochondrial dysfunction, and lung transcriptomes (secondary outcomes) were assessed. Results High-pressure adverse mechanical ventilation reduced the survival of infected animals (0%; 0 of 7) compared with spontaneous breathing (100%; 7 of 7) and protective mechanical ventilation (86%; 6 of 7; both P < 0.001), with higher lung pathology scores (median [interquartile ranges], 5.5 [4.5 to 7.0] vs. 12.6 [12.0 to 14.0]; P = 0.046), interleukin-8 lung concentrations (106 [54 to 316] vs. 804 [753 to 868] pg/g of lung; P = 0.012), and alveolar mitochondrial DNA release (0.33 [0.28 to 0.36] vs. 0.98 [0.76 to 1.21] ng/μl; P < 0.001) compared with infected spontaneously breathing animals. Survival (0%; 0 of 7; control group) was improved by mesenchymal stem cells (57%; 4 of 7; P = 0.001) or ceftaroline alone (57%; 4 of 7; P < 0.001) and improved even more with a combination treatment (86%; 6 of 7; P < 0.001). Mesenchymal stem cells reduced lung pathology score (8.5 [7.0 to 10.5] vs. 12.6 [12.0 to 14.0]; P = 0.043) and alveolar mitochondrial DNA release (0.39 (0.34 to 0.65) vs. 0.98 (0.76 to 1.21) ng/μl; P = 0.025). Mesenchymal stem cells combined with ceftaroline reduced interleukin-8 lung concentrations (665 [595 to 795] vs. 804 [753 to 868] pg/g of lung; P = 0.007) compared to ceftaroline alone. Conclusions In this preclinical study, mesenchymal stem cells improved the outcome of rabbits with pneumonia and high-pressure mechanical ventilation by correcting immune and mitochondrial dysfunction and when combined with the antibiotic ceftaroline was synergistic in mitigating lung inflammation. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Resume Les cellules T a recepteur antigenique chimerique (CAR T-cells) sont une nouvelle classe de traitements anticancereux produits en manipulant genetiquement des lymphocytes T autologues ou allogeniques afin de generer l’expression d’un recepteur chimerique dirige contre un antigene exprime sur la membrane de cellules tumorales. En Europe, le tisagenlecleucel (Kymriah™) dispose d’une autorisation de mise sur le marche (AMM) pour le traitement de la leucemie aigue lymphoblastique B en rechute/refractaire chez l’enfant et le jeune adulte et celui du lymphome B diffus a grandes cellules (LBDGC) en rechute/refractaire. L’AMM de l’axicabtagene ciloleucel (Yescarta™) porte sur le traitement du LBDGC et du lymphome primitif a cellules B du mediastin en rechute/refractaire. Les deux medicaments de therapie genique aujourd’hui autorises en Europe sont constitues de cellules T autologues genetiquement modifiees pour exprimer un CAR dirige contre CD19, un antigene exprime tout au long de la differenciation lymphoide B et sur de nombreuses hemopathies malignes B. Ce travail collaboratif fait partie d’une serie de travaux d’experts et a pour but de proposer des conseils pratiques pour aider les preleveurs a effectuer le prelevement des cellules mononucleees sanguines autologues et fournir le materiel cellulaire de depart a l’industriel fabricant, en vue de la production pharmaceutique de CAR T-cells.

Background Mesenchymal stem/stromal cells (MSC) have immunomodulatory properties, studied in a wide range of diseases. Validated quality controls must confirm this activity in the context of clinical trials. This study presents a method’s validation, assessing MSC’s ability to inhibit lymphocyte proliferation, according to the ICH Q2 standard. Methods MSC were co-cultured with CellTrace™ Violet-labeled Peripheral blood mononuclear cells (PBMC) coming from a bank of ten donors, at seven different ratios for 7 days. Cell trace violet PBMC bank was validated in parallel. Flow cytometry analysis was used to obtain the division percentage of T cells. The percentage of inhibition of lymphocyte proliferation by MSC, for each ratio X, was calculated using the formula: Ratio × percentage of inhibition = (control percentage of division—ratio × percentage of division)/control percentage of division. The inhibition percentage of lymphocyte proliferation function of co-culture ratios was represented in a line graph. The corresponding area under the curve was calculated, representing MSC’s ability to inhibit lymphocyte proliferation. Results Two cell trace violet PBMC banks were compared for bank validation. When compared using four different MSC samples coming each from a different donor, their area under the curve did not show any statistical differences and were correlated. Moreover, the stability of one cell trace violet PBMC bank was confirmed up to 509 days of storage. Analytical parameters were investigated for method validation. Analysis of repeatability and reproducibility respectively showed a standard deviation of 6.1% and 4.6%. The assay was robust regarding PBMC, as no statistical differences were found between inhibitory activities when testing three adjacent concentrations of PBMC. Still, attention is needed on MSC quantity as it can influence results. Linearity was evaluated: the percentage of inhibition of lymphocyte proliferation function of co-culture ratios was linear on the exploited range. Finally, the assay measurement range allowed to differentiate MSC presenting different inhibition activities. Conclusion This quantification method displayed low analytical variability and no inter-bank variability of PBMC. However, MSC quantification should be checked before co-culture to reduce variability. Therefore, it could be used for the qualification of MSC batches’ immunomodulatory activity.

