Increased Urothelial Cell Detection in the Primary Bladder Smooth Muscle Cell Cultures with Dual MACS/qRT-PCR Approach

Abstract

Bladder tissue has been regenerated in humans with neurogenic bladder using an implant produced from autologous urothelial (UC) and smooth muscle cells (SMC) expanded from bladder biopsies seeded onto a biodegradable synthetic scaffold. As the majority of bladder cancers are urothelial carcinomas (aka, transitional cell carcinoma), this 2-cell type autologous sourcing strategy presents significant challenges to product development. Entire bladders have been regenerated in cystectomized animals using a single-cell-type sourcing strategy: implants were seeded with bladder-derived SMC-only. Applying the bladder SMC-only sourcing strategy to produce clinical implants for bladder replacement or urinary diversion in bladder cancer patients requires methods for screening SMC cultures for the presence of potentially cancerous UC cells to provide evidence of SMC culture purity before seeding the scaffold. In this report, we show a 10-fold to 100-fold improvement in the sensitivity of qualitative and quantitative reverse-transcription PCR (qRT-PCR)-based assays for detecting UC positive for Cytokeratin 5 (CK5) in mixed SMC/UC cultures when the cell population was first subjected to magnetic activated cell sorting to enrich for cells expressing the epithelial cell adhesion molecule (known as EPCAM or CD326), a marker known to be present in normal UC and upregulated in the cancerous UC.

Defining protein expression in the urothelium: a problem of more than transitional interest

“…Utilizing epithelialspecific cell surface markers such as uroplakin, epithelial cell adhesion molecule (EpCAM), or E-cadherin, it is possible to obtain a highly enriched fraction of urothelial cells (23). These can be then be examined in a variety of ways from functional studies (if the cells are maintained/cultured) to molecular characterization with RT-PCR and/or immunoblotting (47).…”

Organ specific regenerative markers in peri-organ adipose: kidney

“…In this regard, we have observed induction of established tubular markers within primary cultures of KiSAS cells in response to modulation with known morphogenic agents such as retinoic acid (our unpublished observations). Renal adipose may represent a potentially ideal alternate cell source for treatment of chronic anemia secondary to CKD, since renal adipose can be isolated in much larger quantities than renal primary cells and, as is the case for adipose associated with tubular organs such as bladder, may be unaffected by the occurrence of metastasis in renal cancer patients [46,47]. …”

EpCAM Expression in Lymph Node Metastases of Urothelial Cell Carcinoma of the Bladder: A Pilot Study

“…The expression rates of EpCAM in urothelial cell carcinoma (UCC) of the bladder varies between 27% and 99% in different studies. Besides, EpCAM expression has also been found in normal urothelial cells [ 11 , 13 , 14 ]. Because of the wide variance of EpCAM expression in the primary tumor, the expression of EpCAM in lymph node metastases of urothelial cell bladder cancer has not been determined yet, as far as we know.…”