Oottamasathien Siam, Williams Karin, Franco Omar E, Thomas John C, Saba Katrina, Bhowmick Neil A, Staack Andrea, Demarco Romano T, Brock John W, Hayward Simon W, Pope John C
Department of Urologic Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Dev Dyn. 2006 Oct;235(10):2795-801. doi: 10.1002/dvdy.20886.
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.
组织重组是一种利用宿主啮齿动物的体内环境来评估协调器官发育的旁分泌信号事件的强大方法。研究报告了啮齿动物膀胱尿路上皮原代培养物的成功生成，但尚无研究报道其在组织重组中用于重现膀胱组织。我们提出，原代培养的膀胱尿路上皮与诱导性胚胎膀胱间充质重组时，将在重组模型中形成膀胱组织。分离成年大鼠膀胱并获取尿路上皮。将膀胱尿路上皮片重悬于胶原蛋白中并维持在组织培养中。扩增（>20代）后，将尿路上皮与胚胎第14天的小鼠膀胱间充质重组，然后移植到免疫缺陷小鼠宿主的肾包膜下。28天后收获移植物。分别用单独的膀胱间充质、单独的培养膀胱尿路上皮和单独的胶原蛋白基质进行对照移植。用染色和免疫组织化学（苏木精和伊红染色、Gomori三色染色、广谱尿路上皮蛋白、平滑肌肌动蛋白α和γ）对最终组织进行评估。对培养的尿路上皮进行广谱角蛋白、波形蛋白和广谱尿路上皮蛋白的免疫细胞化学检测证实为纯细胞群，无间充质污染物。重组移植物的染色显示有成熟尿路上皮和基质分化的膀胱组织。对照组织未形成膀胱组织。我们成功证明，由原代培养的膀胱尿路上皮与胚胎膀胱间充质重组可形成嵌合膀胱。这是研究膀胱发育和疾病分子机制的一种强大新工具。未来的应用可能包括尿路上皮的体外基因操作以及在体内环境中研究这些操作对生长和发育的影响。