Chimeric antigen receptor (CAR) T-cells are a new class of cancer treatments manufactured through autologous or allogeneic T cells genetic engineering to induce CAR expression directed against a membrane antigen present at the surface of malignant cells. In Europe, tisagenlecleucel (Kymriah™) has a marketing authorization for the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia in children and young adults and for the relapsed/refractory diffuse large B-cell lymphoma (DLBCL). The marketing authorization for axicabtagene ciloleucel (Yescarta™) is the treatment of relapsed/refractory DLBCL and mediastinal B-cell lymphoma. Both products are "living drugs" and genetically modified autologous T cells directed against CD19 which is an antigen expressed throughout B lymphoid differentiation and on many B malignancies. This collaborative work - part of a series of expert works on the topic - aims to provide practical advice to assist collection facilities that procure the starting material i.e. blood mononuclear cells for autologous CAR T-cell manufacturing.

Background: Umbilical mesenchymal stem/stromal cells (MSCs), and especially those derived from Wharton’s jelly (WJ), are a promising engineering tool for tissue repair in an allogeneic context. This is due to their differentiation capacity and immunological properties, like their immunomodulatory potential and paracrine activity. Hence, these cells may be considered an Advanced Therapy Medicinal Product (ATMP). The purpose of this work was to differentiate MSCs from WJ (WJ-MSCs) into chondrocytes using a scaffold and to evaluate, in vitro, the immunomodulatory capacities of WJ-MSCs in an allogeneic and inflammatory context, mimicked by IFN-γ and TNF-α priming during the chondrogenic differentiation. Methods: Scaffolds were made from hydrogel composed by alginate enriched in hyaluronic acid (Alg/HA). Chondrogenic differentiation, immunological function, phenotype expression, but also secreted soluble factors were the different parameters followed during 28 days of culture. Results: During chondrocyte differentiation, even in an allogeneic context, WJ-MSCs remained unable to establish the immunological synapse or to induce T cell alloproliferation. Moreover, interestingly, paracrine activity and functional immunomodulation were maintained during cell differentiation. Conclusion: These results show that WJ-MSCs remained hypoimmunogenic and retained immunomodulatory properties even when they had undergone chondrocyte differentiation.

International audience; BACKGROUND: The use of mesenchymal stem cells (MSCs) is being extensively studied in clinical trials in the setting of various diseases including diabetes, stroke, and progressive multiple sclerosis. The unique immunomodulatory properties of MSCs also point them as a possible therapeutic tool during sepsis and septic shock, a devastating syndrome associated with 30-35% mortality. However, MSCs are not equal regarding their activity, depending on their tissue origin. Here, we aimed at comparing the in vivo properties of MSCs according to their tissue source (bone marrow (BM) versus Wharton's jelly (WJ)) in a murine cecal ligation and puncture (CLP) model of sepsis that mimics a human peritonitis. We hypothesized that MSC properties may vary depending on their tissue source in the setting of sepsis.METHODS: CLP, adult, male, C57BL/6 mice were randomized in 3 groups receiving respectively 0.25 × 106 BM-MSCs, 0.25 × 106 WJ-MSCs, or 150 μL phosphate-buffered saline (PBS) intravenously 24 h after the CLP procedure.RESULTS: We observed that both types of MSCs regulated leukocyte trafficking and reduced organ dysfunction, while only WJ-MSCs were able to improve bacterial clearance and survival.CONCLUSION: This study highlights the importance to determine the most appropriate source of MSCs for a given therapeutic indication and suggests a better profile for WJ-MSCs during sepsis.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for treatment of some malignant and non-malignant hematological diseases. However, post-HSCT patients are severely immunocompromised and susceptible to viral infections, which are a major cause of morbidity and mortality. Although antiviral agents are now available for most types of viral infections, they are not devoid of side effects and their efficacy is limited when there is no concomitant antiviral immune reconstitution. In recent decades, adoptive transfer of viral-specific T cells (VSTs) became an alternative treatment for viral infection after HSCT. However, two major issues are concerned in VST transfer: the risk of GVHD and antiviral efficacy. We report an exhaustive review of the published studies that focus on prophylactic and/or curative therapy by donor VST transfer for post-HSCT common viral infections. A low incidence of GVHD and a good antiviral efficacy was observed after adoptive transfer of VSTs from HSCT donor. Viral-specific T-cell transfer is a promising approach for a broad clinical application. Nevertheless, a randomized controlled study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with VSTs is still needed to demonstrate efficacy and safety.

