Establishment of Human Bone Marrow-Derived Mesenchymal Stem Cell Lines with SV40 Large T Antigen and Their Differentiation to Chondrocytes

Park, Jeong Ah; Kim, Eun Kyung; Koh, Sang Hyeok; Kang, Hyoung Jin; Shin, Hee Young; Ahn, Hyo Seop

Abstract

PURPOSE
Mesenchymal stem cells (MSC) can be isolated from bone marrow (BM) and when systemically administrated to different species, they undergo site-specific differentiation. In this study, we isolated MSC from human BM and generated a continuously growing colony of cell lines (SNU-hMSC) with SV40 large T antigen. The purposes of this study are to identify whether SNU-hMSC have the characteristics of MSC and their possibility of chondrogenic differentiation. METHODS: MSC were mobilized from BM and cultured in DMEM-LG media for 2 weeks. We obtained SNU-hMSC, by introducing a viral vector of SV40 large T antigen and culturing it in the selected media for 6 months. We identified specific cell markers of MSC via FACS analysis and analyzed expression of cytokines, chemokines and receptors by RT-PCR. To stimulate the proliferation of the cells, we processed the media with FGF, BMP-2 and IL-6. The each medium's cell counts were counted in day 7 and day 14. To differentiate SNU-hMSC, they were cultured in chondrogenic media. After 2 weeks, chondrogenic differentiation was evaluated with safranin-O staining and the expression of COMP, aggrecan and SOX-9. RESULTS: SNU-hMSC exhibited MSC markers. When the IL-6, BMP-2 and FGF were added to each medium, the cell numbers were significantly increased as compared with control. In the study of differentiation, SNU-hMSC exhibited strong safranin-O staining, and chondrogenic gene expression was observed. CONCLUSION: SNU-hMSC expressed markers and cytokines identical with MSC. SNU-hMSC maintained multipotency of differentiation.