Cell-Based Therapy in Minipig Model of Radiation-Induced Xerostomia

该项目将开发一个功能齐全的，可植入的人类唾液腺， 头颈癌放疗后口干/口干。尽管监管要求 用于组织工程器官临床转化的接近人类的动物模型，而大型动物模型则不然 存在测试干细胞的方法，以恢复唾液功能，包括液体分泌和蛋白质生产 有助于消化和口腔健康。啮齿动物模型虽然对测试产品设计有用，但缺乏关键属性 将可植入的生物设备运送到诊所。为了弥补这些不足，我们 一个跨学科团队，包括休斯顿的法拉赫-卡森/哈灵顿团队、帕辛诺实验室 在匹兹堡开发了一种方法，使用辐射猪模型进行遗传操作， 位于安阿伯的Lombaert实验室，以评估植入物整合。我们最近的实验证明， 免疫抑制下的模型耐受性良好，为开发异种移植提供了前所未有的机会 唾液人干/祖细胞（hS/PC）移植研究的模型，并避免需要开发 猪对猪的自体移植这项建议建立在我们令人兴奋的初步结果，开发和表征一种新的 放射免疫抑制的小型猪模型用于测试移植的hS/PC恢复唾液分泌的能力 分泌功能我们假设这个模型将概括辐射人体的环境 唾液床，并提供评估人类S/P细胞功能所需的信息类型， 移植到患有口干症的患者体内。具体目标是：1）建立辐射 免疫抑制的小型猪作为合适的宿主动物，以评价长期稳定性、生物相容性和结局 含有包封的hS/PC的基质改性的透明质酸盐（HA）水凝胶材料; 标准化优化策略以将唾液新组织原型放置在宿主腮腺床中; B）评估 植入的无细胞水凝胶构建体在放置于辐射腮腺中后不同时间的稳定性， 根据需要进行配方变更，以确保长期生物整合; c）使用定量评分系统 为了评估移植到辐射后包封的hS/PC负载的构建体随时间的命运， 唾液床，包括活力、无过度生长的生长、包括脉管系统和神经的宿主整合，以及 分化成表达腺泡、导管和肌上皮标记物的唾液结构。2)评估能力 a）证明从移植组织中递送功能性蛋白质以恢复唾液功能： 修复性基因治疗结合唾液腺植入治疗放射性小型猪的实验研究 新生组织; B）通过评价水通道蛋白5在新生组织和流体中的表达来证明流体流动的恢复 使用直接唾液收集技术生产。有助于唾液腺功能恢复的评价 通过使用遗传修饰来产生分泌的Met-Luc S/P细胞，当植入时， 通过采集宿主血液或唾液中的Luc样本来跟踪移植物存活时间。

This project will develop a fully functional, implantable human salivary gland for patients suffering from xerostomia/dry mouth after radiotherapy for head and neck cancer. Despite the regulatory requirement for a close-to-human animal model for clinical translation of tissue engineered organs, large animal models do not exist to test stem-cell approaches to restore salivary function including fluid secretion and protein production needed for digestion and oral health. Rodent models, while useful to test product designs, lack key attributes needed to deliver an implantable biological device to the clinic. To address these shortcomings, we assembled an interdisciplinary team that includes the Farach-Carson/Harrington team in Houston, the Passineau laboratory in Pittsburgh to develop an approach using a radiated pig model amenable to genetic manipulation, and the Lombaert laboratory in Ann Arbor to assess implant integration. Our recent demonstration that a human-in-pig model under immunosuppression is well tolerated presents an unprecedented opportunity to develop xenograft model for the salivary human stem/progenitor cell (hS/PC) transplantation studies and avoid the need to develop a pig-in-pig autograft. This proposal builds on our exciting preliminary results to develop and characterize a novel radiated immunosuppressed mini-pig model for testing the ability of transplanted hS/PCs to restore salivary secretory function. We hypothesize that this model will recapitulate the environment of the radiated human salivary bed, and provide the type of information needed to evaluate the function of human S/P cells one day to be transplanted into patients suffering from xerostomia. Specific Aims are: 1) Establish the radiated immunosuppressed minipig as a suitable host animal to evaluate the long term stability, biocompatibility and fate of matrix-modified hyaluronate (HA) hydrogel materials containing encapsulated hS/PCs; a) Develop and standardize optimized strategies to place the salivary neotissue prototype in the host parotid bed; b) Evaluate stability of implanted acellular hydrogel constructs at various times after placement in the radiated parotid and make formulational changes as needed to ensure long term biointegration; c) Use a quantitative scoring system to evaluate the fate over time of encapsulated hS/PC-loaded constructs after transplantation into the radiated salivary bed including viability, growth without overgrowth, host integration including vasculature and nerve, and differentiation into salivary structures expressing acinar, ductal and myoepithelial markers. 2) Evaluate the ability of the transplanted tissue to restore salivary function: a) Demonstrate functional protein delivery from the salivary neotissues into the irradiated minipig by combining restorative gene therapy with implanted salivary neotissues; b) Demonstrate restoration of fluid flow by evaluating aquaporin 5 expression in neotissues and fluid production using direct saliva collection techniques. The evaluation of salivary functional restoration will be aided by use of a genetic modification to create a secreted Met-Luc S/P cell that when implanted would provide a noninvasive means of following graft survival over time simply by sampling Luc in host blood or saliva.