International audience; Background & AimMesenchymal Stromal/Stem Cells (MSC) are a heterogeneous population of self-renewal multipotent cells which can be obtained from adult sources (Bone Marrow MSC (BMMSC)) and fetal sources (Wharton Jelly MSC (WJMSC)). MSC are a very interesting tool for tissue engineering because of their differentiation properties and for cell therapy because of their immunomodulation properties. Indeed, MSC secrete soluble factors which can modulate the immune response.Before their clinical use, an in vitro expansion step must be performed in order to obtain the sufficient dose. However, this expansion step could affect MSC quality and need to be controlled in order to increase the therapeutic efficacy.The first challenge was to optimize the culture conditions and to choose the most appropriate media for the MSC culture to ensure that they retain their functional properties and thus their therapeutic potential. The second challenge was to know the impact of in vitro expansion on MSC functional properties.Methods, Results & ConclusionWe firstly studied the impact of culture conditions (normoxia (N), hypoxia (H), fetal calf serum (FCS) supplement, human platelet lysate (hPL) supplement on the behavior of MSC. Our results showed that better proliferation properties were obtained for MSC expanded with human platelet lysate (hPL) in hypoxia for WJMSC and in normoxia for BMMSC. Moreover, we observed that WJMSC and BMMSC expanded with hPL have better clonogenicity and are less senescent. Using neutralizing antibodies, we confirmed the involvement of high concentration of growth factors (PDGF, EGF,…) in hPL in WJMSC proliferation properties. The main surface markers and differentiation capacities were found to be equivalent for WJMSC and BMMSC for the different culture conditions.We secondly studied the impact of in vitro expansion on MSC immunomodulation properties. We showed that WJMSC and BMMSC expanded in hypoxia have better immunosuppressive properties when they are co-cultivated with CD4+ T cells and that senescence and interferon-γ (IFN-γ) stimulation could affect the MSC immunomodulatory properties.To conclude MSC expansion with hPL gives better clonogenicity properties and better proliferation properties in normoxia for BMMSC and in hypoxia for WJMSC without affecting surface markers and differentiation properties. Moreover, WJMSC expanded in hypoxia with hPL with low senescence are more immunosuppressive. This property can be improved by IFN-γ stimulation.

The present study proposed to compare the impact of agitation mode (static, orbital, and mechanical) on the culture of mesenchymal stem cells extracted from the Wharton's jelly of umbilical cords (WJ-MSC), in a clinical grade culture medium, using human platelet lysate and different xeno-free microcarriers. Attachment, expansion, and detachment performances were characterized by a new dedicated tool of microscopic image posttreatment, allowing an in situ cell counting without detachment step. Results showed that performances in static mode were not necessarily representative of those obtained in dynamic mode. Moreover, impacts on nutrient consumptions and metabolite productions were identified, such as a higher glutamine consumption when Cytodex-1 microcarriers were used. The detachment strategy used was relatively efficient for Star-Plus, Plastic-Plus, and Hillex II, but not sufficient for Cytodex-1. Despite Cytodex-1 presented promising attachment and expansion performances, Star-Plus and Plastic-Plus showed a better compromise, respectively, for the orbital and the mechanical agitation modes.

International audience; Mesenchymal stem cells (MSC) have emerged as alternative sources of stem cells for regenerative medicine because of their multipotency and strong immune-regulatory properties. Also, human leukocyte antigen G (HLA-G) is an important mediator of MSC-mediated immunomodulation. However, it is unclear whether MSC retain their immune-privileged potential after differentiation. As promising candidates for cartilage tissue engineering, the immunogenic and immunomodulatory properties of chondro-differentiated MSC (chondro-MSC) require in-depth exploration. In the present study, we used the alginate/hyaluronic acid (Alg/HA) hydrogel scaffold and induced both bone marrow- and adipose tissue-derived MSC into chondrocytes in three-dimensional condition. Then, MSC before and after chondrocyte differentiation were treated or not with interferon γ and tumor necrosis factor α mimicking inflammatory conditions and were compared side by side using flow cytometry, mixed lymphocyte reaction, and immunostaining assays. Results showed that chondro-MSC were hypoimmunogenic and could exert immunosuppression on HLA-mismatched peripheral blood mononuclear cells as well as undifferentiated MSC did. This alloproliferation inhibition mediated by MSC or chondro-MSC was dose dependent. Meanwhile, chondro-MSC exerted inhibition on natural killer cell-mediated cytolysis. Also, we showed that HLA-G expression was upregulated in chondro-MSC under hypoxia context and could be boosted in allogenic settings. Besides, the Alg/HA hydrogel scaffold was hypoimmunogenic and its addition for supporting MSC chondrocyte differentiation did not modify the immune properties of MSC. Finally, considering their chondro-regenerative potential and their retained immunosuppressive capacity, MSC constitute promising allogenic sources of stem cells for cartilage repair.

