Bone Marrow Stromal Cells for Bladder Tissue Engineering

描述（由申请人提供）：当正常膀胱细胞在培养物中生长时，它们保持正常，当患病或异常膀胱细胞在培养物中生长时，它们保持异常。需要膀胱扩大或再生的患者可能没有健康的膀胱细胞用于再生，因此需要替代细胞来源。由于骨髓基质细胞（BMSC）具有培养和分化为平滑肌样细胞的能力，它们可能是组织工程膀胱再生的替代细胞来源。BMSC通过骨髓抽吸获得，并且可以在体外扩增至足够的数量。它们不是干细胞，但显示出干细胞群体的多种特征，包括与原代平滑肌细胞（SMC）培养物相比相似的细胞增殖、组织学外观和收缩表型。本申请的重点是研究BMSC用于膀胱再生的有前途的潜力。第一个具体目的：（1）在体外与膀胱尿路上皮细胞（UC）共培养时表征BMSC。(1.1)分离培养BMSC、UC和SMC，（1.2）用增强型绿色荧光蛋白（GFP）标记BMSC，用于后续鉴定;（1.3）将BMSC与UC在体外SIS上共培养。（1.3.1）评估UC对BMSC细胞生长和密度的影响。（1.3.2）通过表型外观、分子分析和免疫组织化学染色，评估在SIS上的UC共培养物中诱导BMSC分化的最佳时机。第二个具体目标：（2）确定BMSC的体内存活及其对膀胱功能和再生的影响。(2.1)用增强的GFP标记BMSC以供以后鉴定，（2.2）将GFP标记的BMSC与UC在SIS上体外共培养，（2.3）在半膀胱切除后将细胞-支架构建体植入犬膀胱中，（2.3.1）使用尿动力学研究分析膀胱功能和容量，（2.3.3）通过组织学、收缩性测定和免疫组织化学染色评价不同时间点的膀胱再生程度。

DESCRIPTION (provided by applicant): When normal bladder cells are grown in culture they remain normal, and when diseased or abnormal bladder cells are grown in culture they remain abnormal. A patient needing bladder augmentation or regeneration likely does not have healthy bladder cells for regeneration, and therefore needs an alternative cell source. Because Bone Marrow Stromal Cells (BMSC) have the capacity to be cultured and differentiated into smooth muscle-like cells, they may be useful as an alternative cell source for tissue engineered bladder regeneration. BMSC are obtained by bone marrow aspiration and can be expanded to sufficient numbers in vitro. They are not stem cells, but display multiple features of a stem cell population, including similar cell proliferation, histological appearance, and contractile phenotype compared to primary smooth muscle cell (SMC) cultures. This application focuses on researching the promising potential of BMSC for bladder regeneration. First Specific Aim: (1) Characterize BMSC when co-cultured with bladder urothelial cells (UC) in vitro. (1.1) Isolate and culture BMSC, UC, and SMC, (1.2) Label BMSC with enhanced Green Fluorescent Protein (GFP) for later identification, (1.3) Co-culture BMSC with UC on SIS in vitro. (1.3.1) Assess effect of UC on BMSC cell growth and density, (1.3.2) Evaluate optimal timing to induce BMSC differentiation in UC co-culture on SIS through phenotypic appearance, molecular analysis, and immunohistochemical staining. Second Specific Aim: (2) Determine the in vivo survival of BMSC and their impact on bladder function and regeneration. (2.1) Label BMSC with enhanced GFP for later identification, (2.2) Co-culture GFP labeled BMSC and UC on SIS in vitro, (2.3) Implant cell-scaffold construct into a canine bladder after hemicystectomy, (2.3.1) Analyze bladder function and capacity using urodynamic studies, (2.3.2) Evaluate in vivo BMSC survival at different time points, (2.3.3) Evaluate extent of bladder regeneration at different time points through histology, contractility assays, and immunohistochemical staining.