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，例如炎症和复制压力，损害和耗尽血液 造血干细胞（HSC），导致与白血病相关突变的功能障碍和选择。我们 建议研究HSC侮辱性镰状细胞病（SCD），这是一种慢性溶血性贫血， 炎症成分和增加的造血需求。 SCD中的造血异常包括 增加循环造血茎和祖细胞（HSPC），BM HSPC数量增加 骨髓（BM）生态位的扰动表型和损害。老年SCD患者还动员HSPC 差，可能会成为羟基脲的难治性。越来越多的证据表明SCD患者可能有 克隆造血的发生率提高，基线和同种异体HSC的MDS和AML的发生率提高 移植或自体HSC基因治疗。考虑到这些是唯一的治疗疗法 SCD，重要的是要更好地理解和防止SCD引起的对HSC及其微型的侮辱 环境。因此，在这里，我们将研究HSC池在SCD患者中的适应性。飞行员 数据揭示了SCD的中年小鼠模型中表型和功能HSC的巨大丧失。 这些小鼠中的HSC还显示出细胞循环增加的证据。矛盾的是，RNA测序显示 SCD HSC的衰老相关变化。实际上，基本上更多的SCD HSPC对生物标志物是阳性的 相对于年龄匹配的对照组，小鼠和具有SCD的个体的衰老。基于我们 初步发现，我们假设由病理压力驱动的过早衰老 在SCD期间导致HSC功能障碍。在本提案的目标1中，我们将询问HSC的孤立 来自SCD的小鼠，使用高分辨率显微镜，用于衰老的分子和功能标志， HSC功能的单细胞转录组学和定量测定。我们还将测试是否使用 消除衰老细胞的药物可以恢复HSC池的功能。在AIM 2中，我们将询问BM 小儿SCD患者的HSC具有衰老的分子和功能标志，相对于年龄 匹配的控件。我们将更精确地定义这些年轻的HSC池的发作年龄 使用定量限制稀释异种移植和离体单细胞分析的个体 谱系潜力。在plerixafor-Mobilization之前和之后，从外周血中分离出的HSPC也将是 研究了，正如试点研究表明，plerixafor动员了这些患者的衰老HSPC。 AIM 3将集中精力 研究有助于SCD HSC中过早衰老的机制。在这里，我们将雇用 高分辨率显微镜，离体培养和单细胞功能测定，以专门检验假设 这种复制性应力和溶血直接导致SCD HSC中的过早衰老。我们也会 测试消除自由血红素的血红素治疗是否可以减轻HSC衰老并恢复 用SCD的小鼠中的HSC池功能。