Chemically Defined Serum-Free Medium for Undifferentiated Human Mesenchymal Stem Cells

Objectives: Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into mesenchymal cell types such as osteoblasts, chondroblasts, and adipocytes. For research, hMSCs are generally cultured in the presence of fetal bovine serum (FBS). The ill-defined components of FBS hamper analysis of the cell biological mechanisms that control cell behaviors, such as differentiation. Therefore, we have developed a growth factor-defined, serum-free medium for culturing hMSCs. Methods: A human bone marrow-derived hMSC line UE7T-13 (JCRB 1154) was used in this study. The life span was prolonged by infecting them with a retrovirus containing human papillomavirus E7 and hTERT. We first tested the ability of hESF9 medium, which we had developed for use with hES cells, to support the growth of UE7T-13 cells because hMSCs have multipotent properties similar to hES cells and then we speculated that hMSCs should be able to grow in similar culture conditions as hES cells. To assess hMSC marker gene expression, cultured UE7T-13 cells were subjected to quantitative RT-PCR, immunocytochemistry and flow cytometry. Results: Addition of TGF-β1 to hESF9 medium (D-hESF10) supports the robust proliferation of hMSCs. The expanded UE7T-13 cells expressed the human pluripotency markers. The potential to differentiate into osteoblasts and adipocytes was confirmed. Conclusion: The developed medium, D-hESF10 supported the proliferation and undifferentiated state of a hMSC line. This work provides a useful tool to understand the basic biological characteristics of hMSCs.