Regulation of Stem Cell Development

描述(由申请人提供)：对于某些类型的细胞，如造血干细胞(HSC)，来自胚胎干细胞(ES)或“重新编程”的干细胞的治疗应用可能需要在不引入基因改变以诱导自我更新的情况下扩大这些群体。为了解决这个问题，我们建议测试我们的假设，即调节正常发育的途径可以被操纵，以产生反映正常发育状态的扩大的细胞类型群体。作为这种方法的模型，我们重点介绍我们拥有丰富经验的Notch和Wnt途径。因此，ES和诱导的多能干细胞(IPS)将通过操纵Notch(项目1)和Wnt(项目2)途径来评估HSC数量增加的发展。作为每个项目的一部分，计划对基因表达和表观遗传状态进行全球评估，以分别确定体外来源的造血细胞和分离的正常对应细胞之间的关键相似或不同之处。基于我们目前使用Notch配体Delta1的人类CD34+干/祖细胞的生成和临床应用，我们还将测试体外生成的具有良好特性的干/祖细胞群体的体内生物学功能，并评估潜在偏离“正常”分子图谱的意义(项目3)。为了进一步了解Notch和Wnt诱导的自我更新和分化所涉及的机制，我们将在ES/IPS和脐带血来源的CD34+细胞的不断扩大的群体中进行基因组规模的“多路”RNAi筛选(项目4)。组建的团队代表了弗雷德·哈钦森癌症研究中心(FHCRC)和华盛顿大学(UW)在造血干细胞生物学、Wnt和Notch信号、表观遗传学、发育过程中基因表达的进化、基因组规模的RNAi技术以及ES和IPS方面的必要经验方面的多学科专业知识。我们期望通过与评估心脏和肺以及造血祖细胞的干细胞联盟的其他成员的合作，提供专业知识以及调节Notch和Wnt信号的独特试剂。

DESCRIPTION (provided by applicant): Therapeutic application of cells derived from embryonic stem (ES) or "reprogrammed" stem cells may require, for certain cells types such as hematopoietic stem cells (HSC), expansion of these populations without introducing genetic alterations to induce self-renewal. To address this issue, we propose to test our hypotheses that pathways that regulate normal development can be manipulated to generate expanded populations of cell types that reflect normal, developmental states. As a model for this approach, we focus on the Notch and Wnt pathways with which we have extensive experience. Thus, ES and induced pluripotent stem (IPS) cells will be assessed for development of enhanced numbers of HSC by manipulation of the Notch (project 1) and Wnt (project 2) pathways. As part of each of these projects, global assessment of gene expression and epigenetic state is planned to respectively determine key similarities or differences between in vitro derived hematopoietic cells and isolated normal counterparts. Based on our current generation and clinical application of human CD34+ stem/progenitor cells using the Notch ligand Delta1, we will also test the in vivo biological function of a well characterized in vitro generated stem/progenitor population and assess the significance of potential deviations from "normal" molecular profiles (Project 3). To further understand mechanisms involved in Notch- and Wnt-induced affects on self-renewal and differentiation, we will perform genome-scale, "multiplexed" RNAi screens in expanding populations of ES/IPS and cord blood derived CD34+ cells (project 4). The assembled team represents multidisciplinary expertise at the Fred Hutchinson Cancer Research Center (FHCRC) and the University of Washington (UW) in hematopoietic stem cell biology, Wnt and Notch signaling, epigenetics, evolution of gene expression in development, genome-scale RNAi technology, and requisite experience in ES and IPS. We anticipate providing the expertise, as well as unique reagents for modulating Notch and Wnt signaling, in collaborations with other members of the stem cell consortium evaluating heart and lung as well as hematopoietic progenitors.