Probing neural precursor diversity with phage display derived scFv antibodies

描述（由申请人提供）：我们从中枢神经系统分离不同干细胞和祖细胞群的能力取得了快速进展，为中枢神经系统疾病的诊断和治疗开辟了有希望的新途径。这些进步主要依赖于特定标记物的使用，这些标记物使我们能够区分发育和功能相关的细胞类型。利用目前可用的有限数量的细胞类型特异性标记，已经有可能确定中枢神经系统内控制谱系关系的一些关键原则。然而，谱系研究也表明，存在更大程度的细胞多样性，这超出了当今可用标记的范围。在这里，我们建议系统地开发一个标记库，可用于鉴定和纯化不同的神经细胞群，具有前所未有的特异性。为了实现这一目标，我们将把分离中枢神经系统前体种群的能力与噬菌体展示技术的力量结合起来。通过体外选择与离体神经细胞表面结合的单链抗体克隆，特异性选择可用于活细胞表达分析的试剂。该方案将允许快速筛选大量克隆，同时避免需要对动物进行免疫的程序所固有的问题。我们的研究计划将集中于分离标记2个特定中枢神经系统群体的标记物：早期胶质祖细胞和不同的星形胶质细胞群体。目前还没有标记物可以对胶质前体分化的不同阶段进行积极的鉴定。分离这些标记将使我们能够区分少突胶质细胞和星形胶质细胞前体，以及谱系限制和增殖潜力的进展状态，正如在来自中枢神经系统不同区域的胶质前体中观察到的那样。同样，虽然GFAP继续被用作星形胶质细胞的第一定义标记物，但很明显，在发育过程中、急性病变中或胶质瘢痕组织中发现的星形胶质细胞之间，星形胶质细胞表型可能存在显著差异。使用胶质祖细胞的初步数据有力地支持了这种新方法的可行性。Lay声明：拟议的研究旨在产生新的工具，以便更好地识别大脑中不同类型的干细胞及其后代。这些工具还将有助于开发更好的诊断和治疗影响大脑的疾病。

DESCRIPTION (provided by applicant): The rapid progress in our ability to isolate different stem cell and progenitor cell populations from the central nervous system has opened up the door to promising new avenues for the diagnosis and therapy of diseases of the CNS. These advances depend critically on the use of specific markers that allow us to distinguish between both developmentally and functionally related cell types. Using the limited number of cell type specific markers currently available, it has been possible to identify a number of key principles governing lineage relationships within the CNS. However, lineage studies also suggest that a much greater degree of cell diversity exists, that lies beyond the purview of markers available today. Here we propose to systematically develop a library of markers that can be used to identify and purify distinct neural cell populations with unprecedented specificity. To achieve this, we will combine our ability to isolate CNS precursor populations with the power of Phage Display technology. Through in vitro selection of single chain antibody clones that bind to the surface of isolated neural cells, we will specifically select for reagents that can be used for expression analysis in live cells. This protocol will allow rapid screening of large numbers of clones while avoiding problems inherent to procedures requiring the immunization of animals. Our plan of research will focus on the isolation of markers labeling 2 specific CNS populations: early glial progenitors and distinct populations of astrocytes. There are currently no markers that allow a positive identification of distinct stages of glial precursor differentiation. Isolating such markers will allow us to discern between oligodendroglial and astroglial precursors, as well as between progressive states of lineage restriction and proliferative potential, as observed in glial precursors from different regions of the CNS. Similarly, while GFAP continues to be used as the 1 defining marker of astrocytes, it is clear that astrocytic phenotypes can vary dramatically between astrocytes found during development, in acute lesions or in glial scar tissue. Preliminary data using glial progenitors strongly support the feasibility of this novel approach. Lay statement: The proposed research aims to generate new tools that will allow a better identification of different types of stem cells and their offspring in the brain. These tools will also help in developing better diagnostics and treatments for diseases affecting the brain.