Many clinical trials report mesenchymal stem/stromal cells (MSCs) efficacy in various indications. Therefore, standardization of MSC production becomes necessary. MSC properties are impacted by tissue origin, especially if they are from extraembryonic tissue or adult sources. For this reason, we evaluated the impact of MSC tissue origin on production. Methods: Three productions of MSC from Wharton’s Jelly (WJ) or from bone marrow (BM) were performed according to good manufacturing practice. The identity (phenotype, differentiation, and clonogenic capacities), safety (karyotype, telomerase activity, sterility, and donor qualification), and functionality (viability, mixed lymphocyte reaction) of each cell batch were analyzed. Results: Slight differences between MSC sources were observed for phenotype, telomerase activity, and clonogenic capacities. Conclusion: Both sources have made it possible to quickly and easily obtain clinical grade MSC. However, as availability of the source is thought to be essential, WJ seems more advantageous than BM.

Sepsis and septic shock are the leading cause of admission and mortality in non-coronary intensive care units. Currently, however, no specific treatments are available for this syndrome. Due to the failure of conventional treatments in recent years, research is focusing on innovative therapeutic agents, including cell therapy. One particular type of cell, mesenchymal stromal/stem cells (MSCs), has raised hopes for the treatment of sepsis. Indeed, their immunomodulatory properties, antimicrobial activity and capacity of protection against organ failure confer MSCs with a major advantage to treat the immune and inflammatory dysfunctions associated with sepsis and septic shock. After a brief description of the pathophysiology of sepsis and septic shock, the latest advances in the use of MSCs to treat sepsis will be presented.

International audience; The ability of natural killer (NK) cells to kill tumor cells without antigen recognition makes them appealing as an adoptive immunotherapy. However, NK cells are not routinely used in the context of leukemic relapse after hematopoietic stem cell transplantation. Patients who experience relapse can be treated with donor lymphocyte infusions (DLI) based on small-cell fractions frozen at the time of transplantation. Since peripheral blood stem cells (PBSCs) are increasingly used as a stem cell source and as a source of cells for DLI, we aimed to evaluate the impact of G-SCF mobilization on NK cell phenotype, subset repartition, and functionality. Immunomagnetically isolated NK cells from healthy donor blood, donor PBSCs, and patient PBSCs were expanded for 14 days with IL-15. The expansion capacity, phenotype, and functions (cytokine secretion and cytotoxicity) of NK cell subsets based on CD56 and CD16 expression were then evaluated. Mobilized sources showed a significant decrease of CD56brightCD16+ NK cells (28 versus 74%), whereas a significant increase (64 versus 15%) of CD56brightCD16- NK cells was observed in comparison with peripheral blood. Patient-mobilized NK cells showed a significantly decreased cytotoxicity, and antibody-dependent cell cytototoxicity (ADCC) was also observed to a lesser extent in NK cells from healthy donor PBSC. G-CSF-mobilized NK cell TNF-α and IFN-γ secretion was impaired at day 0 compared to healthy donors but was progressively restored after culture. In conclusion, expansion of NK cells from G-CSF-mobilized sources may progressively improve their functionality.

International audience; Virus-specific T-cell (VST) infusion becomes a promising alternative treatment for refractory viral infections after hematopoietic stem cell transplantation (HSCT). However, VSTs are often infused during an immunosuppressive treatment course, especially corticosteroids, which are a first-line curative treatment of graft-versus-host disease (GVHD). We were interested in whether corticosteroids could affect adenovirus (ADV)-VST functions. After interferon (IFN)-γ based immunomagnetic selection, ADV-VSTs were in vitro expanded according to three different culture conditions: without methylprednisolone (MP; n = 7), with a final concentration of MP 1 µg/mL (n = 7) or MP 2 µg/mL (n = 7) during 28 ± 11 days. Efficacy and alloreactivity of expanded ADV-VSTs was controlled in vitro. MP transitorily inhibited ADV-VST early expansion. No impairment of specific IFN-γ secretion capacity and cytotoxicity of ADV-VSTs was observed in the presence of MP. However, specific proliferation and alloreactivity of ADV-VSTs were decreased in the presence of MP. Altogether, these results and the preliminary encouraging clinical experiences of co-administration of MP 1 mg/kg and ADV-VSTs will contribute to safe and efficient use of anti-viral immunotherapy.

