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Mechanisms and consequences of sickle cell disease-induced cycling in hematopoietic stem cells

镰状细胞病诱导造血干细胞循环的机制和后果

基本信息

批准号：
10464657
负责人：
Aditya Shirish Barve
金额：
$ 6.76万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10464657
关键词：
ATAC-seq Abnormal Hemoglobins Age Age-Months Aging Animal Diseases Animals Aspirate substance Autologous Autologous Transplantation Biochemical Biological Assay Bone Marrow Cancer Biology Carrying Capacities Cell Compartmentation Cell Cycle Cell Proliferation Cells Chronic Clinical Clinical Trials DNA Damage Data Analyses Data Set Environment Epigenetic Process Erythrocytes Erythropoiesis Exposure to Flow Cytometry Frequencies Functional disorder Genes Genetic Transcription Glean Goals Health Hematologic Neoplasms Hematological Disease Hematology Hematopoietic Hematopoietic Neoplasms Hematopoietic Stem Cell Mobilization Hematopoietic Stem Cell Transplantation Hematopoietic Stem Cell subsets Hematopoietic stem cells Hemolysis Human Immune Impairment In Vitro Inflammation Inherited Investigation Knowledge Laboratories Lead Learning Life Mediating Mentorship Molecular Molecular Probes Molecular Profiling Morbidity - disease rate Morphology Mus Mutation Myelogenous Organ Output Oxygen Pain Pathway interactions Patients Phenotype Pilot Projects Ploidies Positioning Attribute Postdoctoral Fellow Regimen Research Personnel Resource Development Resources Risk Safety Saint Jude Children&apos s Research Hospital Sickle Cell Sickle Cell Anemia Source Stress Testing Time Training Transgenic Organisms Translating Transplantation Work Xenograft procedure aged career development cell age cell injury chemotherapy curative treatments cytokine disorder control exhaustion fitness gene therapy hematopoietic stem cell differentiation hemoglobin B hemoglobin polymer immune activation improved in vivo insight mortality mouse model novel polymerization post-transplant premature promoter self-renewal sickling stem cell biology stem cell function systemic inflammatory response training opportunity transcriptome sequencing

项目摘要

Project Summary Sickle cell disease (SCD) is a painful debilitating life-long condition resulting from mutations in the gene encoding hemoglobin β subunit, causing abnormal hemoglobin polymerization leading to hemolysis, repeated vasooclusion, and chronic systemic inflammation resulting in substantial global morbidity and early mortality. Curative therapy for SCD relies on hematopoietic stem cell (HSC) transplantation, however the damaging effects of SCD pathophysiology on HSCs remain uncharacterized and we seek to fill this gap in current knowledge. Our preliminary studies show that HSCs display increased cycling in a transgenic SCD mouse model upon aging, and myeloid lineage biased in vitro differentiation of SCD patient derived HSCs. As a postdoctoral fellow in the McKinney-Freeman laboratory, I will investigate the functional consequences and molecular mechanisms underlying SCD mediated HSC cycling in a murine model and translate these findings to human HSC during SCD. In Aim 1, I will use colony formation assays, limiting dilution primary and secondary HSC transplantation, and serial exposure to chemotherapy to assess the detrimental impact of SCD-induced cycling on HSC frequency and function. In Aim 2, I will probe the molecular and epigenetic dysregulation underlying increased HSC cycling during SCD. HSCs isolated from SCD and control mice will be subjected to bulk RNA-seq and ATAC-seq to define transcriptional dysregulation correlated with changes in gene promoter accessibility meditating increased cycling. Finally, I will translate our findings to humans by interrogating cell cycle dysregulation in HSCs isolated from SCD patients (Aim 3). SCD patient-derived bone marrow aspirates will be profiled for frequency of phenotypic HSCs and alterations in cell cycle status by flow cytometry and ex vivo EdU incorporation. Lineage potential and hematopoietic output of SCD HSCs will be analyzed by colony formation and in vitro single HSC differentiation assays. Lastly, SCD HSC repopulating potential and self-renewal will be examined by limiting dilution primary and secondary transplantation into human cytokine expressing immune deficient mice. Together the proposed studies serve to deepen our understanding of a previously unexplored aspect of HSC biology by examining the impact of SCD. Greater understanding of SCD mediated mechanisms of HSC impairment become exceptionally important as autologous HSC transplantation following gene editing or gene therapy for SCD improve and increase in frequency. These aims draw on my prior training in cancer biology of hematologic malignancies but also provide abundant novel training opportunities in the field of HSC biology and for professional career development. The McKinney-Freeman lab and St. Jude Children’s Research Hospital are ideal environments in which to receive training in the study HSC biology, combining state-of-the-art institutional resources, career development resources, and excellent mentorship with the singular goal of advancing to an independent academic primary investigator position.

项目摘要镰状细胞病（SCD）是一种痛苦的衰弱的终身条件所造成的基因编码突变，血红蛋白β亚基，引起异常血红蛋白聚合，导致溶血，反复血管闭塞和慢性全身性炎症，导致大量的全球发病率和早期死亡率。 SCD的治愈性治疗依赖于造血干细胞（HSC）移植，然而， SCD病理生理学在HSC上的作用仍然没有特征化，我们试图填补现有知识中的这一空白。我们初步研究显示在转基因SCD小鼠模型中HSC在老化时表现出增加的循环，和髓系偏向的SCD患者来源的HSC的体外分化。作为一名博士后研究员，麦金尼-弗里曼实验室，我将研究功能后果和分子机制在小鼠模型中，基础SCD介导的HSC循环，并将这些发现转化为人类HSC， SCD。在目标1中，我将使用集落形成试验，有限稀释的初级和次级HSC移植，和连续暴露于化疗以评估SCD诱导的循环对HSC的有害影响频率和功能。在目标2中，我将探讨增加的基因表达的分子和表观遗传失调。 SCD期间的HSC循环。将从SCD和对照小鼠分离的HSC进行批量RNA-seq和测序。 ATAC-seq定义与基因启动子可及性变化相关的转录失调冥想增加了循环。最后，我将通过询问细胞周期，在从SCD患者分离的HSC中的异常调节（目的3）。SCD患者来源的骨髓穿刺液将通过流式细胞术和离体EdU分析表型HSC的频率和细胞周期状态的改变合并。将通过集落形成分析SCD HSC的谱系潜力和造血输出和体外单一HSC分化测定。最后，SCD HSC的再生潜力和自我更新将是通过有限稀释初次和二次移植到表达免疫的人细胞因子中进行检查缺陷小鼠这些拟议的研究有助于加深我们对以前未探索的 HSC生物学方面通过检查SCD的影响。更好地理解SCD介导的机制在基因编辑后自体HSC移植时，HSC损伤变得异常重要或基因治疗对SCD的改善和频率增加。这些目标都是基于我之前在癌症方面所接受的培训血液恶性肿瘤的生物学，但也提供了丰富的新的培训机会，在HSC领域生物学和专业职业发展。麦金尼-弗里曼实验室和圣裘德儿童研究医院是理想的环境中接受培训的研究HSC生物学，结合国家的最先进的机构资源、职业发展资源和优秀的导师，其唯一目标是晋升为独立学术主要研究员。