Background & Aim Mesenchymal 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 & Conclusion We 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.