Rapid Multiplexed Nanoprobe Assays for Pluripotent Stem Cell Differentiation

用于多能干细胞分化的快速多重纳米探针测定

• 批准号: 8592883
• 负责人: Dana Larocca
• 金额: $ 0.74万
• 依托单位: MANDALA BIOSCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2013-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8592883
• 关键词: Antibodies; Automation; Biological Assay; Cell Differentiation process; Cell Line; Cell Therapy; Cell Transplantation; Cells; Code; Color; DNA Integration; Degenerative Disorder; Derivation procedure; Detection; Development; Disease model; Drug toxicity; Ectoderm; Embryo; Endoderm; Excision; Gene Expression Profiling; Genetic Variation; Germ Layers; Germ Lines; Goals; Gold; Government; Hair; Health; Human; In Vitro; Individual; Injury; Label; Life; Marketing; Mesoderm; Methods; Peptides; Phage Display; Phase; Pluripotent Stem Cells; Preclinical Drug Evaluation; Procedures; Production; Property; Quantum Dots; RNA; Regenerative Medicine; Relative (related person); Replacement Therapy; Research; Research Personnel; SCID Mice; Scientist; Sensitivity and Specificity; Series; Services; Skin; Somatic Cell; Source; Staining method; Stains; Stem cells; Technology; Teratoma; Testing; Therapeutic; Time; Toxicity Tests; Toxicology; Validation; Variant; assay development; base; cell bank; cell type; cost; design; drug discovery; drug testing; environmental chemical; human embryonic stem cell; human embryonic stem cell line; in vitro testing; in vivo; induced pluripotent stem cell; innovation; interest; nanoprobe; pluripotency; progenitor; prototype; public health relevance; regenerative; research and development; screening; self-renewal; stem; stem cell differentiation; trend; vector

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.

描述（由申请人提供）：人多能茎（HPS）细胞具有无限期自我更新并分化为许多不同细胞类型的能力。因此，它们有潜在地提供各种人类细胞类型的无限供应，用于发育研究，疾病建模，药物筛查，环境化学测试，预测毒理学以及最终的细胞替代疗法，以治疗许多当前棘手的退行性疾病。 HPS细胞的来源不再限于人类胚胎（HES）细胞。重新编程技术通过易于获得的正常和患病的供体细胞（例如皮肤或头发），从而使HPS细胞（称为诱导多能（IPS）细胞）的常规产生（称为诱导的多能（IPS）细胞）推进了该领域。结果，近年来可用于研发的HPS细胞系的数量和遗传多样性大大增加。这种趋势很可能会继续进行政府和私人计划，以融入数千个IPS细胞系。但是，我们表征和验证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化是一个critcal问题，需要为每个IPS细胞系仔细表征至少6个克隆。当前验证多能性和表征分化的方法涉及对IPS克隆的费力和耗时的免疫细胞化学（ICC）分析，用于三个主要细菌层中每种体外分化的多种标记。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筛选而言是不切实际的。因此，对更有效的体外IPS筛选方法的急需未满足，以评估质量和确定体外分化倾向。在这里，我们建议开发一个肽靶向量子点（PTQD）分析，以分化为每个生殖层。我们将PTQD分析设计为比ICC更简单，更快，更灵敏，更方便。该测定法在单个步骤中使用PTQD进行实时细胞靶向和检测，这使得比ICC更适合多重和自动化。重要的是，该测定可以保留细胞的生存供进一步使用。在第一阶段，我们将开发用于确定内胚层（DE）的多重PTQD分析，并确定外胚层和中胚层测定的其他肽。在证明了DE多路复用PTQD分析的可行性之后，我们将在第二阶段，使用来自阶段的肽进行外胚层和中胚层PTQD测定。体外朝着一条生殖系或另一种生殖系，以便他们可以选择适当的线以适合其个人应用。在为每个种系开发了探针之后，我们以II阶段结束为目标是使用颜色编码的PTQD开发简单的分化倾向测定法，从而为3个细菌层中的每一个都产生不同的颜色读数。