Bladder tissue formation from cultured bladder urothelium

Abstract

Tissue recombination is a powerful method to evaluate the paracrine-signaling events that orchestrate the development of organs using the in vivo environment of a host rodent. Studies have reported the successful generation of primary cultures of rodent bladder urothelium, but none have reported their use to recapitulate bladder tissue with tissue recombination. We propose that primary cultured bladder urothelium, when recombined with inductive embryonic bladder mesenchyme, will form bladder tissue in a recombination model. Adult rat bladders were isolated and urothelium obtained. Sheets of bladder urothelium were re-suspended in collagen and maintained in tissue culture. After expansion (>20 passages), the urothelium was recombined with embryonic day-14 mouse bladder mesenchyme, then grafted beneath the renal capsule of immunocompromised mouse hosts. Grafts were harvested after 28 days. Control grafts were performed with bladder mesenchyme alone, cultured bladder urothelium alone, and collagen matrix alone. Final tissues were evaluated with staining and immunohistochemistry (H&E, Gomori's trichrome, broad-spectrum uroplakin, and smooth muscle actin ␣ and ␥). Immunocytochemistry on cultured urothelium for broad-spectrum keratin, vimentin, and broad-spectrum uroplakin confirmed pure populations, void of mesenchymal contaminants. Staining of recombinant grafts demonstrated bladder tissue with mature urothelium and stromal differentiation. Control tissues were void of bladder tissue formation. We have successfully demonstrated that a chimeric bladder is formed from primary cultured bladder urothelium recombined with embryonic bladder mesenchyme. This is a powerful new tool for investigating the molecular mechanisms of bladder development and disease. Future applications may include the in vitro genetic manipulation of urothelium and examining those effects on growth and development in an in vivo environment. Developmental Dynamics 235:2795-2801, 2006.

Lineage Enforcement by Inductive Mesenchyme on Adult Epithelial Stem Cells across Developmental Germ Layers

“…Under the influence of preputial gland mesenchyme, bladder epithelia retained its urothelial phenotype and expressed Foxa1 (previously reported in adult bladder [30]), compared to adjacent foci of sebaceous epithelial cells that did not express Foxa1, confirming the transformation of epithelia in those select regions (Fig. 6C, 6D).…”

Translocation of NF-κB and Expression of Cyclooxygenase-2 Are Enhanced by Ketamine-Induced Ulcerative Cystitis in Rat Bladder

“…Ultrastructural morphology showed microvilli in stratified urothelium and immunolabeled for cytokeratin 17, which confirmed successful urothelial cell culture. 30 Moreover, human J82 cells were used for examining whether activated NF-kB translocated to nucleus and affected COX-2 mRNA expression. Human J82 cells were cultured in Dulbecco's modified Eagle's medium Q16 (InvitroGen Corp., Carlsbad, CA) with 10% fetal bovine serum.…”

“…After the rat bladder was excised, a modified Roszell's procedure was performed. 29,30 The bladder mucosa was gently scraped from the muscle tissue. The bladder was immersed in 4 mL of 1% collagenase IV (Sigma-Aldrich) at 37 C on a shaker for 40 minutes.…”

“…The urothelial cells were plated at a cell density of 5 Â 10 6 cells/mL in Gibco keratinocyte serum-free medium Q15 (Life Technologies, Grand Island, NY). The culture was supplemented with 5 ng/mL epidermal growth factor, 100 U/mL penicillin, and 1 mg/mL streptomycin (Gibco) 30 for 48 hours. Ultrastructural morphology showed microvilli in stratified urothelium and immunolabeled for cytokeratin 17, which confirmed successful urothelial cell culture.…”

Maintenance of bladder urothelia integrity and successful urothelialization of various tissue-engineered mesenchymes in vitro

“…We may postulate that the induction of the pseudostratified disposition of urothelial cells results from the achievement of the first step of horizontal development, which is promoted by the paracrine signals from BMCs. This hypothesis does not exclude the possibility of a sequential differentiation-specific growth factor release by the BMCs (DiSandro et al 1998, Oottamasathien et al 2006.…”