Establishment of immortalized urine derived stem cell line with SV40Tag gene

Abstract

Objective To establish immortalized urine derived stem cell line with simian vacuolating virus 40 large T antigen (SV40Tag) for providing a source for tissue regeneration. Methods Transfer pMPH-FRT-SV40T with SV40Tag gene and transposase to urine derived stem cell through lipofection gene transfer. SV40Tag gene was knocked out by flippers recombination enzyme (Flp). Hygromycin was used for screening positive clones for cell passaging. Reverse transcription-polymerase chain reaction (RT-PCR) was used for detecting the expressions of SV40Tag. CD73, CD44, CD90(Thy1), CD29(integrin), CD105(endoglin), CD166(ALCAM), BMPRII, SSEA4, CD117 and CD133, all major stem cell markers of immunofluorescent cytochemistry. Cellular morphology and growth were observed and analyzed. Results The induction of bone morphogenetic protein 9 (BMP9) had the potential of differentiating into osteoblasts and adipocytes. After the injection of these cells into nude mice, no tumor was formed after 4 weeks. Conclusions An immortalized urine derived stem cell line has been established with SV40Tag. After differentiate to osteoblast and aidpocyte, cells still possess the stem cell potential. This cell line may be used for tissue regeneration. Key words: Stem cell research; Tissue engineering; Regeneration medicine