Regulation of Hematopoietic Stem Cell Self-renewal and Differentiation

描述（由申请人提供）：提高对干细胞和祖细胞中基因调控网络的理解对于开发用于实验和治疗目的的操纵祖细胞命运的策略至关重要。MicroRNA（miRNAs）是一种小的非编码调节RNA，可降低互补靶信使RNA（mRNA）的翻译和/或稳定性。miRNA代表了控制基因表达和细胞命运特化的关键机制，除了传统上与此作用相关的转录因子（TF）之外。造血系统提供了一个杰出的模型，研究的作用，miRNA的调节干/祖细胞的命运，因为细胞群体在不同阶段的分化得到了很好的表征。先前的研究支持miRNA在调节造血中的关键作用，但仅关注少数候选miRNA。大多数miRNA表达的相对丰度和特异性仍有待研究，表达的miRNA单独或协同调节基因表达和决定细胞命运和功能的作用仍知之甚少。本研究的目的是探讨miRNAs在造血干细胞和祖细胞分化和自我更新中的作用。一个由在造血、RNA生物学、生物工程/纳米技术和生物信息学方面具有专业知识的多学科合作者组成的团队将开发和应用最先进的技术来实现这一重要目标。 项目1将研究造血干细胞和祖细胞中miRNA的表达和基因调控。将使用超高通量测序方法研究精确定义的祖细胞群体中的miRNA表达和转录组组成，并研究不同分化阶段的miRNA、转录因子及其靶基因的调控网络。 项目二研究miRNAs在造血干细胞和祖细胞谱系特化和自我更新中的作用。将开发用于筛选miRNA功能的高通量方法，并通过详细的体外和体内分析验证推定的功能。 项目3将在单细胞水平上研究造血干细胞和祖细胞中miRNA介导的基因调控。一种新开发的微流控生物处理器将用于研究单个祖细胞中miRNA介导的基因调控。所提出的研究具有明确的翻译应用，以操纵干细胞和祖细胞群体用于治疗，包括移植，定向分化和基因治疗。这些研究也将为理解包括恶性肿瘤在内的病理状态下miRNA介导的基因调控的扰动提供基础。为这些研究开发的测序、筛选和生物工程方法具有相当大的潜力，可在该联盟内更广泛地应用于研究miRNA在心脏、血管和肺组织中的祖细胞群体中的细胞命运决定中的作用，以及研究从多能干细胞发育人类组织特异性干细胞。

DESCRIPTION (provided by applicant): Improved understanding of gene regulatory networks in stem and progenitor cells is essential for the development of strategies for manipulating progenitor cell fates for experimental and therapeutic purposes. MicroRNAs (miRNAs) are small non-coding regulatory RNAs that reduce translation and/or stability of complementary target messenger RNAs (mRNAs). miRNA represents a critical mechanism for the control of gene expression and cell fate specification in addition to transcription factors (TF) that are traditionally associated with this role. The hematopoietic system provides an outstanding model for investigation of the role of miRNAs in regulation of stem/progenitor cell fate since cell populations at different stages of differentiation are well characterized. Previous studies support a critical role for miRNA in regulating hematopoiesis but have only focused on a few candidate miRNAs. The relative abundance and specificity of expression for most miRNAs remains to be investigated, and the role of expressed miRNA singly or cooperatively in regulating gene expression and determining cell fate and function remains poorly understood. The goal of the proposed studies is to investigate the role of miRNAs in regulation of hematopoietic stem and progenitor cell differentiation and self-renewal. A multidisciplinary team of collaborators with expertise in hematopoiesis, RNA biology, bioengineering/nanotechnology and bioinformatics will develop and apply state- of-the-art technologies to address this important goal. Project 1 will study miRNA expression and gene regulation in hematopoietic stem and progenitor cells. miRNA expression and transcriptome composition in precisely defined progenitor populations will be studied using ultra-high throughput sequencing approaches and regulatory networks of miRNAs, transcription factors and their target genes at different stages of differentiation will be investigated. Project 2 will study the function of expressed miRNAs in lineage specification and self-renewal of hematopoietic stem and progenitor cells. High-throughput methods for screening miRNA function will be developed and putative function validated by detailed in vitro and in vivo analyses. Project 3 will study miRNA mediated gene regulation in hematopoietic stem and progenitor cells at the single cell level. A newly developed microfluidic bioprocessor will be used to investigate miRNA-mediated gene regulation in individual progenitor cells. The proposed studies have clear translational applications to manipulation of stem and progenitor cell populations for therapeutics, including transplantation, directed differentiation and gene therapy. These studies will also provide a basis for understanding perturbations in miRNA-mediated gene regulation in pathological states including malignancies. The sequencing, screening and bioengineering approaches developed for these studies have considerable potential for broader application within the consortium for investigation of the role of miRNA in cell fate determination in progenitor populations in cardiac, vascular and pulmonary tissues, and for investigating human tissue specific stem cell development from pluripotent stem cells.