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中生长时，它们保持正常，当病变或异常膀胱细胞在培养中生长时，它们保持异常。需要膀胱增强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。骨髓基质细胞（Bone Marrow Stromal Cells， BMSC）具有培养和分化为平滑肌样细胞的能力，可作为组织工程膀胱再生的替代细胞来源。骨髓间充质干细胞是通过骨髓抽吸获得的，可以在体外扩增到足够数量。它们不是干细胞，但表现出干细胞群的多种特征，包括与原代平滑肌细胞（SMC）培养物相似的细胞增殖、组织学外观和收缩表型。本研究旨在研究骨髓间充质干细胞在膀胱再生中的应用前景。第一个特定目的：(1)体外研究BMSC与膀胱尿路上皮细胞（UC）共培养的特性。（1.1）分离培养BMSC、UC和SMC；（1.2）用增强的绿色荧光蛋白（GFP）标记BMSC，以便后续鉴定；（1.3）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UC在SIS上共培养诱导BMSC分化的最佳时机。(2)确定骨髓间充质干细胞的体内存活率及其对膀胱功能和再生的影响。（2.1）用增强的GFP标记BMSC以供后期鉴定，（2.2）在体外SIS上共培养GFP标记的BMSC和UC，（2.3）在半胱氨酸切除后将细胞支架植入膀胱，（2.3.1）通过尿动力学研究分析膀胱功能和容量，（2.3.2）通过组织学、收缩性实验和免疫组织化学染色评估不同时间点BMSC在体内的存活情况，（2.3.3）评估不同时间点膀胱再生的程度。

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.