Attempts to identify tissue-specific stem cells by the LRC strategy have been made on every major organ, often more than once. For the urinary bladder, only one such study exists, and the conclusion is that 9% of the urothelial basal cells retained the BrdU label 1 year after its administration 11
. However, scoring BrdU-LRC is often guesswork as the indicative brown color is very difficult to tell apart from the concomitant purplish-brownish nuclear stain. As such, the present study wished to reassess the urinary bladder’s LRC profile by employing the EdU label whose detection is completely devoid of ambiguity 16
. Also different from the previous study were the use of neonatal rats rather than adult rats, and a maximal time frame of 8 weeks rather than 1 year. Both of these two choices are consistent with most LRC studies. In regard to histology, we examined both the mucosa and detrusor while the previous study focused on the urothelium. In addition, we stained for stem cell markers Lgr5, CD34, SSEA-1, and c-kit while the previous study Bcl, p63, cytokeratin 14, and beta1 integrin.
In the present study, labeling of neonatal rat bladder by EdU occurred at a high rate, but the number of labeled cells dropped sharply within 2 days. These observations are consistent with most LRC studies and generally reflect the rapid cell cycling in most tissues of neonatal animals 12–14
. Although initially (day 1) the urothelium had more than twice the labeling rate as the detrusor, these two compartments eventually (at 8 weeks) had similar rate of LRC. This epithelium versus detrusor difference mimics the epithelium versus stroma difference in the endometrium 14
. Also noteworthy is that the distribution of the labeled cells was mostly random, whether in the urothelium or detrusor, and in all bladder tissues harvested at different time points after EdU injection. Thus, the results differ from the previous study, in which preferential labeling of urothelial basal cells was noted 11
To examine stem cell marker expression in the bladder, particularly in LRC, four well-characterized markers that relate to epithelial, mesenchymal, hematopoietic, and neural stem cells were investigated. Epithelial stem cell marker Lgr5 was scantly detected in the urothelium one to eight weeks after EdU labeling, and some of the expressing cells were EdU-positive. But, there was no correlation between Lgr5 expression and label retaining. Mesenchymal stem cell marker CD34 was detected in the lamina propria and detrusor, mostly in association with blood vessels, at all time points after EdU labeling. While some CD34-expressing cells were EdU-positive, there was no correlation between CD34 expression and label retaining. Neural stem cell marker SSEA-1 was strongly expressed in the superficial layer (umbrella cells) of the urothelium. While this is in agreement with previous studies 9
, no colocalization of SSEA-1 and EdU was found at any time points after EdU labeling.
The expression of HSC marker c-kit in the urinary bladder has been intensely studied. While it is expressed in both the mast cells and the ICCs, most attention has been given to the latter as these cells are believed to be the pacemaker of detrusor contraction 20
. In the present study we detected c-kit expression in the lamina propria and the detrusor as previous studies did. More importantly, we found a high rate of co-localization in c-kit expression and EdU labeling. Specifically, approximately 30–40% of c-kit-expressing cells in either the lamina propria or the detrusor were labeled with EdU at any time points after EdU labeling (). Thus, it appears that c-kit-expressing cells are proliferative in neonatal rat bladder (so as to get labeled) but soon become quiescent in the more matured bladder (so as to retain the label). This property of rapid cycling from proliferation to quiescence appears to be preferentially associated with c-kit expression, as the number of c-kit-expressing cells among EdU-positive cells increased sharply between day 1 and day 3 after EdU labeling and stabilized thereafter (). While the significance of these findings is presently unknown, it should be pointed out that, in bone marrow, c-kit-expressing HSCs have been found to cycle rapidly from proliferative to quiescent state 21
, and the function of c-kit has been shown to regulate the maintenance of quiescent HSC 22
. In addition, c-kit expression has been associated with quiescent hepatic satellite cells 23
, and ICCs in adult small intestine have been found to lack proliferative activity 24
. Thus, the c-kit-expressing LRCs in the urinary bladder are probably quiescent ICCs.
Due to the rarity of tissue-specific stem cells and lack of specific markers, their identification by histological means is usually a first step and should be followed with more definitive approaches such as clonogenicity and plasticity tests. In the study by Kurzrock et al 11
, a clonogenicity test was attempted with the assumption that a single BrdU-labeled cell isolated from rat bladder could give rise to a BrdU-labeled colony in culture. However, since a BrdU-labeled cell loses half of the label with each cell division, it is unlikely that all of the cells in a colony could be BrdU-positive as shown in the graph. Thus, the existence of bladder stem cells remains unsubstantiated by published studies or by the present study.