Investigating hematopoietic stem cell dysfunction during sickle cell disease

研究镰状细胞病期间的造血干细胞功能障碍

• 批准号: 10681829
• 负责人: SHANNON L MCKINNEY-FREEMAN
• 金额: $ 70.86万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681829
• 关键词: AMD3100; Age; Age Months; Age of Onset; Allogenic; Antibodies; Apoptosis; Autologous; Biological Assay; Biological Markers; Blood; Bone Marrow; Bone Marrow Transplantation; Bromodeoxyuridine; CD34 gene; Cell Aging; Cell Cycle; Cell Separation; Cell Size; Cell surface; Cells; Cellular Assay; Childhood; Chronic; DNA Damage; Data; Functional disorder; Gene Expression; Gene Expression Profiling; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heme; Hemolysis; Hemolytic Anemia; Hemopexin; Human; Immune; Impairment; Individual; Inflammation; Inflammatory; Inherited; Microscopy; Molecular; Molecular Disease; Mus; Mutation; Output; Pathologic; Patients; Pharmaceutical Preparations; Phenotype; Physiologic pulse; Physiological; Pilot Projects; Population; Production; RNA; Refractory; Resolution; Sampling; Sickle Cell Anemia; Stains; Stress; Stress Tests; Supporting Cell; TP53 gene; Testing; Time; Transplantation; United States; Xenograft procedure; beta-Galactosidase; cell age; curative treatments; exhaust; experience; fitness; functional restoration; gene therapy; hydroxyurea; in vivo; inhibitor; leukemia; middle age; mouse model; multiorgan damage; peripheral blood; premature; prevent; progenitor; programs; reconstitution; repaired; self-renewal; senescence; stem; stem cell function; stem cell gene therapy; stem cells; telomere; transcriptome sequencing; transcriptomics; uptake; vaso-occlusive pain

Project Summary Chronic insults, such as inflammation and replicative stress, impair and exhaust blood-sustaining hematopoietic stem cells (HSCs), leading to dysfunction and selection for leukemia-associated mutations. We propose to study HSC insults in sickle cell disease (SCD), a chronic hemolytic anemia with a large inflammatory component and increased hematopoietic demand. Hematopoietic abnormalities in SCD include increased circulating hematopoietic stem and progenitors (HSPCs), increased numbers of BM HSPCs with perturbed phenotypes and damage to the bone marrow (BM) niche. Older SCD patients also mobilize HSPCs poorly and can become refractory to hydroxyurea. Mounting evidence indicates that SCD patients may have enhanced rates of clonal hematopoiesis, as well as MDS and AML at baseline and following allogeneic HSC transplantation or autologous HSC gene therapy. Considering that these are the only curative therapies for SCD, it is important to better understand and prevent SCD-induced insults to HSCs and their micro- environment. Thus, here we will examine the fitness of the HSC pool in individuals with SCD. Pilot data reveals a dramatic loss of both phenotypic and functional HSCs in middle-aged mouse models of SCD. HSCs in these mice also display evidence of increased cell cycling. Paradoxically, RNA-sequencing reveals senescence-related changes in SCD HSCs. Indeed, substantially more SCD HSPCs were positive for biomarkers of senescence in both mice and individuals with SCD, relative to age-matched controls. Based on our preliminary findings, we hypothesize that premature senescence driven by pathologic stress contributes to HSC dysfunction during SCD. In Aim 1 of this proposal, we will interrogate HSCs isolated from mice with SCD for molecular and functional hallmarks of senescence using high-resolution microscopy, single cell transcriptomics and quantitative assays for HSC function. We will also test if treating SCD mice with drugs that eliminate senescent cells can restore function to the HSC pool. In Aim 2, we will interrogate BM HSCs from pediatric SCD patients for molecular and functional hallmarks of senescence, relative to age- matched controls. We will define more precisely the age of onset of damage to the HSC pool in these young individuals with SCD using quantitative limiting dilution xenotransplantation and ex vivo single cell assays for lineage potential. HSPCs isolated from peripheral blood before and after plerixafor-mobilization will also be studied, as pilot studies suggest that plerixafor mobilizes senescent HSPCs in these patients. Aim 3 will focus on investigating mechanisms that contribute to premature senescence in SCD HSCs. Here, we will employ high-resolution microscopy, ex vivo culture, and single cell functional assays to specifically test the hypothesis that replicative stress and hemolysis contribute directly to premature senescence in SCD HSCs. We will also test if treatment with hemopexin, which eliminates free heme, can alleviate HSC senescence and restore function to the HSC pool in mice with SCD.

项目摘要 慢性侮辱，如炎症和复制应激，会损害和耗尽维持血液的能力 造血干细胞(HSCs)，导致功能障碍和白血病相关突变的选择。我们 建议研究镰状细胞病(SCD)中的HSC侮辱，SCD是一种慢性溶血性贫血，具有 炎症成分和增加的造血需求。SCD的造血异常包括 循环中的造血干祖细胞(HSPC)增加，BM HSPC数量增加 受扰的表型和对骨髓(BM)生态位的损害。老年SCD患者也动员HSPC 很差，可对羟基尿素产生耐受性。越来越多的证据表明，SCD患者可能患有 在基线和异基因造血干细胞移植后，克隆性造血率以及MDS和AML的增加 移植或自体造血干细胞基因治疗。考虑到这些是唯一的治疗方法 SCD，重要的是要更好地了解和防止SCD对HSC及其微环境的侮辱 环境。因此，在这里我们将检查SCD患者的HSC池的适合性。引航员 数据显示，在SCD的中年小鼠模型中，表型和功能的HSCs都发生了戏剧性的丧失。 这些小鼠体内的HSCs也显示出细胞周期增加的证据。矛盾的是，RNA测序揭示了 SCD HSCs的衰老相关变化。事实上，更多的SCD HSPC的生物标记物呈阳性 与年龄匹配的对照组相比，小鼠和SCD患者的衰老程度都有所提高。基于我们的 初步发现，我们假设病理性应激导致的早衰 在SCD期间导致HSC功能障碍。在本提案的目标1中，我们将审问隔离的HSC 从患有SCD的小鼠身上用高分辨率显微镜观察衰老的分子和功能特征， 单细胞转录和肝星状细胞功能的定量分析。我们还将测试用来治疗SCD小鼠的 消除衰老细胞的药物可以恢复HSC池的功能。在目标2中，我们将审问BM 来自儿童SCD患者的HSC衰老的分子和功能特征，相对于年龄- 配对的对照组。我们将更准确地定义这些年轻人HSC池受损的开始年龄 应用定量限制稀释法异种移植和体外单细胞分析检测SCD患者 血统潜力。动员前后外周血中分离出的HSPC也将被 研究，因为初步研究表明，plerixafor动员这些患者衰老的HSPC。目标3将专注于 关于SCD HSCs早衰机制的研究。在这里，我们将雇用 高分辨率显微镜、体外培养和单细胞功能分析来具体验证这一假设 复制应激和溶血直接导致SCD HSCs的早衰。我们还将 测试消除游离血红素的血凝素治疗是否可以延缓HSC衰老和恢复 对SCD小鼠的HSC池有一定的调节作用。