Kurzrock EA, Lieu DK, deGraffenried LA, Chan CW, Isseroff RR. Label-retaining cells of the bladder: candidate urothelial stem cells. Am J Physiol Renal Physiol 294: F1415-F1421, 2008. First published March 26, 2008 doi:10.1152/ajprenal.00533.2007.-Adult tissue stem cells replicate infrequently, retaining DNA nucleotide label (BrdU) for much longer periods than mature, dividing cells in which the label is diluted during a chase period. Those "labelretaining cells" (LRCs) have been identified as the tissue stem cells in skin, cornea, intestine, and prostate. However, in the urinary tract uroepithelial stem cells have not yet been identified. In this study, BrdU administration identified urothelial LRCs in the rat bladder with 9% of the epithelial basal cells retaining BrdU label 1 yr after its administration. Markers for stem cells in other tissues, Bcl, p63, cytokeratin 14, and ␤1 integrin, were immunolocalized in the basal bladder epithelium in or near urothelial LRCs, but not uniquely limited to these cells. Flow cytometry demonstrated that urothelial LRCs were small, had low granularity, and were uniquely ␤4 integrin bright. Urothelium from long-term labeled bladders was cultured and LRCs were found to be significantly more clonogenic and proliferative, characteristics of stem cells, than unlabeled urothelial cells. Thus, this work demonstrates that LRCs in the bladder localize to the basal layer, are small, low granularity, uniquely ␤4 integrin rich, slowly cycling and demonstrate superior clonogenic and proliferative ability compared with unlabeled epithelial cells. We propose that LRCs represent putative urothelial stem cells.

A human line of spontaneously immortalized keratinocytes (SIK cells) has been derived from ostensibly normal epidermis and has proven useful in dissecting molecular changes associated with immortalization. The original cultures had a normal karyotype and a colony forming efficiency of -3% through 10 passages. At passage 15, after which normal strains ordinarily senesce, these cells continued vigorous growth and gradually increased in colony forming efficiency, stabilizing at '30% by passage 40. During the early stage of increasing colony forming efficiency, the cells acquired a single i(6p) chromosomal aberration and 5-to 10-fold increases in expression of the cell-cycle control proteins cyclin A, cyclin B, and p34Cdc2. Additional chromosomal aberrations accumulated at later passages (i(8q) and +7), but the i(6p) and the increased expression of cell-cycle proteins were maintained, raising the possibility that these features were important for immortalization. Regulation of cell growth and differentiation in the cultures appeared minimally altered compared with normal keratinocytes as judged by their microscopic appearance and generation of abortive colonies, sensitivity to growth suppression by transforming growth factor-,B and tetradecanoylphorbol acetate, and dependence upon epidermal growth factor for progressive growth.

These results demonstrate geographical sequestration of urothelial progenitor cells to the area of the bladder that encompasses the bladder neck and trigone, which may be a factor in pathological disparities between the trigone and remaining bladder.

Rat urothelium can be reliably grown using fibroblast conditioned medium and a 3T3 feeder layer during primary culture. Serially passaged cells can survive cryopreservation and they are able to reconstitute epithelium on an acellular bladder matrix. Cells that are incorporated into the matrix express markers of early differentiation and demonstrate typical morphological characteristics by SEM. These culture techniques and this in vitro organoid model should facilitate the use of rat urothelium.

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