Establishment of immortalized umbilical cord mesenchymal stem cells mediated by hTERT

Objective To establish the immortalized umbilical cord mesenchymal stem cells(UCMSCs) mediated by human telomerase reverse transcriptase (hTERT) so as to offer enough UCMSCs for in vitro or clinical researches. Methods Human UCMSCs were isolated and cultured to passage 4,and then identified by flow cytometry analysis. The pLXSN-hTERT plasmid was transfected into UCMSCs by liposome to establish the immortalized UCMSCs. Expressions of hTERT mRNA and protein were respectively tested by RT-PCR and immunocytochemistry. The cell cycle kinetics of the passage of hTERT-UCMSCs and UCMSCs were detected with flow cytometry. Results Flow cytometry analysis showed that UCMSCs expressed CD29, CD44 and CD 105 strongly, but CD31, CD34 and CD45 slightly; these were right the features of human UCMSCs. RT-PCR showed that hTERT mRNA was expressed low in UCMSCs, and high in pLXSN-hTERT transfected UCMSCs.Immunocytochemistry revealed that fluorescence intensity of cell nuclei was increased significantly after transfection and that hTERT was expressed all in cell nuclei. Flow cytometry analysis suggested that the number of hTERT-UCMSCs at phase S was increased significantly, and the cells would be able to passage stably (more than 35 passages). Conclusions The immortalized UCMSCs can be established by hTERT transfection, and the number of immortalized UCMSCs can meet the need of in vitro or clinical researches. Key words: UCMSCs; hTERT; Immortalization