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

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是当今干细胞生物学中最重大的挑战，即确定人类诱导多能干细胞（hiPSC）是否真正等同于人类胚胎干细胞（hESC），从而可以作为一种稳定、安全、争议较少的基础研究和细胞替代疗法资源。尽管迄今为止的证据表明，小鼠ESC和iPSC在功能上是可互换的，但这种分析是初步的和不完整的，特别是对人类细胞而言，而且越来越多的证据表明，重要的分子差异区分了两种多能干细胞。为了回答是否存在显著的功能和分子差异，我们召集了一群杰出的合作者，每个人都具有独特和互补的专业知识，利用基因组学、表观遗传学、蛋白质组学、计算和病理分析，对多个ESC和iPSC品系进行全面的表型、功能和分子比较。近年来，Daley实验室同时致力于从废弃的体外受精胚胎中衍生出新的hESC，并通过直接体细胞重编程产生hiPSC。此外，我们还收集了大量通过直接重编程（miPSC）或从受精后胚胎（fESC）、孤雌生殖（pESC）和体细胞核移植（ntESC）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测试iPSC在多能性测定中与ESC功能等同的假设，但两类多能干细胞之间的分子差异仍然存在。我们将进一步验证这一假设，即ESC和iPSC之间的差异主要是由于iPSC中残留的转基因表达，一旦去除转基因，这种差异就会消失。然而，初步数据表明，即使不含转基因的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”），重编程过程赋予了iPSC独特的分子特征。除了比较和对比ESC和iPSC，我们的分析将阐明ESC和iPSC独立克隆之间的差异程度。定义功能相似性的程度，评估任何分子差异的本质，以及定义成功重编程状态的生物标志物是本提案的关键目标。在此收集的见解将有助于改进重编程方法，从而促进多能干细胞在疾病研究和细胞治疗中的应用。

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.