Background Allogeneic hematopoietic stem cell transplantation (HSCT), the most widely used potentially curable cellular immunotherapeutic approach in the treatment of hematological malignancies, is limited by life-threatening complications: graft versus host disease (GVHD) and infections especially viral infections refractory to antiviral drugs. Adoptive transfer of virus-specific T cells is becoming an alternative treatment for infections following HSCT. We report here the results of a phase I/II multicenter study which includes a series of adenovirus-specific T cell (ADV-VST) infusion either from the HSCT donor or from a third party haploidentical donor for patients transplanted with umbilical cord blood (UCB). Methods Fourteen patients were eligible and 11 patients received infusions of ADV-VST generated by interferon (IFN)-γ-based immunomagnetic isolation from a leukapheresis from their original donor (42.9%) or a third party haploidentical donor (57.1%). One patient resolved ADV infection before infusion, and ADV-VST could not reach release or infusion criteria for two patients. Two patients received cellular immunotherapy alone without antiviral drugs as a pre-emptive treatment. Results One patient with adenovirus infection and ten with adenovirus disease were infused with ADV-VST (mean 5.83 ± 8.23 × 103 CD3+IFN-γ+ cells/kg) up to 9 months after transplantation. The 11 patients showed in vivo expansion of specific T cells up to 60 days post-infusion, associated with adenovirus load clearance in ten of the patients (91%). Neither de novo GVHD nor side effects were observed during the first month post-infusion, but GVHD reactivations occurred in three patients, irrespective of the type of leukapheresis donor. For two of these patients, GVHD reactivation was controlled by immunosuppressive treatment. Four patients died during follow-up, one due to refractory ADV disease. Conclusions Adoptive transfer of rapidly isolated ADV-VST is an effective therapeutic option for achieving in vivo expansion of specific T cells and clearance of viral load, even as a pre-emptive treatment. Our study highlights that third party haploidentical donors are of great interest for ADV-VST generation in the context of UCB transplantation. (N° Clinical trial.gov: NCT02851576, retrospectively registered). Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0469-0) contains supplementary material, which is available to authorized users.

International audience; Most human tissues do not regenerate spontaneously, which is why "cell therapy" are promising alternative treatments. The Principe is simple: patients' or donors' cells are collected and introduced into the injured tissues or organs directly or in a porous 3D material, with or without modification of their properties. This concept of regenerative medicine is an emerging field which can be defined as "the way to improve health and quality of life by restoring, maintaining, or enhancing tissue and organ functions".There is an extraordinarily wide range of opportunities for clinical applications: artheropathies, diabetes, cartilage defects, bone repair, burns, livers or bladder regeneration, organs reconstruction (lung, heart, liver...) neurodegenerative disorders, sepsis...Different stem cells (SC) with different potential can be used and characterised (totipotent, mesenchymal of different origins, especially those present in tissues...). Today it is undeniable that cells like bone marrow, adipose tissue or Wharton Jelly stem cells, are of potential interest for clinical applications because they are easily separated and prepared and no ethical problems are involved in their use.In this paper some potential clinical applications in the vascular field are considered: peripheral arteriopathy in diabetic patients, cardiac insufficiency, traitment of erectile dysfunction, or organ regeneration with liver as example. But the regeneration of tissue or organ is and will remain a challenge for the future development of cell therapy. Many problems remain to be solved that could lead to the development of innovative strategies to facilitate cell differentiation, increase the yield of cells and ensure a standardised product, overcome the risks of teratogenic effects and/or immune reactions, enable grafting via direct cell or biotissue transplantation and avoid legal issues involved in national regulations.

