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）：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大的挑战是确定人诱导多能干细胞（hiPSC）是否真正等同于人胚胎干细胞（hESC） ），因此可以作为基础研究和细胞替代疗法的稳定、安全且争议较少的资源。尽管迄今为止的证据表明鼠类 ESC 和 iPSC 在功能上是可以互换的，但分析是初步且不完整的，尤其是对于人类细胞，并且越来越多的证据表明区分这两种类型的多能干细胞的重要分子差异。为了回答是否存在显着的功能和分子差异，我们聚集了一批杰出的合作者，每个人都拥有独特和互补的专业知识，利用基因组、表观遗传学、蛋白质组学、计算和病理分析。近年来，Daley 实验室同时致力于从废弃的 IVF 胚胎中衍生出新型 hESC，并通过直接体细胞重编程生成 hiPSC。此外，我们还拥有大量通过直接重编程 (miPSC) 产生的或从受精 (fESC)、孤雌生殖 (pESC) 和体细胞核移植 (ntESC) 后的胚胎分离的小鼠多能干细胞。这套全面的试剂为我们提供了一个独特的机会来检验这一假设：iPSC 在多能性测定中与 ESC 的功能相当，但两类多能干细胞之间仍然存在分子差异。我们将进一步检验这样的假设：ESC 和 iPSC 之间的差异很大程度上是由于 iPSC 中残留的转基因表达造成的，并且一旦转基因被去除，这种差异就会消失。然而，初步数据表明，即使是无转基因的 iPSC 在表观遗传学上也与 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余表观遗传特征（“表观遗传记忆”），并且重编程过程赋予 iPSC 独特的分子特征。除了比较和对比 ESC 和 iPSC 之外，我们的分析还将阐明 ESC 和 iPSC 独立克隆之间的变异程度。定义功能相似性的程度、评估任何分子差异的性质以及定义成功重编程状态的生物标志物是该提案的关键目标。本文收集的见解将有助于改进重编程方法，从而促进 iPSC 在疾病研究和细胞治疗中的应用。 公共健康相关性：多能干细胞作为基础生物医学研究、疾病建模和药物筛选的工具提供了巨大的前景，并提供了一种衍生患者特异性抗排斥细胞的方法，这些细胞可用于大量遗传性疾病的细胞替代疗法。 、恶性和退行性疾病。建立“诱导多能干”或“iPS”细胞的技术实现了长期以来寻求的生成定制干细胞的策略。如果 iPS 细胞在功能和分子上被证明与囊胚来源的人类胚胎干细胞相当，那么它将促进研究，平息有争议的公众辩论，并产生一种新的组织修复和再生方式。