Search

International audience

International audience

International audience; 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; Question: Medulloblastoma is the most common malignant brain tumor in the pediatric population. Although conventional treatments improve survival, the chances of recovery remain low and result in severe neurological sequelae, especially in young children. CD3-CD56 + Natural Killer cells are able to recognize and kill tumor cells, particularly during the loss of class I MHC molecules. Their cytotoxic potential has already been demonstrated in vitro and in vivo with certain brain tumors but few studies concern medulloblastoma. Our aim was to evaluate the cytotoxic effect of allogeneic NK cells on medulloblastoma cells.Methods: Three lines of medulloblastoma subgroups (DAOY, D283 and D341) were analyzed and compared to the K562 leukemia cell line, known to be very sensitive to NK cells. The NK cells were selected from blood of healthy donors by immunomagnetic selection, and amplified in the presence of IL-15. After expansion, their cytotoxic potential was evaluated by different tests targeting each step involved in the process of tumor lysis: the phenotype of tumor cells by flow cytometry (expression of B7 molecules, CD56, 73, PVR and MICA/B), conjugation by flow cytometry and confocal microscopy, the degranulation by granzyme B elispot and finally the death of the target cells, by necrosis, late apoptosis and cell lysis (by flow cytometry and adenylate kinase measurement). The impact of co-culture with tumor cells on the expression of inhibitory or activating receptors was also analyzed. Nk cells were next injected in medulloblastoma xenografted nude mice and the survival was followed.Results: 3 medulloblastoma lines expressed CD56, PVR and CD105. The expression was heterogeneous for B7H1, H3 and H4, CD73, and MICA/B. The 3 lines were able to conjugate with the NK cells and to induce their degranulation. A necrosis percentage of between 30% (D283 and D341) and 38% (DAOY) could be measured after 4 hours of incubation. Percentages of cell lysis between 32 and 77% could be detected for the DAOY and D341 lines respectively. After co-culture, an increase in expression of the activating receptors NKG2D and DNAM1 could be observed while the expression of the inhibitory receptors KIR2DL1 and 2DL5 remained stable. These results suggested that NK cells were able to exert effective cytotoxicity against medulloblastoma. The in vivo survival study of medulloblastoma xenografted nude mice showed an increased survival of the NK cell treated group.

International audience

International audience

International audience; Endogenous and exogenous stresses could induce senescence, a phenomenon through which the capacity of cell division, growth, and function is lost. Even if senescence is considered a barrier against cancer since it blocks the proliferation of transformed cells, it contributes to organismal aging. Recently, senescence has been proposed to support other biological processes such as development and tissue repair. A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype.Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This calls for the investigation of the mechanisms of senescence and the development of efficient means to reverse it.

Background The umbilical cord is becoming a notable alternative to bone marrow (BM) as a source of mesenchymal stromal cells (MSC). Although age-dependent variations in BM-MSC are well described, less data are available for MSC isolated from Wharton’s jelly (WJ-MSC). We initiated a study to identify whether obstetric factors influenced MSC properties. We aimed to evaluate the correlation between a large number of obstetric factors collected during pregnancy and until peripartum (related to the mother, the labor and delivery, and the newborn) with WJ-MSC proliferation and chondrogenic differentiation parameters. Methods Correlations were made between 27 obstetric factors and 8 biological indicators including doubling time at passage (P)1 and P2, the percentage of proteoglycans and collagens, and the relative transcriptional expression of Sox-9, aggrecans, and total type 2 collagen (Coll2T). Results Amongst the obstetric factors considered, birth weight, the number of amenorrhea weeks, placental weight, normal pregnancy, and the absence of preeclampsia were identified as relevant factors for cell expansion, using multivariate linear regression analysis. Since all the above parameters are related to term, we concluded that WJ-MSC from healthy, full-term infants exhibit greater proliferation capacity. As for chondrogenesis, we also observed that obstetric factors influencing proliferation seemed beneficial, with no negative impact on MSC differentiation. Conclusions Awareness of obstetric factors influencing the proliferation and/or differentiation of WJ-MSC will make it possible to define criteria for collecting optimal umbilical cords with the aim of decreasing the variability of WJ-MSC batches produced for clinical use in cell and tissue engineering. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0609-z) contains supplementary material, which is available to authorized users.

