Label retaining and stem cell marker expression in the developing rat urinary bladder

Abstract

Objective: To identify potential stem cells in urinary bladder by label-retaining cell (LRC) strategy and immunostaining for putative stem cell markers.

Methods: Newborn rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine (EdU) and their bladders harvested at 4 different time points afterward. The bladders were processed for EdU staining and immunofluorescence staining for stem cell markers Lgr5, CD34, SSEA-1, and c-kit. EdU-positive cells were counted and colocalization with stem cell markers determined.

Results: At day 1 post-EdU injection, 1804.0 ± 227.7 bladder cells were labeled in each cross-section. As time increased, fewer bladders remained labeled, dropping to 236.5 ± 53.0 cells per field. In the 1-day bladders, 27.5% ± 4.9% of the epithelial cells were labeled as compared to 12.1% ± 2.8% in the detrusor. The labeling rates in these 2 tissue compartments gradually equalized, reaching at approximately 5.5% in the 8-week samples. Distribution of LRC was random, without preferential labeling of basal cells. Lgr5 and SSEA-1 were detectable in the urothelium, and CD34 and c-kit in the lamina propria and detrusor. Approximately 30%-40% of c-kit-positive cells were EdU positive.

Conclusion: Labeling of bladder cells by EdU occurred randomly, and label retaining was not associated with expression of Lgr5, CD34, or SSEA-1. The strong association between label retaining and c-kit expression appears to relate to interstitial cells of Cajal, not stem cells.