Comparative phenotypic, functional, and molecular analysis of ESC and iPSC

ESC 和 iPSC 的比较表型、功能和分子分析

• 批准号: 7941821
• 负责人: GEORGE M CHURCH
• 金额: $ 168.4万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941821
• 关键词: Basic Science; Biological Assay; Biological Markers; Biomedical Research; Biotechnology; Blood; Cell Line; Cell Therapy; Cells; Chimerism; Church; Collection; Data; Degenerative Disorder; Derivation procedure; Disease; Disease model; Embryo; Epigenetic Process; Fertilization; Foundations; Future; Gene Dosage; Generations; Genetic; Genomics; Germ Lines; Glean; Goals; Grant; Histologic; Human; In Vitro; Karyotype; Left; Malignant - descriptor; Memory; Mesenchymal; Messenger RNA; Methods; Methylation; Modality; Molecular; Molecular Analysis; Mus; Natural regeneration; Nature; Parthenogenesis; Pathologic; Patients; Pluripotent Stem Cells; Polymorphism Analysis; Positioning Attribute; Preclinical Drug Evaluation; Process; Production; Property; Proteomics; Protocols documentation; Public Health; Publications; RNA Interference; Reagent; Replacement Therapy; Research; Research Personnel; Residual state; Resources; Single Nucleotide Polymorphism; Somatic Cell; Stem cells; Techniques; Teratoma; Testing; Therapeutic Studies; Time; Tissues; Transgenes; United States National Institutes of Health; Variant; Wound Healing; base; blastocyst; chromatin modification; cohort; comparative; comparative genomic hybridization; human embryonic stem cell; improved; induced pluripotent stem cell; insight; novel; pluripotency; programs; protein expression; public health relevance; relating to nervous system; somatic cell nuclear transfer; stem; stem cell biology; tool; transgene expression

DESCRIPTION (provided by applicant): Comparative phenotypic, functional, and molecular analysis of pluripotent stem cells arguably the most significant challenge in stem cell biology today is determining whether human induced pluripotent stem cells (hiPSC) are truly equivalent to human embryonic stem cells (hESC), and thus can serve as a stable, safe, and less controversial resource for basic research and cell replacement therapies. Although the evidence to date suggests that murine ESC and iPSC are functionally interchangeable, the analysis is preliminary and incomplete, especially for human cells, and evidence is accumulating that important molecular differences distinguish the two types of pluripotent stem cells. To answer whether significant functional and molecular differences exist, we have assembled an outstanding cohort of collaborators, each with unique and complementary expertise, to perform a comprehensive phenotypic, functional, and molecular comparison of multiple ESC and iPSC lines using genomic, epigenetic, proteomic, computational, and pathologic analysis. In recent years, the Daley lab has simultaneously pursued derivation of novel hESC from discarded IVF embryos and generation of hiPSC by direct somatic cell reprogramming. Moreover, we have an extensive collection of murine pluripotent stem cells generated by direct reprogramming (miPSC) or isolated from embryos after fertilization (fESC), parthenogenesis (pESC), and somatic cell nuclear transfer (ntESC). This comprehensive set of reagents affords us a unique opportunity to test the hypothesis that iPSC are the functional equivalents of ESC in assays of pluripotency, but that molecular differences persist between the two classes of pluripotent stem cells. We will further test the hypothesis that differences between ESC and iPSC are largely due to residual transgene expression in iPSC, and will resolve once transgenes are removed. However, preliminary data suggests that even transgene-free iPSC are epigenetically distinct from ESC. Thus, an alternative hypothesis is that factor-based reprogramming leaves a residual epigenetic signature of the tissue of origin ("epigenetic memory"), and that the reprogramming process confers unique molecular features on iPSC. In addition to comparing and contrasting ESC and iPSC, our analysis will illuminate the degree of variation among independent clones of ESC and iPSC. Defining the extent of functional similarity, assessing the nature of any molecular differences, and defining biomarkers of the successfully reprogrammed state are key goals of this proposal. Insights gleaned herein will contribute to improved reprogramming methods, thereby facilitating the application of iPSCs to disease research and cell therapies. PUBLIC HEALTH RELEVANCE: Pluripotent stem cells offer tremendous promise as tools for basic biomedical research, disease modeling and drug screening, and provide a means of deriving patient-specific rejection-proof cells that might be used in cell replacement therapies for a large number of genetic, malignant, and degenerative diseases. Techniques for establishing "induced pluripotent stem" or "iPS" cells fulfill the long-sought strategy for generating customized stem cells. If proven equivalent-both functionally and molecularly-to blastocyst-derived human embryonic stem cells, iPS cells will facilitate research, quell contentious public debate, and yield a new modality for tissue repair and regeneration.

描述（由申请人提供）：对多能干细胞的比较表型，功能和分子分析可以说，当今干细胞生物学中最重要的挑战是确定人类诱导的多能干细胞（HIPSC）是否真正等同于人类胚胎干细胞（HESC），因此可以作为稳定的，安全的，可用于稳定的，可作为稳定的细胞和基础研究。尽管迄今为止的证据表明，鼠ESC和IPSC在功能上是可以互换的，但该分析是初步和不完整的，尤其是对于人类细胞，并且有证据表明重要的分子差异区分了两种类型的多能干细胞。为了回答是否存在明显的功能和分子差异，我们组装了出色的合作者队列，每个合作者都具有独特的和互补的专业知识，以使用基因组，表观遗传学，蛋白质组学，计算和病理学分析对多个ESC和IPSC线进行全面的表型，功能和分子比较。近年来，戴利实验室（Daley Lab）同时通过直接的体细胞再编程从丢弃的IVF胚胎中推导了新的hESC和HIPSC的产生。此外，我们收集了广泛的鼠多能干细胞（通过直接重编程（MIPSC）产生的鼠多能干细胞）或受精后从胚胎（FESC），单次生殖（PESC）和体细胞核转移（NTESC）产生的鼠多能干细胞。这组全面的试剂为我们提供了一个独特的机会，可以测试IPSC在多能性测定中是ESC的功能等效物的假设，但是两种多能干细胞之间的分子差异持续存在。我们将进一步检验以下假设：ESC和IPSC之间的差异主要是由于IPSC中的残留转基因表达，并且一旦去除转基因，将解决。但是，初步数据表明，即使是不含转基因的IPSC也与ESC不同。因此，另一种假设是，基于因子的重编程留下了原始组织的残留表观遗传学特征（“表观遗传记忆”），并且重编程过程赋予IPSC上独特的分子特征。除了比较和对比ESC和IPSC外，我们的分析还将阐明ESC和IPSC独立克隆之间的变化程度。定义功能相似性的程度，评估任何分子差异的性质，并定义成功重编程状态的生物标志物是该建议的关键目标。本文收集的洞察力将有助于改进重编程方法，从而促进IPSC在疾病研究和细胞疗法中的应用。 公共卫生相关性：多能干细胞作为基本生物医学研究，疾病建模和药物筛查的工具提供了巨大的希望，并提供了一种可能用于大量遗传，恶性疾病和退化性疾病的细胞替代疗法中的患者特异性拒绝细胞的方法。建立“诱导多能茎”或“ IPS”细胞的技术符合生成定制干细胞的长期策略。如果在功能和分子上被证明是胚泡衍生的人类胚胎干细胞，IPS细胞将促进研究，平息有争议的公众辩论，并为组织修复和再生产生新的方式。