Rapid Multiplexed Nanoprobe Assays for Pluripotent Stem Cell Differentiation

描述（由申请人提供）：人多能干（hPS）细胞具有无限自我更新和分化成许多不同细胞类型的能力。因此，它们可能为发育研究、疾病建模、药物筛选、环境化学测试、预测毒理学和最终细胞替代疗法提供各种人类细胞类型的无限供应，以治疗许多目前难治的退行性疾病。hPS细胞的来源不再局限于人类胚胎干细胞（hES）。重编程技术通过从容易获得的正常和患病供体细胞（如皮肤或毛发）常规生产hPS细胞（称为诱导多能（iPS）细胞）来推进该领域。因此，近年来可用于研究和开发的hPS细胞系的数量和遗传多样性大大增加。这一趋势可能会继续与政府和私人的倡议，银行数以千计的iPS细胞系。然而，我们表征和验证hPS细胞的能力并没有跟上生产速度的进步。iPS细胞系质量和分化倾向的变化是一个关键问题，需要仔细表征每个iPS细胞系的至少6个克隆。目前验证多能性和表征分化的方法涉及对iPS克隆进行费力且耗时的免疫细胞化学（ICC）分析，以获得3个初级胚层中每一个的体外分化的多个标志物。在SCID小鼠中进行畸胎瘤形成的体内测试需要6-12周，因此对于初始iPS筛选是不实际的。因此，对于更有效的体外iPS筛选方法来评估质量和确定体外分化倾向存在关键的未满足的需求。在这里，我们建议开发一个肽靶向量子点（PTQD）检测分化到每个胚层。我们设计的PTQD测定比ICC更简单、更快速、更灵敏、更方便。该检测试剂盒使用PTQD在一个步骤中进行活细胞靶向和检测，使该检测试剂盒比ICC更适合多路复用和自动化。重要的是，该测定保留了细胞的活性以供进一步使用。在I期，我们将开发用于定形内胚层（DE）的多重PTQD测定，并鉴定用于外胚层和中胚层测定的其他肽。在证明了DE的多重PTQD测定的可行性后，我们将在II期中使用I期肽开发外胚层和中胚层PTQD测定。对于干细胞研究人员来说，了解每个iPS细胞系在体外向一个生殖系或另一个生殖系分化的倾向是非常有用的，这样他们就可以选择合适的细胞系来适应他们的个人应用。在为每个种系开发了探针之后，我们的目标是在第二阶段结束时使用颜色编码的PTQD开发一种简单的分化倾向测定法，其导致3个胚层中的每一个的不同颜色读数。

DESCRIPTION (provided by applicant): Human pluripotent stem (hPS) cells have the ability to self-renew indefinitely and to differentiate into many different cell types. They therefore potentially offer an unlimited supply of a variety of human cell types for developmental research, disease modeling, drug screening, environmental chemical testing, predictive toxicology and ultimately cell replacement therapies to treat many currently intractable degenerative diseases. The source of hPS cells is no longer limited to human embryonic stem (hES) cells. Reprogramming technology has advanced the field by enabling the routine production of hPS cells, known as induced pluripotent (iPS) cells, from easily obtained normal and diseased donor cells such as skin or hair. As a result, the number and genetic diversity of hPS cell lines available for research and development has greatly increased in recent years. This trend is likely to continue with government and private initiatives to bank thousands of iPS cell lines. However, our ability to characterize and validate hPS cells has not kept pace with advances in the rate of production. Variation in iPS cell line quality and differentiation propensity is a critcal issue that requires careful characterization of at least 6 clones for each iPS cell line. Current methods of verifying pluripotency and characterizing differentiation involve laborious and time consuming immunocytochemical (ICC) analysis of iPS clones for multiple markers of in vitro differentiation for each of 3 primary germ layers. In vivo testing in SCID mice for teratoma formation takes 6-12 weeks and is therefore not practical for initial iPS screening. Thus there is a critical unmet need for more efficient in vitro iPS screening methods to assess quality and to determine in vitro differentiation propensity. Here we propose to develop a peptide targeted quantum dot (PTQD) assay for differentiation to each germ layer. We have designed the PTQD assay to be simpler, faster, more sensitive and more convenient than ICC. The assay uses PTQDs for live cell targeting and detection in a single step, making the assay more amenable to multiplexing and automation than ICC. Importantly, the assay preserves the cells live for further use. In phase I, we will develop a multiplexed PTQD assay for definitive endoderm (DE) and identify additional peptides for ectoderm and mesoderm assays. Having demonstrated the feasibility of a multiplexed PTQD assay for DE, we will in phase II, develop ectoderm and mesoderm PTQD assays using peptides from phase I. It is very useful for stem cell researchers to know the propensity of each iPS cell line to differentiate in vitro toward one germ line or another so that they can choose an appropriate line to fit their individual application. Having developed probes for each germ line, we aim by the end of phase II to develop a simple differentiation propensity assay using color coded PTQDs that results in a different color readout for each of the 3 germ layers.