International audience; n the framework of an ANR project obtained in 2015, we propose to develop a new advancedtherapy medicinal product for cartilage defect treatment with extemporaneous association of ascaffold and Wharton’s jelly mesenchymal stromal cells (WJ-MSC).Due to their capacity of proliferation and differentiation, MSC appear to be currently one of themost promising ways of obtaining cartilage cells. The conjunctive tissue of the umbilical cordor Wharton’s jelly is an abundant and promising source of MSC for clinical applications. Theypresent immunomodulatory properties and have a higher proliferation potential than MSC fromadult tissues. All these advantages enable this source to represent a virtually inexhaustiblesource of stem cells especially for allogeneic tissue engineering therapies.The first part of this work made it possible to perform a technological transfer from researchtowards the clinic. The transfer consisted in adjusting MSC isolation, production andconservation methods developed by research lab in clinical grade conditions.Eight umbilical cords were obtained after the signing of an informed consent form by pregnantmothers. In parallel, for each umbilical cord collected, well identified obstetric factors wererecorded. We developed a fast, simple and efficient explant method for the isolation of MSCfrom human umbilical cords requiring minimal manipulation and thus reducing the risk ofcontamination. MSC isolation, production and conservation were performed in a controlledatmosphere zone. All reagents and materials were well-defined and controlled. Cell culture wasperformed in culture containers (CellSTACK®, Macopharma) in a closed system. MSC werecultivated in hypoxia until passage 2 and then frozen. Quality controls were carried out duringproduction: serological and microbiological controls, cell number, viability, clonogeniccapacity, immunophenotype, mesodermic differentiation potential and karyotype.This isolation method showed a 100% success rate. We were able to obtain a large number ofviable and secured cells with an average number of 50.106 cells per CellSTACK and a doublingtime of about 1.5 days. During the culture, WJ-MSC exhibited a sustained clonogenic capacityand a specific mesenchymal phenotype.These results made it possible to assess the feasibility and to validate the clinical gradeamplification technique as well as to obtain precisely characterized and secured WJ-MSC.

International audience; PURPOSE:Review of various publications on stem cell therapy to treat erectile dysfunction of diabetic origin.MATERIAL AND METHODS:Bibliographic search in PUBMED performed using the keywords cell therapy strain/erectile dysfunction associated with diabetes. Among the 51 articles obtained from the PUBMED research, we selected 16 articles for their specificity of studying erectile dysfunction (DE) related to diabetes.RESULTS:Different types of stem cells have been studied: adipose derived mesenchymal stem cells/bone marrow derived mesenchymal stem cells as well as progenitor endothelial cells. The experimental protocols are quite similar from one study to the next with nevertheless some specifications concerning the studied cells and the monitoring of the latter. Intracavernous pressure (ICP) measured after the injection of stem cells into the corpus cavernosum was always significantly higher than the control populations. The addition of certain growth factors to stem cells by gene transfection improve the efficacy of the cells. No ideal tracking markers of the cells have been identified.CONCLUSION:The positive effect of the injection of stem cells on the ICP belongs to the cellular trans-differentiation effect but especially to the paracrine effects which have not yet been completely elucidated.; ObjectifRevue des différentes publications concernant l’utilisation des cellules souches pour traiter la dysfonction érectile d’origine diabétique.Matériel et méthodesRecherche bibliographique dans PUBMED réalisée en utilisant les mots clés : stem cell therapy/diabetes associated erectile dysfunction. Parmi les 51 articles obtenus de la recherche PUBMED, nous avons retenu 16 articles pour leur spécificité d’étude de la dysfonction érectile (DE) liée au diabète.RésultatsDifférents types de cellules souches ont été étudiés : mésenchymateuses adipeuses, ou issues de la moelle osseuse ainsi que des cellules endothéliales progénitrices. Les protocoles expérimentaux sont assez similaires d’une étude à l’autre avec néanmoins quelques spécificités concernant les cellules étudiées et le suivi de ces dernières. La pression intracaverneuse (PIC) mesurée après l’injection des cellules souches au sein des corps caverneux a toujours été significativement supérieure par rapport aux populations témoins. L’adjonction aux cellules souches de certains facteurs de croissance par transfection génique semble accroître leur efficacité. Aucun marqueur idéal de suivi n’a pu être identifié.ConclusionL’effet de l’injection de cellules souches sur la PIC semble être lié non pas uniquement à l’effet cellulaire propre mais surtout à des effets paracrines qui restent à élucider.

Histological analysis of chondrogenic differentiation. Pellets slides were cut and stained with Alcian Blue and Red Kernechtrot (A–C) or Sirius Red (D–F) to determine proteoglycan and collagen synthesis, respectively. A semiquantitative study of the distribution of stained descriptors was processed using Image J (National Institutes of Health, Bethesda, MD, USA). A custom-written Image J program was used to measure the percentage area from the whole section of the pellet. A chromatic segmentation of each histological stain was performed using the hue, saturation, and brightness properties of the images with the «Color Thresholder ImageJ» function. (DOCX 2164 kb)

Online supplement. (DOCX 21 kb)

Maternal factors. Table S2. Labor and delivery factors. Table S3 Newborn factors. Table S4. Impact of pre-thawing parameters on proliferation and chondrogenic differentiation. (DOCX 47 kb)

International audience