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：
10676740
负责人：
Aditya Shirish Barve
金额：
$ 7.18万
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/10676740
关键词：
ATAC-seq Abnormal Hemoglobins Age Age Months Aging Animal Diseases Animals Autologous Autologous Transplantation Biochemical Biological Assay Bone Marrow Bone Marrow Aspiration Cancer Biology Carrying Capacities Cell Compartmentation Cell Cycle Cell Proliferation Cell Separation 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 Hemoglobin Hemolysis Human Immune Impairment In Vitro Inflammation Inherited Institution Investigation Knowledge Laboratories Lead Learning Mediating Meditation 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 Rag1 Mouse 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 exhaustion fitness gene therapy hematopoietic stem cell differentiation hemoglobin polymer immune activation improved in vivo insight mortality mouse model novel 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.

项目摘要镰状细胞疾病（SCD）是由于编码基因突变而导致的痛苦的衰弱终身状况血红蛋白β亚基，导致异常血红蛋白聚合导致溶血，重复血管肿块和慢性全身性炎症导致了大量的全球发病率和早期死亡率。 SCD治疗疗法依赖于造血干细胞（HSC）移植，但是有害影响 HSC上的SCD病理生理学仍然没有表征，我们试图在当前知识中填补这一空白。我们的初步研究表明，HSC在衰老后的转基因SCD小鼠模型中显示出增加的循环。 SCD患者衍生的HSC的体外分化偏差。作为博士后家伙麦金尼 - 弗里曼实验室，我将研究功能后果和分子机制鼠模型中的基础SCD介导的HSC循环，并将这些发现转化为人类HSC scd。在AIM 1中，我将使用菌落形成测定法，限制稀释初级和次级HSC移植，以及连续暴露于化学疗法，以评估SCD诱导的循环对HSC的有害影响频率和功能。在AIM 2中，我将探测增加的分子和表观遗传失调的增加 SCD期间的HSC骑自行车。从SCD和对照小鼠中分离出的HSC将受到散装RNA-seq和 ATAC-SEQ定义转录失调与基因启动子可及性的变化相关冥想增加的循环。最后，我将通过询问细胞周期将我们的发现转化为人类从SCD患者分离出的HSC的失调（AIM 3）。 SCD患者衍生的骨髓抽吸将是介绍了表型HSC的频率以及通过流式细胞仪和EX VIVO EDU改变细胞周期状态的频率并入。 SCD HSC的谱系潜力和造血输出将通过菌落形成分析和体外单HSC分化测定法。最后，SCD HSC重新流动潜力和自我更新将是通过将稀释原发性和继发移植限制为表达免疫的人类细胞因子进行检查不足的小鼠。拟议的研究共同加深了我们对以前出乎意料的理解 HSC生物学的方面通过检查SCD的影响。对SCD介导的机制的更多了解由于基因编辑后自体HSC移植，HSC损伤变得异常重要或用于SCD的基因疗法改善和增加频率。这些目标借鉴了我先前在癌症方面的培训血液学恶性肿瘤的生物学，但在HSC领域也提供了丰富的新型培训机会生物学和职业发展。麦金尼 - 弗里曼实验室和圣裘德儿童研究医院是接受研究HSC生物学培训的理想环境，结合了最先进的环境机构资源，职业发展资源和出色的心态，其目标是晋升为独立的学术主要研究者职位。