Background & Aim Mesenchymal 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 & Conclusion To 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.