Novel antibodies for mesenchymal tissue stem cells

描述（由申请人提供）：目前用于克隆分离/富集具有间充质组织分化能力的干细胞群的试剂有限。在本申请中，我们提出了一个系统的努力，以产生这些细胞的细胞表面标记抗体。这些抗体将提供分选和纯化不同细胞群的方法，从而允许有意义的基因表达研究发现对维持不同发育阶段的干细胞/祖细胞重要的分子。它们还将允许干细胞在组织中的定位。此外，这些标记物可以允许分离足够的干细胞用于离体组织工程，或甚至在手术室内，使得具有分化能力的细胞的浓度可以用于骨骼组织的修复或再生，而不需要进一步的体外分离和扩增。我们建议使用的技术组合，包括消减免疫方案，杂交瘤生长方法，允许管理大量的克隆和筛选方法，应该允许相关抗体的离散选择。我们假设，这种方法将大大增加产生特异性成体干细胞抗体的可能性超过以前使用的方法。我们的具体目标是使用消减免疫来产生针对成体干细胞的单细胞悬浮液的单克隆抗体。将进行大规模筛选，以确定定义重要干细胞类别的抗体。分类种群的特征在于它们的可扩展性和自我更新的特性。将鉴定针对感兴趣的干细胞群体的新型抗体。干/祖细胞分化成间充质组织的能力的特异性抗体标记物是缺乏的。该提案的目的是开发新的抗体，其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用于在治疗性移植之前或在分化和随后的移植之前纯化细胞。

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.