Reagents for Targeted Ablation of Residual Contaminating Pluripotent Stem Cells

描述（由申请人提供）：人多能干细胞（hPS）作为药物发现、疾病建模和移植以替代因退行性疾病或损伤而丢失的细胞的可再生细胞来源具有巨大潜力。一个关键的障碍， hPS细胞研究的成功应用是缺乏产生hPS细胞衍生物的纯群体的方法。污染细胞对于药物发现和移植都是一个问题，在药物发现中需要纯的细胞群用于测定再现性，在移植中由于它们可能形成肿瘤或分化为不适当的细胞类型而存在安全性问题。具体来说，我们建议在这里解决残留污染的未分化的hPS细胞，有可能形成畸胎瘤的问题。我们建议开发hPS细胞靶向毒素（hPS-CTT），杀死残留的hPS细胞，分化的细胞群体的损伤或改变最小。为了实现选择性细胞靶向毒性，我们将联合收割机与植物毒素皂草素结合， 只有在细胞内化时才有毒。这种方法比以前提出的需要细胞操作的遗传修饰和物理分离方法简单得多，这些方法可能会改变或胁迫所需的细胞群。它也比先前提出的细胞毒性抗体灵活得多，因为任何内化抗体或肽都可以适用于靶向细胞杀伤。此外，hPS-CTT被设计为与死细胞一起去除，从而减少其对存活细胞的潜在影响。为了证明I期的可行性，我们将筛选用于选择性hPS细胞靶向的抗体和肽。将细胞靶向剂（CTA）间接缀合至毒素，并测试其在含有掺入未分化hPS细胞的分化hPS细胞的混合物的模型细胞系统中去除未分化hS细胞的能力。我们将通过流式细胞术、PluriTest和定量RT-PCR测量治疗前后的hPS细胞含量。将使用体内畸胎瘤测定来测试最佳hPS-CTT去除hPS细胞的能力。将通过蛋白质印迹分析和功能测定来测量处理群体中hPS-CTT的持久性以检测残留毒性。将各种靶向剂与二级毒素缀合物组合的2组分系统将用于I期以显示可行性。在第二阶段，我们将开发直接与毒素结合的细胞靶向剂，并进一步优化hPS细胞去除方案。

DESCRIPTION (provided by applicant): Human pluripotent stem (hPS) cells have great potential as a renewable source of cells for drug discovery, disease modeling and for transplantation to replace cells lost to degenerative diseases or injury. A critical barrier to the successful application of hPS cell research is the lack of methods for producing pure populations of hPS cell derivatives. Contaminating cells are both an issue for drug discovery where pure populations are needed for assay reproducibility and for transplantation where they present a safety issue because of their potential for tumor formation or differentiation to inappropriate cell types. Specifically, we propose here to address the problem of residual contaminating undifferentiated hPS cells that have the potential to form teratomas. We propose to develop hPS cell targeted toxins (hPS-CTTs) that kill residual hPS cells with minimal damage or alteration of the differentiated cell population. To achieve selective cell targeted toxicity, w will combine an internalizing hPS selective targeting agent with the plant toxin, saporin, which is only toxic upon cellular internalization. This approach is much simpler than previously proposed genetic modification and physical separation methods that require cell manipulations that could alter or stress the desired cell population. It is also much more flexible than the previously proposed cytotoxic antibody because any internalizing antibody or peptide can be adapted for targeted cell killing. Moreover, the hPS-CTT is designed to be removed with the dead cells thus reducing its potential impact on the surviving cells. To demonstrate feasibility in phase I, we wil screen antibodies and peptides for selective hPS cell targeting. The cell targeting agents (CTAs) will be indirectly conjugated to a toxin, and tested for their ability to remove undifferentiated hS cells in a model cell system containing admixtures of differentiated hPS cells spiked with undifferentiated hPS cells. We will measure hPS cell content before and after treatment by flow cytometry, the PluriTest, and quantitative RT-PCR. The ability to the optimal hPS-CTT to remove hPS cells will be tested using an in vivo teratoma assay. The persistence of the hPS-CTT in the treated population will be measured by western blot analysis and a functional assay to detect residual toxicity. A 2 component system that combines various targeting agents with secondary toxin conjugates will be used in phase I to show feasibility. In phase II, we will develop cell targeting agents directly conjugated to the toxin and further optimize protocols for hPS cell removal.