SELF-RENEWAL OF PRIMATE ES CELLS

灵长类 ES 细胞的自我更新

• 批准号: 6224381
• 负责人: James A Thomson
• 金额: $ 12.55万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6224381
• 关键词: antibody; bacteriophage M13; bacteriophage T7; cell biology; cell cell interaction; cell differentiation; cell proliferation; cell type; complementary DNA; embryonic stem cell; fibroblast growth factor; fibroblasts; genetic library; growth factor receptors; immunocytochemistry; ligands; macrophage; membrane proteins; molecular cloning; muscle cells; peptides; plasmids; single cell analysis; tissue /cell culture; vascular endothelium

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.

描述（改编自申请人的摘要）：申请人最近的 NHP的推导（恒河猴和普通果棒）和人类ES细胞的推导已有 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。 灵长类动物ES细胞的要求 对于成纤维细胞馈线是最重要的限制因素 常规的NHP ES细胞大规模扩展，并识别特定 介导NHP ES细胞自我更新的成纤维细胞产生因子是一个 批判性研究需求。 申请人的初步结果表明 成纤维细胞产生的基本因素不是分泌的，因此 膜结合或紧密连接到细胞外细胞外 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了以下事实的阻碍 分子不分泌；因此需要新颖的方法。 这 申请人的基本假设是介导的配体和受体 NHP ES细胞自我更新是成纤维细胞和ES细胞整体膜 蛋白质。 为了识别这些基本信号分子，研究人员 将完成以下特定目标：1）他们将使用噬菌体T7 显示成纤维细胞cDNA以筛选成成纤维细胞产生的多肽 特异性结合了细胞表面分子。 多肽 特异性结合恒河ES细胞将被定量测定 刺激对恒河类细胞自我更新的影响。 克隆的成纤维细胞 - 产生的多肽将用于识别ES细胞表面受体 它们约束。 2）调查人员将使用吞噬（M13）库来 分离高亲和力单链FV抗体，该抗体特异性结合 细胞表面的恒河体细胞。 结合恒河ES细胞的抗体将是 定量测定对恒河类细胞自我更新的抑制作用。 充当拮抗剂的抗体将用于识别ES细胞 它们结合的表面受体。