Optimización de la técnica de aislamiento y proliferación de células mesenquimales procedentes de la gelatina de Wharton

Mesenchymal stem cells are multipotent adult stem cells that can be isolated from different tissues. The umbilical cord, and more precisely, the Wharton’s Jelly, offers a low-cost and pain-free collection method of MSCs. MSCs are self-renewing and non-specialized cells, with the potential to differentiate in several distinct mesenchymal lineages. They also have immunosuppressive and anti-inflammatory properties that have made them of interest for a wide range of cellular therapies in clinic application. In 2006, the International Society for Cellular Therapy (ISCT) published minimal guidelines to define MSC’s identity. They must be plastic-adherent when maintained in standard culture conditions, must express CD73, CD90, CD105, and lack expression of CD34, CD45 and HLA-DR surface molecules. Finally, regarding to their plasticity, they must have the potential to differentiate in adipocytes, chondrocytes and osteoblasts. In the present work we optimize the isolation and proliferation technique of Wharton’s Jelly-MSCs in order to obtain a large number of stem cells with therapeutic potential. For achieving the main objective, we isolated MSCs from the umbilical cord following two different methods: enzymatic digestion and explant culture. The proliferation and expansion procedures were carried out in incubators. When MSCs reached confluence, we characterized the immunophenotypic profile of both adherent and non-adherent cells and finally, we studied the senescence associated β-galactosidase. We also explored the possibility of reducing the concentration of FBS in the culture medium. Instead, we added xeno-free autologous plasma from the umbilical cord blood. It contains growth factors and cytokines, among others, that contribute to the proliferation and expansion of stem cells. Regarding the isolation method, enzymatic digestion has been more efficient than the explant method. MSCs isolated by enzymatic digestion have been successfully cultured in most of the umbilical cords using a panel of collagenase and hyaluronidase enzymes. Expansion and proliferation of MSCs have been measured under microscope. There have not been any substantial differences between culture medium with FBS or autologous plasma. The population doubling time of Wharton’s Jelly-MSCs was calculated as (31.57 ± 0.49) h. The adherent cells displayed different types of morphologies, but most of them were fibroblast-like cells that lack the expression of hematopoietic surface markers (CD34, CD45). Non adherent cells were characterized as progenitor endothelial cells (CD34+ CD45-). Finally, the senescent associated β-galactosidase revealed that MSCs entered in replicative senescence too early, preventing them for a clinical application. Nevertheless, MSCs possess heterogeneous phenotypes and functionalities, heavily influenced by source of isolation and culture conditions. Therefore, MSCs from the Wharton Jelly require a deeper characterization