SELF-RENEWAL OF PRIMATE ES CELLS

描述(改编自申请人的摘要)：申请人的近况 NHP(恒河猴和普通绒猴)和人类ES细胞的衍生 对人类发育生物学、药物发现、药物的广泛影响 测试和移植医学。灵长类胚胎干细胞的需求 对于成纤维细胞，饲养层是最重要的限制因素 常规大规模扩增NHP ES细胞，并鉴定其特异性 介导NHP ES细胞自我更新的成纤维细胞产生因子(S)是一种 关键的研究需要。申请者的初步结果表明， 成纤维细胞产生的必需因子(S)不分泌，因此是 膜与细胞外结合或紧密附着在细胞外 矩阵。识别基本信号的功能策略 成纤维细胞产生的分子受到以下事实的阻碍 分子不是分泌的，因此需要新的方法。这个 申请者的基本假设是，配体和受体介导 NHP ES细胞的自我更新是成纤维细胞和ES细胞的整合膜 蛋白质。为了识别这些重要的信号分子，研究人员 将实现以下具体目标：1)它们将使用噬菌体T7 展示成纤维细胞的cDNA以筛选成纤维细胞产生的多肽 特异性结合恒河猴ES细胞表面分子的分子。多肽 特异性结合的恒河猴ES细胞将被定量检测 对恒河猴ES细胞自我更新的刺激作用。克隆成纤维细胞- 产生的多肽将用于识别ES细胞表面受体以 他们把它们捆绑在一起。2)调查人员将使用噬菌体(M13)文库 分离高亲和力单链抗体，特异性结合 恒河猴胚胎干细胞的细胞表面。结合恒河猴ES细胞的抗体将是 定量检测对恒河猴ES细胞自我更新的抑制作用。 作为拮抗剂的抗体将被用来识别ES细胞 它们与之结合的表面受体。

DESCRIPTION (Adapted from the applicant's abstract): The applicants' recent derivation of NHP (rhesus monkey and common marmoset) and human ES cells has widespread implications for human developmental biology, drug discovery, drug testing, and transplantation medicine. The requirement by primate ES cells for a fibroblast feeder layer is the most important limiting factor in the routine large-scale expansion of NHP ES cells, and identifying the specific fibroblast-produce factor(s) that mediate NHP ES cell self-renewal is a critical research need. The applicants' preliminary results demonstrate that the essential factor(s) produced by fibroblasts are not secreted, and thus are either membrane bound or are tightly attached to the cell extracellular matrix. Functional strategies for identifying the essential signaling molecules produced by the fibroblasts are hampered by the fact that the molecules are not secreted; therefore novel approaches are required. The applicants' underlying hypothesis is that the ligands and receptors mediating NHP ES cell self-renewal are fibroblast and ES cell integral membrane proteins. To identify these essential signaling molecules, the investigators will accomplish the following specific aims: 1) They will use phage T7 display of fibroblast cDNAs to screen for fibroblast-produced polypeptides that specifically bind rhesus ES cell surface molecules. Polypeptides that specifically bind rhesus ES cells will be quantitatively assayed for stimulatory effects on rhesus ES cell self-renewal. Cloned fibroblast- produced polypeptides will be used to identify the ES cell surface receptor to which they bind. 2) The investigators will use a phagemid (M13) library to isolate high affinity single chain Fv antibodies that specifically bind to the cell surface of rhesus ES cells. Antibodies that bind rhesus ES cells will be quantitatively assayed for inhibitory effects on rhesus ES cell self-renewal. Antibodies that act as antagonists will be used to identify the ES cell surface receptors to which they bind.