Antibodies Targeting Novel Surface Antigens on Pluripotent Stem Cell Derivatives

描述(由申请人提供)：通过在受体细胞中转移和表达4个基因，将人类成纤维细胞和其他类型的细胞重新编程为完全的胚胎干细胞潜力的能力是干细胞研究和开发的主要技术进步。重新编程的iPS细胞可以自我更新并分化为所有类型的细胞，如人类胚胎干细胞(HES)，但因为它们来自成人皮肤细胞，所以它们可以从患者特定的细胞中制造出来，从而为基因匹配的细胞替代疗法和药物毒性测试提供了可能性。此外，患者特有的iPS细胞来源将使许多疾病的人类细胞发育模型首次得以建立。随着多能干细胞系数量的迅速增加，开发有效的定向分化方案变得更加迫切。一个重要的瓶颈是我们目前对表面标记的有限了解，这些标记定义了从多能干细胞分化出来的各种细胞类型。随着多能干细胞的分化，无论是在空间上，在形成的细胞类型上，还是在时间上，它们沿着其命运细胞谱系的进展程度都存在着异质性。即使细胞培养条件被操纵以引导向特定谱系分化，在培养中仍然存在不需要的、可能形成肿瘤的未成熟细胞和其他谱系的细胞。需要通过表面标记鉴定和细胞分选来确定细胞谱系和分化阶段的能力，以开发出分离更多同质的iPS和其他多能细胞群体的方法，用于疾病建模、药物测试和发现，并最终用于制备安全有效的细胞疗法。它还将提供对人类发展的更好理解。我们建议通过在iPS上识别新的表面标记来满足这一需求，并比较HES、衍生细胞，并开发相应的抗体用于细胞鉴定和阳性和阴性细胞选择。我们将使用融合到耐药基因的基于细胞存活的选择策略，从分化的HIPS和HES cDNA文库中选择编码cDNA的表面蛋白。这些cDNA将为免疫提供重组抗原和DNA表达载体。鸡体内会产生抗体，因为与哺乳动物宿主相比，鸡更有可能对高度保守的抗原产生反应。由此产生的抗体将在分化的iPS和HES细胞上进行测试，以确定其暂时和特殊的表达以及谱系特异性。第一阶段将是开发这些技术和制备多达15种抗体的可行性研究。第二阶段将扩大抗体的开发，并从谱系和阶段特异的cDNA文库和疾病特异的iPS细胞中鉴定额外的表面抗原。这些抗体将商业化，供干细胞研究和开发界使用，用于定义用于疾病建模、药物测试和为临床前和临床研究准备细胞的多能细胞衍生物。。公共卫生相关性：人类胚胎干细胞(HES)通过提供治疗几乎任何退行性疾病或损伤的细胞，以及作为药物测试和发现的人类细胞来源，有可能极大地造福公共健康。胚胎干细胞样细胞是最近通过病毒基因传递从成人皮肤细胞中获得的。这些诱导多能(IPS)细胞是潜在的无限来源，所有类型的人类细胞在基因上与患者匹配，可以用于药物开发、疾病建模和发育研究，最终如果它们可以在没有当前使用的病毒载体的情况下安全地制造出来的话。目前分化多能细胞的方法导致混合群体，其中可能包含潜在的肿瘤形成细胞。需要确定诱导干细胞和胚胎干细胞来源的特定亚群的试剂和方法。我们建议开发针对iPS和HES来源细胞表面新抗原的抗体，作为促进干细胞研究的试剂，并使药物和细胞治疗开发所需的细胞分离、放大和生产成为可能。

DESCRIPTION (provided by applicant): The ability to reprogram human fibroblasts and other cell types to full embryonic stem cell potential by transfer and expression of 4 genes in the recipient cells is a major technical advance for stem cell research and development. The reprogrammed iPS cells can self renew and differentiate to all cell types like human embryonic stem (hES) cells but because they are derived from adult skin cells they can be made from patient specific cells and thus offer the potential for genetically matched cell replacement therapies and drug toxicity testing. Moreover, patient specific iPS cell derivation will allow human cellular developmental models of many diseases to be made for the first time. With the number of pluripotent stem cell lines now increasing much more rapidly, it is even more urgent to develop efficient directed differentiation protocols. An important bottleneck is our current limited knowledge of surface markers that define the various cell types that differentiate from pluripotent stem cells. As pluripotent stem cells differentiate there is heterogeneity both spatially, in the cell types formed and temporally, in their degree of progression along their fated cell lineage. Even when cell culture conditions are manipulated to direct differentiation toward specific lineages there are still unwanted potentially tumor forming immature cells and cells of other lineages in the culture. The ability to define cell lineage and stage of differentiation by surface marker identification and cell sorting is needed to develop ways to isolate more homogeneous populations of iPS and other pluripotent cells for disease modeling, drug testing and discovery, and ultimately for preparing safe and effective cell therapies. It will also provide better understand of human development. We propose here to address this need by identifying new surface markers on iPS, and for comparison hES, derived cells and developing corresponding antibodies for cell identification and positive and negative cell selection. We will select surface protein encoding cDNAs from differentiating hiPS and hES cDNA libraries, using a cell survival based selection strategy by fusion to a drug resistance gene. The cDNAs will provide recombinant antigen and DNA expression plasmids for immunization. Antibodies will be raised in chickens because they are more likely to react to highly conserved antigens than mammalian hosts. The resulting antibodies will be tested on differentiated iPS and hES cells for their temporal and special expression and lineage specificity. Phase 1 will be a feasibility study to develop the techniques and prepare up to 15 antibodies. Phase 2 will expand the antibody development and identify additional surface antigens from lineage and stage specific cDNA libraries and disease specific iPS cells. The antibodies will be commercialized for use by the stem cell research and development community for defining pluripotent cell derivatives for disease modeling, drug testing and preparing cells for preclinical and clinical studies. . PUBLIC HEALTH RELEVANCE: Human embryonic stem (hES) cells have the potential to profoundly benefit public health by providing cells for treating virtually any degenerative disease or injury and as a source of human cells for drug testing and discovery. Embryonic stem cell-like cells have recently been derived from adult human skin cells using viral gene delivery. These induced pluripotent (iPS) cells are a potentially unlimited source of human cells of all types that are genetically matched to patients and can be used drug discovery, disease modeling and developmental studies, and ultimately cell therapies if they can be made safely without currently used viral vectors. Current methods of differentiating pluripotent cells result in mixed populations that can contain potentially tumor forming cells. Reagents and methods of defining specific subpopulations of induced and embryonic stem cell derived cells are needed. We propose here to develop antibodies to novel surface antigens on iPS and hES derived cells as reagents that will facilitate stem cell research and enable the isolation, scale up and production of cells for drug and cell therapy development.