Cryopreservation as a way to maintain extracorporeal photopheresis regimen for GvHD treatment while circumventing patient temporary inability to undergo apheresis

International audience; Adoptive antiviral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is becoming an alternative treatment for viral infection after hematopoietic stem cell transplantation. The T memory stem cell (TSCM) subset was recently described as exhibiting self-renewal and multipotency properties which are required for sustained efficacy in vivo. We wondered if such a crucial subset for immunotherapy was present in VSTs. We identified, by flow cytometry, TSCM in adenovirus (ADV)-specific interferon (IFN)-γ+ T cells before and after IFN-γ-based immunomagnetic selection, and analyzed the distribution of the main T-cell subsets in VSTs: naive T cells (TN), TSCM, T central memory cells (TCM), T effector memory cell (TEM), and effector T cells (TEFF). In this study all of the different T-cell subsets were observed in the blood sample from healthy donor ADV-VSTs, both before and after IFN-γ-based immunomagnetic selection. As the IFN-γ-based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets, we observed that TEM was always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation and especially after expansion in vitro. Comparing T-cell subpopulation profiles before and after in vitro expansion, we observed that in vitro cell culture with interleukin-2 resulted in a significant expansion of TN-like, TCM, TEM, and TEFF subsets in CD4IFN-γ T cells and of TCM and TEM subsets only in CD8IFN-γ T cells. We demonstrated the presence of all T-cell subsets in IFN-γ VSTs including the TSCM subpopulation, although this was weakly selected by the IFN-γ-based immunomagnetic selection system.

5th China-France International Symposium, Kunming, PEOPLES R CHINA, DEC 12-14, 2013; International audience; Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have ability to proliferate and differentiate in different tissues. Different stem cells (SC) with different potential can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue or MSC of Wharton Jelly, which have limited potential, are of interest for clinical applications in regenerative medicine because they are easily separated and prepared and no ethical problems are involved in their use. During the last 10 years, these multipotent cells have generated considerable interest and in particular have been shown to escape allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone, cartilage, diabetes, ophthalmology, urology, liver, organ's reconstruction ...).

International audience; Mesenchymal Stromal/Stem Cells from human Wharton's jelly (WJ-MSC) are an abundant and interesting source of stem cells for applications in cell and tissue engineering. Their fetal origin confers specific characteristics compared to Mesenchymal Stromal/Stem Cells isolated from human bone marrow (BM-MSC). The aim of this work was to optimize WJ-MSC culture conditions for their subsequent clinical use. We focused on the influence of oxygen concentration during monolayer expansion on several parameters to characterize MSC. Our work distinguished WJ-MSC from BM-MSC in terms of proliferation, telomerase activity and adipogenic differentiation. We also showed that hypoxia had a beneficial effect on proliferation potential, clonogenic capacity and to a lesser extent, on HLA-G expression of WJ-MSC during their expansion. Moreover, we reported for the first time an increase in chondrogenic differentiation when WJ-MSC were expanded under hypoxia. In an allogeneic therapeutic context, production of clinical batches requires generating high numbers of MSC whilst maintaining the cells' properties. Considering our results, hypoxia will be an important parameter to take into account. In addition, the clinical use of WJ-MSC would provide significant numbers of cells with maintenance of their proliferation and differentiation potential, particularly their chondrogenic potential. Due to their chondrogenic differentiation potential, WJ-MSC promise to be an interesting source of MSC for cell therapy or tissue engineering for cartilage repair and/or regeneration.

International audience; Le sommaire de ce numéro http://www.john-libbey-eurotext.fr/fr/ revues/medecine/hma/sommaire.md?type= text.html Montrouge, le 14/08/2012 Loïc Reppel Vous trouverez ci-après le tirétiré`tiréà part de votre article au formatélectroniqueformat´formatélectronique (pdf) : Cellules souches et nouvelle loi de bioéthique française paru dans Hématologie, 2012, Volume 18, Numéro 3 John Libbey Eurotext Ce tirétiré`tiréà part numérique vous est délivré pour votre propre usage et ne peutêtrepeutêtre transmisàtransmis`transmisà des tiers qu'` a des fins de recherches personnelles ou scientifiques. En aucun cas, il ne doit faire l'objet d'une distribution ou d'une utilisation promotionnelle, commerciale ou publicitaire. Tous droits de reproduction, d'adaptation, de traduction et de diffusion réservés pour tous pays. Résumé. En juillet 2011, la loi de bioéthique française a fait l'objet d'une révision apportant, dans le domaine des cellules souches, quelques évolutions par rapport à la loi précédente. Concernant les cellules souches embryonnaires (CSE), la nouvelle loi de bioéthique confirme la possibilité d'effectuer des recherches sur l'embryon et sur les CSE uniquement sur dérogations strictement encadrées et délivrées par l'Agence de la biomédecine. En revanche, l'avancée majeure de cette loi repose, à présent, sur un encadrement spécifique des cellules souches hématopoïétiques du sang placentaire en vue d'une utilisation thérapeutique ou scientifique. Ce dernier n'est plus considéré comme un déchet opératoire mais bien comme une source à part entière de cellules souches hématopoïétiques au même titre que la moelle osseuse et le sang périphérique. Abstract. In July 2011, the French bioethics law was the subject of a revision bringing few remarkable changes compared to the previous law, especially in the field of stem cells. Regarding Embryonic stem cells (ESC), the new law confirms the possibility to carry out research on the embryo and ESC exclusively after obtention of a derogation strictly regulated and delivered by the French Biomedicine Agency. On the other hand, the main advance of this law concerns a specific regulating of cord blood hematopoietic stem cells for therapeutic or scientific uses. This collection site is no longer considered as a medical waste but as a source of hematopoietic stem cells as well as bone marrow and peripheral blood.

