Generating Rho-0 Cells using Human Mesenchymal Stem Cell Lines

Introduction: Rho-0 generation requires immortalization process or selecting tumor cells, following by culture in presence of EtBr. The aim of this work was generate Rho-0 cells using human MSC (hMSC) lines and reagents with the ability to remove the mtDNA in a more safety way than EtBr. Methodology: Immortalized hMSCs (3a6) was used and 143B.TK-Rho-0 like control. mtDNA content was measured by RT-PCR and IF. 3a6Rho-0 was evaluated by phenotypic characterization, ROS production, Apoptotic levels and Δψm by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse. Differentiation capacity was evaluated by comparing gene expression of genes involved in, adipogenesis osteogenesis and chondrogenesis. Results: The results showed 3a6 capacity to deplete their mtDNA and survive in presence of uridine. Phenotypic characterization demonstrated that 3a6Rho-0 showed a cell-surface receptor pattern typical of MSC. The functional analysis reflected that 3a6Rho-0 has similar behavior that 143B.TK-Rho-0. 3a6Rho-0 has adipogenic capacity, but lower osteogenic and chondrogenic capacity that 3a6-wt. Conclusion: We suggest that d4t is the best option to get Rho-0 cells from hMSC. Rho-0 cells obtained have similar response that typical Rho-0 used in the literature, and our cell line can be used to study the role of mitochondria in hMSC differentiation.