Objective:To culture and characterize urine derived stem cells（USC）obtained from the urine

and to establish the immortalized stem cells. Methods:Fresh urine was collected. After urine samples being centrifuged

cell pellets were re-suspended and plated in 24-well tissue culture plates. Only single cells that attached to culture wells by day 2 after plating were used. The morphology of i USC was observed by inverted microscope. Stem cell surface mark CD73

CD90

CD146

SSEA-4 were detected with immunofluorescence and flow cytometry. Urothelial cell surface mark uroplakin Ⅰa

Ⅲ

cytokeratin（CK）-7 and 13 were detected with reverse transcription-polymerase chain reaction（RT-PCR）. Smooth muscle cell surface mark desmin and α-smooth muscle actin were detected with RT-PCR. Part of the cells were selected to induce differentiation of urothelial cells and smooth muscle cells. The human telomerase reverse transcriptase（h TERT）gene was transfected into urine derived stem cell to get the immortalized stem cell. RT-PCR and Western blot were used to detect h TERT. The morphology and the growth curve were observed. Uroplakin Ia and desmin were detected with immunofluorescence. Results:Single

small

compact "rice-grain"like cells was observed. The expression of stem cell surface markers

such as CD73

CD90

CD146

SSEA-4 was positive. RT-PCR findings confirmed that urothelial cell surface marker（uroplakinⅠa

cytokeratin（CK）-7

13）and smooth muscle cell surface marker（desmin

α-smooth muscle actin）were present in USC.The cells were induced to urothelial cells and smooth muscle cells successfully. The expression of h TERT gene detected by RT-PCR and Western blot confirmed that h TERT gene was transfected into USC successfully. The growth curves of USC and i USC showed a "S"shape

but i USC has a faster growth speed compared with USC

and has a statistically significant difference（P=0.043

P=0.000

P=0.006

P=0.001

P=0.025）. The expression of stem cell surface markers

such as CD73

CD90

CD146

SSEA-4 was positive as it presents in USC. i USC had the ability to differentiate into the cell lineages expressing urothelial and smooth muscle cell markers（uroplakin Ⅰa and Desmin）by immunofluorescence analysis. Conclusion:The urine-derived stem cells are successfully isolated and immortalized called immortalized urine-derived stem cells（i USC）. It provides seed cells of stable and safe for regeneration and tissue engineering in vitro studies.