Identification Of Urothelial Stem Cells

Purpose:To establish an animal model of "label-retaining cells", and to identify the in vivo location of urothelial stem cells. Methods:Transgenic mice Krt5-rtTa-VP16 and TRE-H2B-GFP were used to cross with each other, in order to obtain bi-transgenic mice. Krt5 and GFP expression level in these mice were evaluated by RT-PCR. Pulse-chase experiment was carried out when they grew up to 4 weeks old. They were then fed with doxycycline food and were injected with cyclophosphamide to stimulate urothelial proliferation. After 2 weeks, all the urothelial basal cells would express GFP protein. In the chase period, serial sections and whole mount samples were prepared. Epifluorescence microscopy, stereo microscopy, deconvolution microscopy, and 2-photon confocal microscopy were used to observe the distribution of "lable-retaining cells" in mouse urinary system. Image J and Volocity softwares were used to reconstitute the images and captured data. Results:Bi-transgenic mice Krt5-rtTa/TRE-H2B-GFP were obtained after the crossing of Krt5-rtTa-VP16 and TRE-H2B-GFP, the urothelial basal cells of which express GFP protein after been fed with doxycycline food. The serial sections data showed that the GFP label-retaining cells are clustered in the bladder trigone, bladder neck, ureteropelvic junction and ureterovesical junction. Our observations on the location of urothelial LRCs in the whole mounts were in great agreement with those determined from serial histological sections. Conclusion:The present study established a successful method to identify the urothelial stem cells. It was observed that the urothelial stem cells locate in the basal layer in bladder trigone, bladder neck, ureteropelvic junction and ureterovesical junction. This study provides foundation for further studies on epithelial stem cells, and theoretic basis for the study of urinary tumorigenesis as well.