Investigating hematopoietic stem cell dysfunction during sickle cell disease

项目摘要 慢性损伤，如炎症和复制应激，损害和耗尽血液维持 造血干细胞（HSC），导致功能障碍和白血病相关突变的选择。我们 我建议研究镰状细胞病（SCD）中的HSC损伤，SCD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 循环造血干细胞和祖细胞（HSPC）增加，BM HSPC数量增加， 扰乱表型和骨髓（BM）生态位的损害。老年SCD患者也可动员HSPC 并且可能变得对羟基脲不敏感。越来越多的证据表明，SCD患者可能有 在基线和同种异体HSC后，克隆造血以及MDS和AML的发生率增加 移植或自体HSC基因治疗。考虑到这些是唯一的治疗方法， 因此，更好地理解和预防SCD诱导的对HSC及其微环境的损伤是重要的。 环境因此，在这里，我们将检查健康的HSC池在个人与SCD。试点 数据揭示了SCD的中年小鼠模型中表型和功能性HSC的显著损失。 这些小鼠中的HSC也显示出细胞周期增加的证据。巧合的是，RNA测序显示 SCD HSC中的衰老相关变化。事实上，更多的SCD HSPC对生物标志物呈阳性， 相对于年龄匹配的对照组，SCD小鼠和个体的衰老。基于我们 初步发现，我们假设病理应激导致的早衰 导致SCD期间HSC功能障碍。在本提案的目标1中，我们将询问分离的HSC， 使用高分辨率显微镜从患有SCD的小鼠中观察衰老的分子和功能标志， 单细胞转录组学和HSC功能的定量测定。我们还将测试是否用以下药物治疗SCD小鼠： 消除衰老细胞的药物可以恢复HSC库的功能。在目标2中，我们将审问BM 来自儿科SCD患者的HSC，用于相对于年龄的衰老的分子和功能标志- 匹配的对照。我们将更精确地定义这些年轻人HSC池损伤的发病年龄， 使用定量有限稀释异种移植和离体单细胞测定， 血统潜力在普乐沙福动员前后从外周血中分离的HSPC也将被 初步研究表明，普乐沙福可动员这些患者中衰老的HSPC。目标3将重点 研究导致SCD HSC过早衰老的机制。在这里，我们将采用 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 复制应激和溶血直接导致SCD HSC的过早衰老。我们还将 测试用清除游离血红素的血红素结合素治疗是否可以减轻HSC衰老并恢复 在SCD小鼠中对HSC池起作用。

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.