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.
项目概要
镰状细胞病(SCD)是世界上最常见的血红蛋白病之一。它是由突变引起的
血红蛋白β基因引起血红蛋白聚合,导致慢性溶血、炎症、损伤
器官,并增加早期发病率。 SCD 病理生理学会对骨髓微环境造成损害和应激
和驻留造血干细胞 (HSC),我们的初步研究表明,骨骼中功能性 HSC 较少
小鼠和患有 SCD 的个体的骨髓。 SCD 骨髓中的正弦和动脉血管网络也
显着扰乱。此外,SCD 的特点是间充质基质细胞 (MSC) 数量减少,这是一种
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 转录谱发生显着变化,并鉴定出三种异常的 SCD MSC 表型:1) 增加
SCD MSC 细胞周期,2) 炎症细胞因子的分泌增加,3) HSC 支持减少。转录
早期 B 细胞因子 3 (EBF3) 是 SCD MSC 中突出的差异表达基因,也是已知的细胞调节因子
循环。 MSC 亚群中 EBF3 的缺失会极大地影响骨髓组成和 HSC 支持。累计起来,
这些发现使我推测 SCD 期间骨髓 MSC 中 EBF3 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 下调是如何进行的
通过在 SCD 和对照 MSC 中过度表达和敲低 Ebf3,驱动我们在小鼠 SCD MSC 中观察到的变化;
然后,我将通过离体原代培养物和体内移植来量化其 HSC 支持能力的变化
研究。在目标 2 中,我将通过蛋白鉴定 EBF3 转录靶标及其在对照和 SCD MSC 中的细胞功能
染色质相互作用研究、基因本体分析、基因集富集分析以及机制
Ebf3 下调的询问。在目标 3 中,我将研究 EBF3 下调对人类的转化影响
SCD MSC 再次采用过度表达和敲低系统来扰乱 EBF3 表达并量化这种影响
影响他们对人类 HSC 的支持。我还将阐明 EBF3 在人类 MSC 中的转录靶标,它已
以前从未做过。这项研究将是更好地理解转录功能障碍如何发生的重要的第一步
SCD 中的 MSC 会影响 HSC 的支持,而这反过来又可能对 SCD 治疗的改进至关重要。
项目成果
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