Cranial neural crest stem cells were the main source of cranio-facial tissue and organ. It is essential to immortalize these cells. The frequency of spontaneous immortalization of human cells is low, but some immortalization genes can significantly increase this frequency.OBJECTIVE:
To investigate the effect of human telomerase reverse transcriptase (hTERT) in the immortalization of rat cranial neural crest stem cells.DESIGN, TIME AND SETTING:
The observational experiment was performed at the Fourth Military Medical University of Chinese PLA from September 2007 to June 2008.MATERIALS:
Three embryonic 8.5 d rats (6 weeks, 180 g), and congenital cellular immune defects male Balb/c nude mice (18-21 g) were obtained from Experimental Animal Center of Fourth Military Medical University of Chinese PLA. Plasmid PCIneo-hTERT was supplied by Department of Endodontics, Stomatology Hospital, Fourth Military Medical University of Chinese PLA.METHODS:
Primary rat cranial crest stem cells were cultured and transfected with eurayotic expressing plasmid PCIneo-hTERT encoding hTERT gene. Positive clones were selected by G418 and expanded for continuous culture. Expression of hTERT and P75 were detected in stable clone by immunocytochemical method. Telomerase activity was also measured. Growth curves were depicted to show proliferation of clones. Nude mice with transfected cells were used observe the biology features of cell clones.MAIN OUTCOME MEASURES:
Cell clone, specific protein P75 expression, telomerase activity, proliferation, tumorigensis test were measured.RESULTS:
Following 24 hours PCIneo-hTERT transfection, a few cells died. After adding G418 for 48 hours, cells gradually died in a large number. At day 12, cell clone appeared, and about 3 cell clones grew well, which were named clones 1, 2 and 3. Following 20-25 passages, clones 1 and 2 died. However, clone 3 grew well in vitro, and stably expressed hTERT and P75. Following transfection, telomerase activity obviously increased, and continuously expressed. After hTERT transfection, the proliferation of three clones was strong in an early phase. Subsequently, the proliferation of clones 1 and 2 gradually became slow, and clones 1 and 2 died. Clone 3 passed the senescence phase, and with no oncogenicity.CONCLUSION:
Rat cranial neural crest stem cells can be immortalized by hTERT transfection and can be used as seed cells in the further study of cranial-facial cell differentiation and tissue engineering.