Novel Markers on Human Embryonic Stem Cells

描述(申请人提供)：胚胎干细胞(ES)是一个主要的研究重点，因为它们的实验用途和作为治疗组织再生和基因替代的巨大希望。ES细胞是一种快速生长的多能细胞，具有分化为多种细胞类型的能力。尽管人类胚胎干细胞(HES)可以在生物医学应用中发挥重要作用，但研究面临一些障碍。例如，只有数量有限的HES细胞株被批准进行研究。此外，很少有HES细胞系是完全稳定的，亚群通常发展为部分分化为不同的细胞系。HES细胞的分化阶段往往伴随着一种或几种质膜抗原的表达，如细胞表面标志物SSEA-4。针对HES细胞表面新型PMAs的单抗作为监测和规范HES细胞发育阶段的研究试剂具有重要的生物医学意义，在HES细胞的诊断和治疗干细胞的开发中也具有潜在的应用前景。Abeome Inc.开发了一种基于杂交瘤的高通量平台DISH(直接选择杂交瘤)，用于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抗。DISH之所以有效，是因为一种专有的、转基因的骨髓瘤融合伙伴允许产生大量分泌和表面呈现Ig的杂交瘤。这一创新消除了限制稀释克隆的劳动密集型和问题缠身的步骤，因为这些杂交瘤可以通过荧光激活细胞分选(FAC)来选择，培养可以自动化。该第一阶段方案的目标是从hESBGN-01或hESBGN-02(NIH注册编号BG-1和BG-2)细胞系衍生的多能HES细胞以及从这些细胞系分化而来的较少多能细胞中分离出一组针对新型PMAs的单抗。总之，福尔马林固定的HES细胞系低传代SSEA-4+和SSEA-4-群体将用于免疫两组小鼠。DISH将被用来分离从这些小鼠身上制备的单个杂交瘤。PMA特异性单抗将被进一步筛选，以识别那些对SSEA-4+细胞表面有反应的单抗，但不与SSEA-4-干细胞反应，反之亦然。这个第一阶段的项目将带领申请者开发其DISH技术的这一新变种，并可能导致mAbs的快速商业化生产到数千种干细胞PMAs。

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.