Immunological purification of rat precartilaginous stem cells and construction of the immortalized cell strain

Precartilaginous stem cells (PCSC) are adult stem cells that control limb growth of animals and can differentiate directionally. In a previous study, PCSC was reported to begin differentiating at the fifth passage; therefore, sufficient uni-phenotype PCSC cannot be harvested from primary cell culture. The purpose of this study was to examine whether simian virus 40 large T antigen gene (SV40Tag) could induce rat PCSC to immortalize. Immunomagnetic separation was used to isolate PCSC labeled with fibroblast growth factor receptor-3 (FGFR-3). Plasmid pCMVSV40T/PUR containing SV40Tag was transfected into PCSC by liposome transfection method. One anti-puromycin cell clone was obtained, which was confirmed as FGFR-3 positive, and expanded to immortalized cell strain. Results from RT-PCR and immunocytochemistry demonstrated that SV40Tag was highly expressed at both mRNA and protein levels after stable transfection. The cells transfected with SV40Tag were expanded to immortalized cell strain, which could maintain its characteristics for 30 passages, named immortalized precartilaginous stem cells (IPCSC). IPCSC were short fusiform or triangular cells with two or three short axons. Immunocytochemistry results of FGFR-3 and Collagen II demonstrated that IPCSC retained the characteristics of PCSC and the high proliferation capability of IPCSC was confirmed by methyl thiazolyl tetrazolium assay. Therefore, we concluded that rat precartilaginous stem cells were purified and immortalized precartilaginous stem cell strain was established. It may provide a stable cell resource for basic research and cell transplantation therapies.