Novel Markers on Human Embryonic Stem Cells

描述（由申请人提供）：胚胎干细胞（ES）是研究的主要焦点，因为它们的实验效用和作为治疗性组织再生和基因替代剂的巨大前景。胚胎干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。尽管人类胚胎干细胞（hES）在生物医学应用中具有重要作用，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he细胞系是完全稳定的，并且亚群经常向不同的细胞系部分分化。hES细胞的分化阶段通常伴随着一种或几种质膜抗原（pma）的表达，如细胞表面标记物SSEA-4。针对hES细胞上新型PMAs的单克隆抗体（mab）作为监测和规范hES细胞发育阶段的研究试剂具有重要的生物医学意义，也具有作为诊断试剂和治疗性干细胞开发的潜力。Abeome公司开发了一种高通量的基于杂家瘤的平台，DISH（直接选择杂家瘤），以前所未有的速度和比任何其他方法低得多的成本生产单克隆抗体。DISH之所以有效，是因为一种专有的、基因修饰的骨髓瘤融合伙伴可以产生杂交瘤，这些杂交瘤可以强烈地分泌和表面呈现Ig。这一创新消除了限制稀释克隆的劳动密集型和问题困扰的步骤，因为这些杂交瘤可以通过荧光活化细胞分选（FACS）选择，并且可以自动镀。该I期计划的目标是从hESBGN-01或hESBGN-02 （NIH注册编号为BG-1和BG-2）细胞系衍生的多能性hES细胞以及从这些细胞系分化出来的多能性较低的细胞中分离出一系列单克隆抗体。总之，将使用福尔马林固定的低传代SSEA-4+和来自hES细胞系的SSEA-4-群体对两组小鼠进行免疫。DISH将用于分离从这些小鼠中制备的单个杂交瘤。对pma特异性单克隆抗体进行亚筛选，鉴定对SSEA-4+细胞表面有反应的单克隆抗体，而对SSEA-4-干细胞表面无反应的单克隆抗体，反之亦然。该I期项目将带领申请人通过其DISH技术的这种新变体的开发，并可能导致数千种干细胞pma的单克隆抗体的快速商业化生产。

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.