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(SCD)是世界上最常见的血红蛋白疾病之一。它是由基因突变引起的 血红蛋白β基因可引起血红蛋白聚合，导致慢性溶血、炎症、损伤 器官，并增加了早期发病率。SCD的病理生理学可以破坏和应激骨髓微环境 和常驻造血干细胞(HSCs)，正如我们的初步研究所证明的那样，骨骼中功能较少的HSCs SCD小鼠和个体的骨髓。SCD骨髓中的血窦和动脉血管网络也是 严重受阻。此外，SCD的特点是间充质基质细胞(MSCs)数量减少， 作为HSC自我更新和分化的关键调节因子的异质细胞群。我们观察到 SCD中MSC转录谱的显著变化并确定了三种异常的SCD MSC表型：1)增加 SCD MSC细胞周期改变，2)炎性细胞因子分泌增加，3)HSC支持减少。抄写 早期B细胞因子3(EBF3)是SCD MSCs中显著差异表达的基因，是已知的细胞调节因子 周而复始。在MSC亚群中EBF3的丢失显著影响骨髓成分和对HSC的支持。累积起来， 这些发现使我假设在SCD期间骨髓间充质干细胞中EBF3的下调导致 降低其HSC支持能力的转录变化。在目标1中，我将确定EBF3如何下调监管 通过EBF3在SCD和对照MSCs中的过度表达和敲除来驱动我们观察到的小鼠SCD MSCs的变化； 然后，我将通过体外原代培养和体内移植来量化他们的HSC支持能力的变化 学习。在目标2中，我将鉴定EBF3转录靶点及其在对照和SCD间充质干细胞中的细胞功能。 染色质相互作用研究、基因本体论分析、基因集丰富分析以及机理 审问EBF3下调。在目标3中，我将研究EBF3下调对人类的翻译影响 SCD MSCs通过再次使用过度表达和敲除系统干扰EBF3的表达并量化这一过程 影响他们对人类造血干细胞的支持。我还将阐明EBF3在人MSCs中的转录靶点，它已经 以前从未有人这么做过。这项研究将是更好地理解转录功能障碍的重要第一步 在SCD中，MSCs影响HSC的支持，这反过来可能在SCD治疗的改进中起关键作用。