Novel antibodies for mesenchymal tissue stem cells

描述（由申请人提供）：用于克隆分离/富集具有间充质组织分化能力的干细胞群的试剂目前是有限的。在这个应用中，我们提出了一个系统的努力来生产这些细胞的细胞表面标记抗体。这些抗体将提供分类和纯化不同细胞群的方法，允许有意义的基因表达研究，以发现在不同发育阶段维持干细胞/祖细胞的重要分子。它们还将使干细胞在组织中的定位成为可能。此外，这些标记可以分离出足够的干细胞用于离体组织工程，甚至在手术室内，这样具有分化能力的细胞浓度就可以用于骨骼组织的修复或再生，而无需进一步的体外分离和扩增。我们建议使用多种技术，包括减法免疫方案、允许管理大量克隆的杂杂瘤生长方法和允许离散选择相关抗体的筛选方法。我们假设，这种方法将大大增加产生特异性成人干细胞抗体的可能性比以前使用的方法。我们的具体目标是使用减法免疫来产生针对成人干细胞单细胞悬液的单克隆抗体。将进行大规模筛选，以确定确定重要干细胞类别的抗体。分类种群以其可扩展性和自我更新的特性为特征。将发现针对有趣干细胞群的新型抗体。缺乏干细胞/祖细胞分化为间充质组织的特异性抗体标记物。这项提议的目的是开发新的抗体，可用于鉴定和分选这些细胞，用于干细胞生物学的研究，以及在治疗性移植前或分化和随后的移植前纯化细胞。

DESCRIPTION (provided by applicant): Reagents for the clonal isolation/enrichment of stem cell populations with mesenchymal tissue differentiation capacity are currently limiting. In this application, we propose a systematic effort to produce cell surface marker antibodies for these cells. These antibodies would provide the means to sort and purify distinct cell populations allowing meaningful gene expression studies to discover molecules important for the maintenance of stem/progenitor cells at different developmental stages. They will also allow the localization of stem cells in tissues. In addition, these markers may allow isolation of sufficient stem cells for ex vivo tissue engineering, or even within the operating room, such that a concentration of cells with differentiation capacity can be used for repair or regeneration of skeletal tissues without need for further in vitro isolation and expansion. We propose the use of a combination of techniques including a subtractive immunization protocol, a hybridoma growth method that allows management of large numbers of clones and screening methods that should allow discrete selection of relevant antibodies. We hypothesize that this approach will greatly increase the likelihood of generating specific adult stem cell antibodies over previously used methods. Our specific aim is to use subtractive immunizations to generate monoclonal antibodies against single cell suspensions of adult stem cells. Large scale screening will be done to identify antibodies that define important classes of stem cells. Sorted populations will be characterized for their properties of expandability and self-renewal. Novel antibodies to interesting stem cell populations will be identified. Specific antibody markers for stem/progenitor cells with the capacity to differentiate into mesenchymal tissues are lacking. The aim of this proposal is to develop novel antibodies that can be used to identify and sort these cells for the study of stem cell biology as well as for the purification of cells before therapeutic transplantation or before differentiation and subsequent transplantation.