Identification of Human Mesenchymal Stromal Cells and Culturing Media Effects on Proliferation, Differentiation, and Cell Surface Markers

Abstract: The mesenchymal stromal cell (MSC) is of great interest for its immunomodulatory and regenerative properties. However, to research and use these MSCs it is essential to identify and characterize them as such. They need to fulfill the MSCs' minimal criteria which assess the differentiation potential, cell surface markers, and adherence. In this study, cells donated from human bone marrow were identified as MSC according to the minimal criteria. Methods used were flow cytometry, immunofluorescent staining, and ELISA. Furthermore, the population was cultured in three different media (DMEM-LG with either 10% FBS, 2% FBS, or 10% FBS supplemented with 10% conditioned media from human urinary bladder carcinoma cells (T24)) for 21 days whereupon tested for the mesenchymal characteristics, cells were counted and size measured at every passage. All cultures maintained their mesenchymal character, however, cells grown in 2% FBS became a considerably more heterogenous population regarding cell size and granularity, perhaps because of senescence. Additionally, these cells somewhat decreased in proliferation and resulted in 1 x 106 cells after 21 days, however, this was not a significant decrease when compared to the 10% FBS culture which had 2.16 x 106 cells after 21 days (p=0.061). On the contrary, the culture supplemented with T24 conditioned media resulted in a significantly higher cell count with 4.75 x 106 cells (p=0.008). Further studies could investigate which components in the conditioned media contributed to the proliferation. Moreover, the cell population in this study could not be characterized as MSC with certainty as additional cell surface markers should be tested.