Investigating dysregulation of hematopoietic stem cell support in sickle cell disease mesenchymal stromal cells
研究镰状细胞病间充质基质细胞中造血干细胞支持的失调
基本信息
- 批准号:10752050
- 负责人:
- 金额:$ 4.37万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-08-01 至 2026-07-31
- 项目状态:未结题
- 来源:
- 关键词:ATAC-seqAcute Myelocytic LeukemiaAdultAffectAntibodiesAutomobile DrivingB-LymphocytesBioinformaticsBlood VesselsBone MarrowBone Marrow DiseasesCandidate Disease GeneCell CompartmentationCell CycleCell ProliferationCell SeparationCell physiologyCellsChromatinChronicCoculture TechniquesDataDiseaseDown-RegulationEarly identificationEpigenetic ProcessFunctional disorderGene Expression ProfileGene StructureGene set enrichment analysisGenesGenetic TranscriptionGoalsHealthHematological DiseaseHematologyHematopoieticHematopoietic Stem Cell TransplantationHematopoietic stem cellsHemoglobinHemoglobinopathiesHemolysisHemolytic AnemiaHumanI-Cell DiseaseImmunoglobulin GImpairmentIndividualInflammationInflammatoryInheritedInstitutionLeadLongevityMolecularMorbidity - disease rateMusMutationOntologyOrganPainPatientsPersonsPhenotypePilot ProjectsPopulationPrincipal InvestigatorProliferatingRecoverySWI/SNF Family ComplexSickle Cell AnemiaSmall Interfering RNAStressSupporting CellSystemTechniquesTestingTissuesTranscription Factor 3TransplantationUnited States National Institutes of HealthUp-RegulationVertebratesWorkcareerchromatin proteincurative treatmentscytokinedifferential expressiongene repressionhematopoietic stem cell self-renewalhemoglobin polymerimprovedin vivoinnovationknock-downmesenchymal stromal cellmortalitymouse modelnucleaseoverexpressionpost-transplantretroviral transductionsuccesstranscription factortranslational impact
项目摘要
Project Summary
Sickle cell disease (SCD) is one of the most common hemoglobinopathies in the world. It results from a mutation in the
hemoglobin β gene which causes hemoglobin polymerization, leading to chronic hemolysis, inflammation, damage to
organs, and increased early morbidity. SCD pathophysiology can damage and stress the bone marrow microenvironment
and resident hematopoietic stem cells (HSCs), as evidenced in our pilot studies revealing fewer functional HSCs in the bone
marrow of both mice and individuals with SCD. Sinusoidal and arterial vascular networks in the SCD bone marrow are also
significantly disrupted. Furthermore, SCD is marked by decreased numbers of mesenchymal stromal cells (MSCs), a
heterogeneous cell population that functions as critical regulators of HSC self-renewal and differentiation. We observed
significant changes to MSC transcriptional profiles in SCD and identified three aberrant SCD MSC phenotypes: 1) increased
SCD MSC cell cycling, 2) increased secretion of inflammatory cytokines, and 3) decreased HSC support. The transcription
factor Early B Cell Factor 3 (EBF3) is a standout differentially expressed gene in SCD MSCs and a known regulator of cell
cycle. Loss of EBF3 in MSC subpopulations dramatically impacts bone marrow composition and HSC support. Cumulatively,
these findings lead me to hypothesize that EBF3 downregulation in bone marrow MSCs during SCD causes
transcriptional changes that decrease their HSC support capacity. In Aim 1, I will determine how Ebf3 downregulation is
driving the changes we observe in mouse SCD MSCs via over-expression and knockdown of Ebf3 in SCD and control MSCs;
I will then quantify changes to their HSC support capacity through ex vivo primary cultures and in vivo transplantation
studies. In Aim 2, I will identify EBF3 transcriptional targets and their cellular functions in control and SCD MSCs via protein-
chromatin interaction studies, gene ontology analysis, and gene set enrichment analysis as well as mechanistic
interrogation of Ebf3 downregulation. In Aim 3, I will investigate the translational impact of EBF3 downregulation in human
SCD MSCs by again employing over-expression and knockdown systems to perturb EBF3 expression and quantify how this
impacts their support of human HSCs. I will also elucidate the transcriptional targets of EBF3 in human MSCs, which has
never been done before. This study will be an important first step in better understanding how transcriptional dysfunction
in SCD MSCs influences HSC support, which, in turn, may be critical in the improvement of SCD curative therapies.
项目摘要
镰状细胞病(SCD)是世界上最常见的血红蛋白疾病之一。它是由基因突变引起的
血红蛋白β基因可引起血红蛋白聚合,导致慢性溶血、炎症、损伤
器官,并增加了早期发病率。SCD的病理生理学可以破坏和应激骨髓微环境
和常驻造血干细胞(HSCs),正如我们的初步研究所证明的那样,骨骼中功能较少的HSCs
SCD小鼠和个体的骨髓。SCD骨髓中的血窦和动脉血管网络也是
严重受阻。此外,SCD的特点是间充质基质细胞(MSCs)数量减少,
作为HSC自我更新和分化的关键调节因子的异质细胞群。我们观察到
SCD中MSC转录谱的显著变化并确定了三种异常的SCD MSC表型:1)增加
SCD MSC细胞周期改变,2)炎性细胞因子分泌增加,3)HSC支持减少。抄写
早期B细胞因子3(EBF3)是SCD MSCs中显著差异表达的基因,是已知的细胞调节因子
周而复始。在MSC亚群中EBF3的丢失显著影响骨髓成分和对HSC的支持。累积起来,
这些发现使我假设在SCD期间骨髓间充质干细胞中EBF3的下调导致
降低其HSC支持能力的转录变化。在目标1中,我将确定EBF3如何下调监管
通过EBF3在SCD和对照MSCs中的过度表达和敲除来驱动我们观察到的小鼠SCD MSCs的变化;
然后,我将通过体外原代培养和体内移植来量化他们的HSC支持能力的变化
学习。在目标2中,我将鉴定EBF3转录靶点及其在对照和SCD间充质干细胞中的细胞功能。
染色质相互作用研究、基因本体论分析、基因集丰富分析以及机理
审问EBF3下调。在目标3中,我将研究EBF3下调对人类的翻译影响
SCD MSCs通过再次使用过度表达和敲除系统干扰EBF3的表达并量化这一过程
影响他们对人类造血干细胞的支持。我还将阐明EBF3在人MSCs中的转录靶点,它已经
以前从未有人这么做过。这项研究将是更好地理解转录功能障碍的重要第一步
在SCD中,MSCs影响HSC的支持,这反过来可能在SCD治疗的改进中起关键作用。
项目成果
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