International audience; Mesenchymal stem cells (MSCs) are useful multipotent stem cells that are found in many tissues. While MSCs can usually be isolated from adults via bone marrow aspiration (BM-MSCs), MSCs derived from the discarded umbilical cord, more precisely from Wharton's jelly (WJ), offer a low-cost and pain-free collection method of MSCs that may be cryogenically stored, and are considered extremely favorable for tissue engineering purpose. The aim of this study was to analyze the harvested number of cells per centimeter of human umbilical cord (UC) and carry out the phenotype of these WJ-MSCs after explant or enzymatic methods. Fresh UCs were obtained from full-term births, and processed within 6 hours from partum to obtain the WJ-MSCs. UC sections were analyzed in confocal microscopy to analyze cells phenotype in situ. Others UC components were treated either by enzymatic method or by explant method to obtain isolated cells and to analyze cells phenotype until the end of the first passage. We have successfully generated MSCs from UC by using explant and enzymatic methods. Using microscopy confocal, we identified the expression of some MSCs markers in situ of Wharton's jelly tissue as well as in perivascular region. Our comparative study, between explant and enzymatic digestion, indicated, that WJ expressed most of MSCs markers in both conditions, but a remarkable variation of cell phenotype expression was distinguished after primary culture comparing to directly isolated cells by enzymatic digestion. We also studied the expression of CD271, which showed to be weakly expressed in situ on fresh fragment of WJ.

L. Reppel , T. Margossian , P. Moreau , N. Mercier , J.-F. Stoltz , C. Huselstein , D. Bensoussan . CNRS UMR 7561, Biopole-Campus Sante, Vandoeuvre-les-Nancy, France; 2 Lorraine Univ., 54000 Nancy, France; Commissariat a l'Energie Atomique/DSV/I2BM/Service de Recherches en Hemato-Immunologie, IUH, Hopital Saint-Louis, 75010 Paris, France; 4 INSERM U684, Faculte de Medecine, Vandoeuvre-les-Nancy, France; CHU de Nancy Brabois, Unite de Therapie Cellulaire et Tissulaire, 54511 Vandœuvre-les-Nancy, France

Background Due to their intrinsic properties, stem cells are promising tools for new developments in tissue engineering and particularly for cartilage tissue regeneration. Although mesenchymal stromal/stem cells from bone marrow (BM-MSC) have long been the most used stem cell source in cartilage tissue engineering, they have certain limits. Thanks to their properties such as low immunogenicity and particularly chondrogenic differentiation potential, mesenchymal stromal/stem cells from Wharton’s jelly (WJ-MSC) promise to be an interesting source of MSC for cartilage tissue engineering. Methods In this study, we propose to evaluate chondrogenic potential of WJ-MSC embedded in alginate/hyaluronic acid hydrogel over 28 days. Hydrogels were constructed by the original spraying method. Our main objective was to evaluate chondrogenic differentiation of WJ-MSC on three-dimensional scaffolds, without adding growth factors, at transcript and protein levels. We compared the results to those obtained from standard BM-MSC. Results After 3 days of culture, WJ-MSC seemed to be adapted to their new three-dimensional environment without any detectable damage. From day 14 and up to 28 days, the proportion of WJ-MSC CD73+, CD90+, CD105+ and CD166+ decreased significantly compared to monolayer marker expression. Moreover, WJ-MSC and BM-MSC showed different phenotype profiles. After 28 days of scaffold culture, our results showed strong upregulation of cartilage-specific transcript expression. WJ-MSC exhibited greater type II collagen synthesis than BM-MSC at both transcript and protein levels. Furthermore, our work highlighted a relevant result showing that WJ-MSC expressed Runx2 and type X collagen at lower levels than BM-MSC. Conclusions Once seeded in the hydrogel scaffold, WJ-MSC and BM-MSC have different profiles of chondrogenic differentiation at both the phenotypic level and matrix synthesis. After 4 weeks, WJ-MSC, embedded in a three-dimensional environment, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins. Today, WJ-MSC represent a real alternative source of stem cells for cartilage tissue engineering.

