Novel Markers on Human Embryonic Stem Cells

人类胚胎干细胞的新标记

• 批准号: 6832961
• 负责人: PAUL W PRICE
• 金额: $ 10万
• 依托单位: ABEOME CORPORATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-17 至 2005-03-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6832961
• 关键词: biomarker; cell differentiation; cell membrane; enzyme linked immunosorbent assay; flow cytometry; human embryonic stem cell line; laboratory mouse; monoclonal antibody; technology /technique development

DESCRIPTION(provided by applicant): Embryonic stem cells (ES) are a major focus of research, because of their experimental utility and great promise as therapeutic tissue regeneration and gene replacement agents. ES cells are rapidly growing pluripotent cells with the capacity to differentiate into multiple cell types. Despite the important role that human ES (hES) cells could have in biomedical applications, research faces some obstacles. For example, only a limited number of hES cell lines have been approved for study. Additionally, few hES cell lines are completely stable and subpopulations often develop that are partially differentiated toward different cell lineages. The stage of differentiation of hES cells is often accompanied by the expression of one or a few plasma membrane antigens (PMAs) such as the cell surface marker SSEA-4. Monoclonal antibodies (mAbs) to novel PMAs on hES cells are of great biomedical interest as research reagents to monitor and standardize the developmental stage of hES cells and also have potential as diagnostic reagents and in the development of therapeutic stem cells. Abeome Inc. has developed a high-throughput hybridoma based platform, DISH (Direct Selection of Hybridomas), for producing mAbs with unprecedented speed and at substantially lower costs than by any other method. DISH works because a proprietary, genetically modified myeloma fusion partner allows the generation of hybridomas that robustly secrete and surface present Ig. This innovation eliminates the labor intensive and problem-plagued steps of limiting dilution cloning because these hybridomas can be selected by Fluorescent Activated Cell Sorting (FACS) and plating can be automated. The goal of this Phase I proposal is to isolate of a battery of mAbs to novel PMAs from pluripotent hES cells derived from the hESBGN-01 or hESBGN-02 (NIH registry #BG-1 and BG-2) cell lines as well as to less pluripotent cells that have differentiated from these lines. In brief, formalin fixed low passage SSEA-4+ and SSEA-4- populations derived from hES cell lines will be used to immunize two sets of mice. DISH will be used to isolate individual hybridomas prepared from these mice. PMA-specific mAbs will be sub-screened to identify those reactive for the surface of SSEA-4+ cells, but not with the SSEA-4- stem cells and vice-versa. This Phase I project will take the Applicant through the development of this novel variation on its DISH technology and could lead to the rapid commercial production of mAbs to thousands of stem cell PMAs.

描述（由申请人提供）：胚胎干细胞（ES）是研究的主要焦点，因为它们具有实验实用性以及作为治疗性组织再生和基因替代剂的巨大前景。 ES细胞是快速生长的多能细胞，具有分化成多种细胞类型的能力。尽管人类 ES (hES) 细胞在生物医学应用中可能发挥重要作用，但研究仍面临一些障碍。例如，只有有限数量的 hES 细胞系被批准用于研究。此外，很少有 hES 细胞系是完全稳定的，并且通常会形成部分分化为不同细胞谱系的亚群。 hES细胞的分化阶段常常伴随着一种或几种质膜抗原（PMA）的表达，例如细胞表面标志物SSEA-4。 hES 细胞上新型 PMA 的单克隆抗体 (mAb) 作为监测和标准化 hES 细胞发育阶段的研究试剂具有巨大的生物医学意义，并且还具有作为诊断试剂和治疗性干细胞开发的潜力。 Abeome Inc. 开发了一种基于高通量杂交瘤的平台 DISH（杂交瘤直接选择），能够以前所未有的速度生产单克隆抗体，且成本远低于任何其他方法。 DISH 之所以有效，是因为专有的转基因骨髓瘤融合伙伴可以产生能够强劲分泌 Ig 并在表面呈现 Ig 的杂交瘤。这项创新消除了有限稀释克隆的劳动密集型和问题多的步骤，因为这些杂交瘤可以通过荧光激活细胞分选（FACS）进行选择，并且平板接种可以自动化。该第一阶段提案的目标是从来自 hESBGN-01 或 hESBGN-02（NIH 注册#BG-1 和 BG-2）细胞系的多能 hES 细胞以及从这些细胞系分化而来的低多能细胞中分离出一组 mAb 和新型 PMA。简而言之，来自 hES 细胞系的福尔马林固定低传代 SSEA-4+ 和 SSEA-4- 群体将用于免疫两组小鼠。 DISH 将用于分离从这些小鼠中制备的个体杂交瘤。 PMA 特异性单克隆抗体将进行二次筛选，以识别那些对 SSEA-4+ 细胞表面具有反应性的单克隆抗体，但对 SSEA-4- 干细胞不具有反应性的单克隆抗体，反之亦然。该第一阶段项目将带领申请人开发其 DISH 技术的这种新颖变体，并可能导致针对数千个干细胞 PMA 的 mAb 的快速商业化生产。