title: 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 alpha and gamma). 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.
authors.affiliation: Department of Urologic Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA. siam.oottamasathien@vanderbilt.edu
authors.forename: Siam
authors.initials: S
authors.lastname: Oottamasathien
authors.forename: Karin
authors.initials: K
authors.lastname: Williams
authors.forename: Omar E
authors.initials: OE
authors.lastname: Franco
authors.forename: John C
authors.initials: JC
authors.lastname: Thomas
authors.forename: Katrina
authors.initials: K
authors.lastname: Saba
authors.forename: Neil A
authors.initials: NA
authors.lastname: Bhowmick
authors.forename: Andrea
authors.initials: A
authors.lastname: Staack
authors.forename: Romano T
authors.initials: RT
authors.lastname: Demarco
authors.forename: John W
authors.initials: JW
authors.lastname: Brock
authors.forename: Simon W
authors.initials: SW
authors.lastname: Hayward
authors.forename: John C
authors.initials: JC
authors.lastname: Pope
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D007150:Immunohistochemistry; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D051379:Mice; D008819:Mice, Nude; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering / Q000379:methods; D014183:Transplantation, Heterologous / Q000379:methods*; D001743:Urinary Bladder / Q000166:cytology / Q000502:physiology*; D060171:Uroplakin III; D019459:Urothelium / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation
chemical_list: D008562:Membrane Glycoproteins; D060171:Uroplakin III