La maladie de Crohn est une maladie inflammatoire chronique de l'intestin évoluant par poussées. Cette pathologie à l'étiologie encore imparfaitement identifiée provoque le plus souvent une diarrhée et des douleurs abdominales, et touche un nombre croissant de personnes à travers le monde. Aucun traitement curatif n'existe pour soigner la maladie de Crohn : sa prise en charge repose sur l'utilisation de molécules anti-inflammatoires que sont les corticoïdes et les dérivés aminosalicylés, et d'immunosuppresseurs visant à empêcher la réaction auto-immune afin de limiter la progression ou l'apparition de lésions muqueuses. Ces thérapeutiques classiques ne permettent cependant pas un contrôle efficace de la pathologie chez tous les patients, et elles exposent à la survenue d'évènements indésirables. C'est dans ce contexte qu'ont été développées les premières biothérapies pour traiter la maladie de Crohn. L'objectif de ce travail est de présenter les quatre biothérapies actuellement commercialisées dans la prise en charge de la maladie de Crohn (infliximab, adalimumab, védolizumab et ustékinumab), et de recenser les biothérapies en cours de développement, qu'il s'agisse d'anticorps monoclonaux, de protéines recombinantes ou encore de thérapie cellulaire.

Septic shock, equal to the myocardial infraction, is currently the tenth cause of death in the world. The pathophysiological complexity of this syndrome, with a simultaneous pro and anti- inflammatory state, results in the failure of conventional treatments. In this sense, research is focusing on innovative therapeutics agent, including mesenchymal stem cells (MSC). Indeed, murine studies of septic shock showed that MSC improve organ injuries, bacteremia and survival by notably a paracrine mechanism. However, MSC properties vary according to the source tissue, especially if they are derived from a fetal tissue (Wharton’s jelly (WJ), placenta amniotic fluid) or an adult tissue (bone marrow (BM), adipose tissue...). Our first objective was to compare, in a septic shock murine model, the effect of BM-MSC with that of WJ-MSC. Although some differences were observed, the same efficiency was demonstrated between these two sources. However, WJ-MSC present large advantages in comparison to BM-MSC due to their important proliferation capacities and potential quantities of umbilical cord donation. Consequently, our second objective was to investigate the effect of WJ-MSC administration in a relevant pig model of peritonitis in order to better mimic a clinical approach in humans. This study, conducted in double-blind and in presence of an experimented intensivist, showed that WJ-MSC produced in clinical grade and used immediately after thawing, improve survival, hemodynamic parameters and organ injuries by another action than that described in murine studies, Le choc septique est actuellement la dixième cause de mortalité à travers le monde à égalité avec les infarctus du myocarde. Sa physiopathologie extrêmement complexe, entrelaçant un état pro-inflammatoire et anti-inflammatoire, rend caduque l’action des thérapeutiques conventionnelles. En ce sens, les recherches s’orientent vers les thérapeutiques innovantes et notamment les cellules souches/stromales mésenchymateuses (CSM). En effet, les études murines ont mis en évidence que les CSM étaient en mesure, notamment par leurs actions paracrines, d’améliorer la survie, la défaillance d’organes mais également la bactériémie de souris soumises à un choc septique. Cependant, les propriétés des CSM varient en fonction du tissu dont elles sont issues et particulièrement selon qu’elles proviennent de tissus fœtaux (cordon ombilical, placenta, liquide amniotique) ou adultes (moelle osseuse, tissu adipeux...). Ainsi, notre premier objectif a été de comparer, dans un modèle murin de choc septique, l’action des CSM issues de la moelle osseuse (MO) à celle des CSM issues de la gelée de Wharton (GW) du cordon ombilical. Cette étude murine a permis de mettre en évidence une action quelque peu différente, entre les CSM-GW et les CSM-MO, sur la physiopathologie du choc septique sans que pour autant, l’une des deux sources de CSM, ne se dégage significativement de l’autre en termes d’efficacité. Cependant, en raison de leur importante capacité de prolifération et de l’accessibilité du tissu source, les CSM-GW apparaissent comme étant nettement plus avantageuses que les CSM-MO. En conséquence, notre deuxième objectif a été d’évaluer l’action des CSM-GW dans un modèle porcin de péritonite afin de se rapprocher un peu plus près de la clinique humaine. Cette étude, menée en double aveugle et en présence continue d’un médecin réanimateur expérimenté, a permis de mettre en évidence que les CSM-GW, produites en grade clinique et utilisées juste après décongélation, étaient en mesure d’améliorer la survie, les paramètres hémodynamiques ainsi que les défaillances d’organes, selon un mécanisme d’action différent de celui rapporté par les études murines