Rapid Multiplexed Nanoprobe Assays for Pluripotent Stem Cell Differentiation

描述(申请人提供)：人类多能干细胞(HPS)具有无限自我更新和分化为多种不同细胞类型的能力。因此，它们可能提供无限的各种人类细胞类型，用于发展研究、疾病建模、药物筛选、环境化学测试、预测毒理学，并最终提供细胞替代疗法来治疗许多目前难以治愈的退行性疾病。HPS细胞的来源不再局限于人类胚胎干细胞(HES)。通过重新编程技术，可以从皮肤或头发等容易获得的正常和疾病供体细胞中常规生产HPS细胞，即所谓的诱导多能(IPS)细胞，从而推动了该领域的发展。因此，可用于研究和开发的HPS细胞系的数量和遗传多样性近年来大大增加。这一趋势可能会继续下去，政府和私人倡议银行数以千计的iPS细胞系。然而，我们鉴定和验证HPS细胞的能力并没有跟上生产率的进步。IPS细胞系质量和分化倾向的变异是一个关键问题，需要为每个iPS细胞系仔细鉴定至少6个克隆。目前验证多能性和鉴定分化的方法包括费时费力地对iPS克隆进行免疫细胞化学(ICC)分析，以寻找3个初级生殖层中每一层的体外分化的多个标记。在SCID小鼠体内进行畸胎瘤形成的测试需要6-12周，因此对于最初的iPS筛查是不现实的。因此，迫切需要更有效的体外iPS筛选方法来评估质量和确定体外分化倾向。在这里，我们建议建立一种多肽靶向量子点(PTQD)分析方法，用于区分每一种胚层。我们设计的PTQD方法比ICC更简单、快速、灵敏和方便。该分析使用PTQD在单一步骤中进行活细胞靶向和检测，使该分析比ICC更易于多路传输和自动化。重要的是，该检测保存了活细胞以供进一步使用。在第一阶段，我们将建立一种用于确定性内胚层(DE)的多重PTQD分析方法，并为外胚层和中胚层分析鉴定其他多肽。在证明了用于DE的多重PTQD分析的可行性后，我们将在第二阶段使用来自第一阶段的多肽开发外胚层和中胚层PTQD分析。对于干细胞研究人员来说，了解每个iPS细胞系在体外向一种或另一种胚系分化的倾向非常有用，以便他们可以选择适合其个人应用的系。在为每一种胚系开发了探针后，我们的目标是在第二阶段结束前开发一种简单的分化倾向分析方法，使用颜色编码的PTQD来导致3个胚层中的每一个都有不同的颜色读数。

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.