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International audience; Background & AimMesenchymal stromal/stem cells (MSCs) are a heterogeneous population characterized by self-renewing and ability to differentiate into mesodermal cell origin. Wharton Jelly MSC (WJ-MSC) are a promising candidate for cell therapy due to their ease of isolation and their tissue regeneration capabilities. Moreover, they can regulate the immune response by secreting a set of soluble factors and by establishing direct interactions with the cells of immune system. These immunomodulation properties are used in the treatment of different pathologies such as graft versus host disease. In order to use WJ-MSCs for clinical applications, an in vitro expansion step is critical to obtain the sufficient dose. This step imposes several physicochemical and hydrodynamic constraints that can influence the quality and functional properties of cells.The objective of this work was to identify and validate predictive markers related to immunomodulatory properties of WJ-MSCs. For this purpose, CD119, CD200 and Notch1 were chosen and studied.Methods, Results & ConclusionAt first, markers distribution was studied by flow cytometry. Secondly, immunomodulatory potential of WJ-MSC was evaluated by a coculture of these cells with CD4, CD8 lymphocytes, followed by the study of lymphocytes proliferation by flow cytometry. These experimentss were performed under different conditions: WJ-MSCs cultivated in normoxia (N) and hypoxia (H), early passage and senescent cells, and under stimulation with gamma interferon (IFN-γ).Our results showed a heterogeneity of distribution of the markers studied. In addition, our study revealed that WJ-MSC cultivated in H was more immunosuppressive than those cultivated in N. Moreover, we showed that the cellular senescence induced a reduction of the immunosuppressive properties, and that IFN-γ stimulation increased WJ-MSC immunosuppression potential. Finally, our study highlighted a trend towards a correlation between the expression of CD119 and Notch1 by WJ-CSM and their immunomodulatory properties.To conclude, the culture conditions and cell senescence affect the immunomodulatory properties of WJ-CSM hence the interest of verifying these properties after the cell expansion procedures. Furthermore CD119 and Notch1 receptors may be predictive markers of the immunomodulatory properties of WJ -CSM to be checked during in vitro amplification.

International audience; Background & AimMesenchymal stromal/stem cells (MSCs) are fibroblast-like multipotent cells capable of proliferation, self-renewal and differentiation into different mesodermal cell-lineages. They are known to be implicated in many key biological processes like tissue regeneration, secretion of bioactive factors which give them immunomodulatory and trophy properties. MSCs isolated from umbilical cord Wharton's Jelly (WJ-MSC) aroused much attention due to several advantages: multipotency capacity, painless, safety for allogenic therapeutic purposes and efficiency for treating certain diseases. In order to use MSCs for therapeutic purpose, these cells must be expanded in long-term, which inevitably triggers cell senescence, a process of cell proliferation arrest that occurs in vivo and in vitro as a response of cells to excessive stresses. This phenomenon is characterized by several markers and features and it can affect cell behavior.Because of senescent cells influence the outcome of a variety of physiological and pathological processes, and because of WJ-MSCs are used in cell therapy, the aim of the study was to characterize senescence WJ-MSCs after in vitro expansion, to control the quality of these cells in terms of function and phenotype.Methods, Results & ConclusionTo induce senescence process, we used in vitro model called “replicative senescence”. Cells were cultured in normoxia (N) and hypoxia (H) conditions and analyzed in early and late passages. Proliferation analysis of MSCs demonstrated a high rate of proliferation in H condition. In addition, senescence analysis using a Beta-Galactosidase test indicated that MSCs in H condition senesce slower than those in N. On the other hand, cell-surface biomarker analysis revealed that all MSCs derived from WJ expressed the common surface markers among which CD44, CD105 without great changes during expansion. However other markers presented changes during expansion (like a decrease in CD146). Senescent cells presented variable immunosuppressive potential compared to MSCs in early passage. Known that senescent cells are characterized by changes in chromatin structure, we studied proteins implicated in chromatin remodeling like HMGB1. Immunofluorescence study showed a translocation of HMGB1 from MSC nucleus in senescent cells. All these results will improve the characterization of MSCs senescence and may provide new parameters for establishing the quality of MSCs prior to their therapeutic application.

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.