Rapid Multiplexed Nanoprobe Assays for Pluripotent Stem Cell Differentiation

描述（由申请人提供）：人类多能干细胞（hPS）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、预测毒理学以及最终治疗许多目前难以治疗的退行性疾病的细胞替代疗法提供各种人类细胞类型的无限供应。hPS细胞的来源不再局限于人类胚胎干细胞（hES）。重编程技术使得从容易获得的正常和患病的供体细胞（如皮肤或头发）中常规生产hPS细胞，即诱导多能（iPS）细胞，从而推动了该领域的发展。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。这一趋势很可能会随着政府和私人机构将数千个iPS细胞系储存起来而持续下去。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。诱导多能干细胞系质量和分化倾向的变化是一个关键问题，需要对每个诱导多能干细胞系至少6个克隆进行仔细的表征。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个初级胚层中每个胚层的多个体外分化标记。在SCID小鼠中进行畸胎瘤形成的体内试验需要6-12周，因此不适合用于初始iPS筛选。因此，迫切需要更有效的体外iPS筛选方法来评估质量并确定体外分化倾向。在此，我们提出了一种肽靶向量子点（PTQD）检测方法，用于向每个胚层分化。我们设计的PTQD分析比ICC更简单、更快、更敏感、更方便。该分析使用PTQDs在单一步骤中进行活细胞靶向和检测，使该分析比ICC更适合多路复用和自动化。重要的是，该试验保存了细胞以供进一步使用。在第一阶段，我们将开发一种用于确定内胚层（DE）的多重PTQD测定方法，并为外胚层和中胚层测定确定额外的肽。在证明了DE的多重PTQD测定的可行性之后，我们将在II期使用i期的肽开发外胚层和中胚层PTQD测定。这对干细胞研究人员了解每个iPS细胞系在体外向一种生殖系分化的倾向非常有用，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。在开发了每个生殖系的探针后，我们的目标是在II期结束时开发一种简单的分化倾向试验，使用颜色编码的PTQDs，对3个胚层中的每个胚层产生不同的颜色读数。

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.