Interrogation of individual cells to identify progenitors and their niches

询问单个细胞以识别祖细胞及其生态位

• 批准号: 8115224
• 负责人: IRVING L. WEISSMAN
• 金额: $ 113.3万
• 依托单位: STANFORD UNIVERSITY
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115224
• 关键词: Adult; Applications Grants; Bioinformatics; Biological Assay; Biological Process; Blood Cells; Bone Marrow; Bone Marrow Diseases; Candidate Disease Gene; Cell Death; Cell Lineage; Cell Transplants; Cells; Cessation of life; Chromatin; Color; Commit; Complex; Coupled; Development; Disease; Engraftment; Environment; Epigenetic Process; Event; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; Growth; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneity; Homologous Gene; Human; In Situ; In Situ Hybridization; In Vitro; Individual; Investigation; Knowledge; Label; Libraries; Ligands; Masks; Membrane Proteins; Methods; Microfluidics; Molecular; Molecular Profiling; Multipotent Stem Cells; Mus; National Heart, Lung, and Blood Institute; Organ; Pathway interactions; Patients; Population; Principal Investigator; Process; Property; Proteins; Regulation; Research; Resolution; Resources; Site; Small Interfering RNA; Stem cells; Technology; Transplantation; Umbilical Cord Blood; Update; Work; Xenograft procedure; base; body system; cell population study; cell type; epigenomics; genome sequencing; in vivo; insight; interest; progenitor; prospective; receptor; reconstitution; self-renewal; stem; tool

DESCRIPTION (provided by applicant): Hematopoiesis is organized as a cellular hierarchy initiated by rare self-renewing hematopoietic stem cells (HSC), which in turn produce lineage-committed progenitors and eventually all the mature blood cells. It is important to identify the molecular events and pathways regulating hematopoietic stem and progenitor self-renewal, growth, death, and differentiation. However, it has become increasingly clear that studying cell populations only provides average values, and that interrogation of individual cells could reveal the presence of subpopulations and provide insight into these biological processes that are masked at the population level. The main goals for this grant application are to combine the power of microfluidic genomic and epigenomic analysis with the ability to isolate purified hematopoietic stem and progenitor populations to develop a high resolution understanding of hematopoiesis. This proposal aims to use single cell microfluidic PCR to determine the heterogeneity of known populations, and to functionally characterize new intermediate populations. Next, this proposal aims to investigate hematopoiesis in situ to define and molecularly characterize hematopoietic niches. Finally, this proposal aims to use the gene signatures of defined mouse progenitors to identify human homologues. The surface proteins expressed within each cell type will be used to separate human cells by FACS, and transplantation into NSG mice will indicate their function. Deep molecular analyses of these mouse and human progenitors to identify candidate gene loci that regulate critical biological functions. The analyses will include definition of open versus closed chromatin-associated genome sequences, definition of the methylome for each progenitor and comparison with the RNA expression profiles, and development of new bioinformatic tools to aid in the identification of critical molecular factors. Functional assays will be conducted with siRNA to investigate the most interesting genes, and will also include microfluidic nanoculture to assess the effect of soluble factors on progenitor functions. Finally, within the NHLBI Progenitor Consortium human bone marrow disorders will be examined from the standpoint of an expanded knowledge of human hematopoietic progenitors.

描述（由申请人提供）： 造血是由罕见的自我更新造血干细胞（HSC）启动的细胞层次结构，后者又产生了谱系合作的祖细胞，最终产生了所有成熟的血细胞。重要的是要确定调节造血茎和祖细胞自我更新，生长，死亡和分化的分子事件和途径。但是，越来越清楚的是，研究细胞种群仅提供平均值，并且对单个细胞的询问可以揭示出亚群的存在，并洞悉在种群水平上掩盖的这些生物学过程。该赠款应用的主要目标是将微流体基因组和表观基因组分析的能力与隔离纯化的造血茎和祖细胞种群隔离的能力，以发展对造血的高分辨率理解。该建议旨在使用单细胞微流体PCR来确定已知种群的异质性，并在功能上表征新的中间种群。接下来，该提案旨在调查原位造血症，以定义和分子表征造血壁ni。最后，该建议旨在使用定义的小鼠祖细胞的基因特征来识别人类同源物。在每种细胞类型中表达的表面蛋白将用于通过FACS分离人类细胞，而将其移植到NSG小鼠中将表明其功能。对这些小鼠和人类祖细胞的深层分子分析，以鉴定调节关键生物学功能的候选基因基因座。分析将包括开放与封闭染色质相关的基因组序列的定义，每个祖细胞的甲基组的定义以及与RNA表达谱的比较，以及开发新的生物信息学工具以帮助鉴定关键分子因子。功能分析将与siRNA进行研究以研究最有趣的基因，还将包括微流体纳米培养物，以评估可溶性因子对祖细胞功能的影响。最后，将从人类造血祖细胞的扩展知识的角度检查NHLBI祖细胞联盟人类骨髓疾病。