Regulation and function of hematopoietic stem cell niches

造血干细胞生态位的调节和功能

• 批准号: 10507236
• 负责人: Kira Gritsman
• 金额: $ 5.64万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10507236
• 关键词: ATAC-seq; Adhesions; Affect; Alleles; Amino Acids; Area; Award; Binding Proteins; Bone Marrow; Bone Transplantation; CD34 gene; CRISPR/Cas technology; Cell Count; Cell Culture Techniques; Cell Maintenance; Cells; Collaborations; Complex; Cues; Development; Distant; Engineering; Engraftment; Enhancers; Erythroid Progenitor Cells; Exhibits; Funding; Genes; Genetic; Genetic Screening; Genetic Transcription; German population; Grant; Hematopoietic Stem Cell Mobilization; Hematopoietic stem cells; Homing; Human; IRF3 gene; Immunodeficient Mouse; Knowledge; Label; Laboratories; Lead; Light; LoxP-flanked allele; Marrow; Mass Spectrum Analysis; Mediating; Mesenchymal; Methods; Modeling; Mus; Muscle Cells; NCI Center for Cancer Research; Natural regeneration; Nerve; OSTF1 gene; Play; Process; Regulation; Role; Signal Transduction; Stable Isotope Labeling; Stem Cell Factor; Structure; Sympathetic Nervous System; Testing; Thrombopoietin; Transplantation; Umbilical Cord Blood; XBP1 gene; bone; circadian; cytokine; embryonic stem cell; erythroid differentiation; genetic analysis; hematopoietic stem cell niche; hormonal signals; human embryonic stem cell; in vivo; insight; interferon regulatory factor-7; irradiation; mathematical model; mesenchymal stromal cell; nestin protein; novel; progenitor; restraint; slug; stem; stem cell function; stem cells; transcription factor; transcriptome sequencing; transplant model

PROJECT SUMMARY: The hematopoietic stem cell (HSC) niche is a complex structure thought to play an important role in regulating HSC function and numbers. Among the niche constituents, MSCs express the highest levels of the major niche factors. Yet, their ability to maintain HSCs ex vivo has been limited thus far. MSCs indeed rapidly loose expression of major niche factors in culture. We have tested the hypothesis that MSCs’ transcriptional machinery was altered in culture conditions and that reinstating the expression of key transcription regulators would revitalize niche activity. We have carried out a genetic screen using 28 transcription regulators that were expressed at high levels in Nestin-GFP+ niche cells in vivo but downregulated in culture, and identified a combination of 5 genes (Ostf1, Xbp1, Irf3, Irf7 and Klf7) that can revitalize MSCs. Revitalized MSCs (rMSCs) exhibit higher niche factor expression and capacity to expand either murine or human HSCs compared to control MSCs. Using RNA-seq and ATAC-seq analyses, we have identified Mef2c as downstream effector of the revitalization process. This continuation proposal will evaluate the hypothesis that HSCs are finely regulated by a niche programmed to maintain their numbers. In Specific Aim 1, we will assess the function of human orthologues of the 5 factors in revitalization of human MSCs. We will use stable isotope labeling with amino acids in cell culture (SILAC) and labeling by azidohomoalanine (AHA) to identify by mass spectrometry novel soluble factors derived from rMSCs. We will test the function of rMSCs in the maturation of embryonic stem (ES) cell-derived HSCs to promote their engraftment in immunodeficient mice. In Specific Aim 2, we will evaluate the role of key transcription regulators in HSC niche activity, focusing our studies on Snai2, Ostf1 and Mef2c using conditional deletions using floxed mouse lines. In Specific Aim 3, we will assess whether HSC numbers are regulated by local niche availability or systemic sensing using transplantation, local irradiation, and mathematical modeling. The proposed studies will shed new light on mechanisms by which HSC numbers are regulated and provide new methods toward their ex vivo expansion.

项目摘要： 造血干细胞（HSC）生态位是一种复杂的结构，被认为起着重要作用 在调查HSC功能和数字时。在利基构成中，MSC表达 主要利基因素的最高水平。然而，它们维持HSC的能力是 迄今为止有限。 MSC确实在培养中迅速松散了主要利基因素的表达。我们有 检验了以下假设，即MSC的转录机械在培养条件下发生了改变和 恢复关键转录调节因子的表达将振兴利基活动。我们 使用28个转录调节器进行了遗传筛选 Nestin-GFP+小众细胞体内的水平，但在培养中下调，并确定了组合 可以振兴MSC的5个基因（OSTF1，XBP1，IRF3，IRF7和KLF7）。复兴的MSC（RMSC） 表现出更高的生态位因子表达和扩展鼠或人HSC的能力 与对照MSC相比。使用RNA-SEQ和ATAC-SEQ分析，我们已经确定了MEF2C 作为振兴过程的下游效应子。该延续建议将评估 假设HSC受到编程以维持其数量的利基市场的细胞调节。 具体目的1，我们将评估5个因素的人类直系同源物的功能 人类MSC。我们将在细胞培养（SILAC）和 通过叠氮杂氨酸（AHA）标记以通过质谱识别新型固体因子 源自RMSC。我们将测试RMSC在胚胎茎成熟（ES）中的功能 细胞来源的HSC促进其在免疫缺陷小鼠中的植入。在特定的目标2中，我们 将评估关键转录调节因子在HSC利基活动中的作用，将我们的研究重点放在 SNAI2，OSTF1和MEF2C使用条件缺失使用Floxed Mouse系列。在特定的目标3中 我们将评估HSC数字是由本地利基可用性或全身感应的调节 使用移植，局部照射和数学建模。拟议的研究将 阐明了对HSC数字进行调节并提供新方法的机制的新启示 向他们的体内扩展。