Investigating dysregulation of hematopoietic stem cell support in sickle cell disease mesenchymal stromal cells

研究镰状细胞病间充质基质细胞中造血干细胞支持的失调

• 批准号: 10752050
• 负责人: Terri Cain
• 金额: $ 4.37万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL GRADUATE SCHOOL OF BIOMEDICAL SCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752050
• 关键词: ATAC-seq; Acute Myelocytic Leukemia; Adult; Affect; Antibodies; Automobile Driving; B-Lymphocytes; Bioinformatics; Blood Vessels; Bone Marrow; Bone Marrow Diseases; Candidate Disease Gene; Cell Compartmentation; Cell Cycle; Cell Proliferation; Cell Separation; Cell physiology; Cells; Chromatin; Chronic; Coculture Techniques; Data; Disease; Down-Regulation; Early identification; Epigenetic Process; Functional disorder; Gene Expression Profile; Gene Structure; Gene set enrichment analysis; Genes; Genetic Transcription; Goals; Health; Hematological Disease; Hematology; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobin; Hemoglobinopathies; Hemolysis; Hemolytic Anemia; Human; I-Cell Disease; Immunoglobulin G; Impairment; Individual; Inflammation; Inflammatory; Inherited; Institution; Lead; Longevity; Molecular; Morbidity - disease rate; Mus; Mutation; Ontology; Organ; Pain; Patients; Persons; Phenotype; Pilot Projects; Population; Principal Investigator; Proliferating; Recovery; SWI/SNF Family Complex; Sickle Cell Anemia; Small Interfering RNA; Stress; Supporting Cell; System; Techniques; Testing; Tissues; Transcription Factor 3; Transplantation; United States National Institutes of Health; Up-Regulation; Vertebrates; Work; career; chromatin protein; curative treatments; cytokine; differential expression; gene repression; hematopoietic stem cell self-renewal; hemoglobin polymer; improved; in vivo; innovation; knock-down; mesenchymal stromal cell; mortality; mouse model; nuclease; overexpression; post-transplant; retroviral transduction; success; transcription factor; translational impact

Project Summary Sickle cell disease (SCD) is one of the most common hemoglobinopathies in the world. It results from a mutation in the hemoglobin β gene which causes hemoglobin polymerization, leading to chronic hemolysis, inflammation, damage to organs, and increased early morbidity. SCD pathophysiology can damage and stress the bone marrow microenvironment and resident hematopoietic stem cells (HSCs), as evidenced in our pilot studies revealing fewer functional HSCs in the bone marrow of both mice and individuals with SCD. Sinusoidal and arterial vascular networks in the SCD bone marrow are also significantly disrupted. Furthermore, SCD is marked by decreased numbers of mesenchymal stromal cells (MSCs), a heterogeneous cell population that functions as critical regulators of HSC self-renewal and differentiation. We observed significant changes to MSC transcriptional profiles in SCD and identified three aberrant SCD MSC phenotypes: 1) increased SCD MSC cell cycling, 2) increased secretion of inflammatory cytokines, and 3) decreased HSC support. The transcription factor Early B Cell Factor 3 (EBF3) is a standout differentially expressed gene in SCD MSCs and a known regulator of cell cycle. Loss of EBF3 in MSC subpopulations dramatically impacts bone marrow composition and HSC support. Cumulatively, these findings lead me to hypothesize that EBF3 downregulation in bone marrow MSCs during SCD causes transcriptional changes that decrease their HSC support capacity. In Aim 1, I will determine how Ebf3 downregulation is driving the changes we observe in mouse SCD MSCs via over-expression and knockdown of Ebf3 in SCD and control MSCs; I will then quantify changes to their HSC support capacity through ex vivo primary cultures and in vivo transplantation studies. In Aim 2, I will identify EBF3 transcriptional targets and their cellular functions in control and SCD MSCs via protein- chromatin interaction studies, gene ontology analysis, and gene set enrichment analysis as well as mechanistic interrogation of Ebf3 downregulation. In Aim 3, I will investigate the translational impact of EBF3 downregulation in human SCD MSCs by again employing over-expression and knockdown systems to perturb EBF3 expression and quantify how this impacts their support of human HSCs. I will also elucidate the transcriptional targets of EBF3 in human MSCs, which has never been done before. This study will be an important first step in better understanding how transcriptional dysfunction in SCD MSCs influences HSC support, which, in turn, may be critical in the improvement of SCD curative therapies.

项目摘要 镰状细胞病是世界上最常见的血红蛋白病之一。它是由一种突变引起的， 血红蛋白β基因引起血红蛋白聚合，导致慢性溶血、炎症、损伤， 器官，并增加早期发病率。SCD的病理生理学可损害和压迫骨髓微环境 和常驻造血干细胞（HSC），正如我们的初步研究所证明的那样， 小鼠和患有SCD的个体的骨髓。SCD骨髓中的窦状和动脉血管网也是 严重中断。此外，SCD的标志是间充质基质细胞（MSC）数量减少， 在HSC自我更新和分化中起关键调节作用的异质细胞群。我们观察到 SCD中MSC转录谱的显著变化，并鉴定了三种异常SCD MSC表型：1）增加 SCD MSC细胞循环，2）增加炎性细胞因子分泌，和3）减少HSC支持。转录 因子早期B细胞因子3（EBF 3）是SCD MSC中突出的差异表达基因，并且是已知的细胞增殖的调节因子。 周期MSC亚群中EBF 3的缺失显著影响骨髓组成和HSC支持。累积起来， 这些发现使我推测，SCD期间骨髓MSC中EBF 3的下调导致 转录改变降低了HSC的支持能力。在目标1中，我将确定Ebf 3下调是如何发生的。 通过在SCD和对照MSC中过量表达和敲低Ebf 3来驱动我们在小鼠SCD MSC中观察到的变化; 然后，我将通过体外原代培养和体内移植来量化HSC支持能力的变化 问题研究在目标2中，我将通过蛋白质-RNA结合鉴定EBF 3转录靶点及其在对照和SCD MSC中的细胞功能。 染色质相互作用研究，基因本体分析，基因集富集分析以及机制 询问Ebf 3下调。在目标3中，我将研究EBF 3下调在人类中的翻译影响。 SCD MSC通过再次采用过表达和敲低系统来干扰EBF 3表达并定量这种干扰如何影响EBF 3的表达。 影响其对人类HSC的支持。我还将阐明EBF 3在人MSC中的转录靶点， 这是前所未有的这项研究将是更好地了解转录功能障碍如何 在SCD中，MSC影响HSC支持，这反过来可能在SCD治愈性疗法的改进中是关键的。