Optimization and characterization of calcium phosphate transfection in mesenchymal stem cells. Tissue engineering. Part C, Methods Lo, C. W., Lin, T. H., Ueno, M., Romero-Lopez, M., Maruyama, M., Kohno, Y., Rhee, C., Yao, Z., Pérez-Cruz, M., Meyer, E., Goodman, S. B. 2019


Mesenchymal stem cells (MSCs) have been used as a therapy to modulate diverse biological processes. To fulfill the requirements for different MSC therapies, safe and effective gene transfer methods for MSCs are critical. Calcium phosphate transfection is an inexpensive and well-described method without discernible biosafety issues; however, an optimal protocol has not been developed for MSCs. In this report, we optimized the protocol of calcium phosphate transfection for murine MSCs, and compared this protocol with other gene transfer methods in different strains of mice and in human cells. We found that transfection efficiency and cell viability showed an inverse relationship depending on serum concentration during the process of calcium phosphate transfection, in which 2% serum was chosen in the optimized protocol. The optimized protocol of calcium phosphate transfection showed a fine balance between efficiency (about 70-80%) and viability (doubling original cell number) compared to other methods. Human MSCs were more resistant to this protocol (about 30% efficiency) compared with murine MSCs. Moreover, MSC potential for osteogenesis, adipogenesis, and chondrogenesis was not affected by calcium phosphate transfection. Finally, MSCs transfected with the luciferase gene were injected into the murine distal femoral bone marrow cavity to monitor gene expression overtime in vivo. MSCs in the bone marrow environment showed extended expression of the luciferase that was transfected by calcium phosphate. This report provides an optimized protocol for calcium phosphate transfection for murine MSCs and characterizes gene over-expression in MSCs in the in vitro and in vivo environments.

View details for DOI 10.1089/ten.TEC.2019.0147

View details for PubMedID 31441373