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病之一。它是由突变中的 血红蛋白β基因引起血红蛋白聚合，导致慢性溶血，炎症，损害 器官，增加了早期发病率。 SCD病理生理学会损害和压力骨髓微环境 和居民造血干细胞（HSC），正如我们的试点研究中证明的那样，骨骼中的功能性HSC较少 小鼠和患有SCD的个体的骨髓。 SCD骨髓中的正弦和动脉血管网络也是 严重破坏。此外，SCD的特征是间充质基质细胞数量减少（MSC），A 作为HSC自我更新和分化的关键调节剂的异质细胞种群。我们观察到 SCD中MSC转录曲线的重大变化，并确定了三个异常SCD MSC表型：1）增加 SCD MSC细胞循环，2）增加炎症细胞因子的分泌以及3）改善HSC支持。转录 因子早期B细胞因子3（EBF3）是SCD MSC中脱颖而出的基因，并且是已知的细胞调节剂 循环。 MSC亚群中EBF3的丢失显着影响骨髓组成和HSC支持。累计， 这些发现使我假设在SCD原因中骨髓MSC中的EBF3下调 转录变化可降低其HSC支持能力。在AIM 1中，我将确定EBF3下调的方式 通过SCD和控制MSC中的EBF3的过表达和敲除鼠标SCD MSC中观察到的变化； 然后，我将通过体内原始培养和体内移植来量化其HSC支持能力的更改 研究。在AIM 2中，我将通过蛋白质 - 在对照和SCD MSC中的EBF3转录靶标及其细胞功能 - 染色质相互作用研究，基因本体分析和基因集富集分析以及机械 EBF3下调的询问。在AIM 3中，我将研究EBF3下调人类的翻译影响 SCD MSC通过再次使用过表达和敲低系统来扰动EBF3表达并量化如何 影响他们对人类HSC的支持。我还将阐明人类MSC中EBF3的转录目标 从来没有做过。这项研究将是更好地了解转录功能障碍的重要第一步 在SCD中，MSC会影响HSC支持，这反过来在改善SCD治疗疗法方面至关重要。