Gprasp2 expression identifies a deeply quiescent hematopoietic stem cells subset with superior stemness and self-renewal

Gprasp2 表达鉴定出具有卓越干性和自我更新能力的深度静止造血干细胞亚群

• 批准号: 10751567
• 负责人: Alanna V Van Huizen
• 金额: $ 6.95万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10751567
• 关键词: 3' Untranslated Regions; Ablation; Bioinformatics; Biological Assay; Blood; Bone Marrow; Cell Cycle; Cell Surface Receptors; Cells; Cellular Assay; Chimeric Proteins; Colony-Forming Units Assay; Cues; Data; Daughter; Disease; Emergency Situation; Environment; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Gene Expression; Genetic Transcription; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cell heterogeneity; Hematopoietic Stem Cell subsets; Hematopoietic stem cells; Homeostasis; Immunofluorescence Immunologic; In Vitro; Infection; Kinetics; Label; Laboratories; Life; Lymphoid; Mass Spectrum Analysis; Measures; Molecular; Mus; Outcome; Output; Pathway Analysis; Patients; Pattern; Peer Review; Photobleaching; Population; Production; Protein Sortings; Proteins; Proteomics; Publications; Recording of previous events; Regulation; Reporter; Research Personnel; Role; Saint Jude Children' s Research Hospital; Stimulus; Stress; Testing; Time; Tomatoes; Training; Transplantation; career; cell type; curative treatments; dimer; exhaustion; flexibility; hematopoietic hierarchy; hematopoietic stem cell expansion; hematopoietic stem cell fate; hematopoietic stem cell quiescence; hematopoietic stem cell self-renewal; in vivo; insight; knock-down; molecular marker; mouse model; multipotent cell; novel; novel marker; overexpression; peripheral blood; post-transplant; preservation; programs; prospective; reconstitution; response; self-renewal; single cell technology; single-cell RNA sequencing; stem cell engraftment; stem cell function; stem cells; stemness; trafficking; transcriptome sequencing; 3' Untranslated Regions; Ablation; Bioinformatics; Biological Assay; Blood; Bone Marrow; Cell Cycle; Cell Surface Receptors; Cells; Cellular Assay; Chimeric Proteins; Colony-Forming Units Assay; Cues; Data; Daughter; Disease; Emergency Situation; Environment; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Gene Expression; Genetic Transcription; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cell heterogeneity; Hematopoietic Stem Cell subsets; Hematopoietic stem cells; Homeostasis; Immunofluorescence Immunologic; In Vitro; Infection; Kinetics; Label; Laboratories; Life; Lymphoid; Mass Spectrum Analysis; Measures; Molecular; Mus; Outcome; Output; Pathway Analysis; Patients; Pattern; Peer Review; Photobleaching; Population; Production; Protein Sortings; Proteins; Proteomics; Publications; Recording of previous events; Regulation; Reporter; Research Personnel; Role; Saint Jude Children' s Research Hospital; Stimulus; Stress; Testing; Time; Tomatoes; Training; Transplantation; career; cell type; curative treatments; dimer; exhaustion; flexibility; hematopoietic hierarchy; hematopoietic stem cell expansion; hematopoietic stem cell fate; hematopoietic stem cell quiescence; hematopoietic stem cell self-renewal; in vivo; insight; knock-down; molecular marker; mouse model; multipotent cell; novel; novel marker; overexpression; peripheral blood; post-transplant; preservation; programs; prospective; reconstitution; response; self-renewal; single cell technology; single-cell RNA sequencing; stem cell engraftment; stem cell function; stem cells; stemness; trafficking; transcriptome sequencing

Project Summary Hematopoietic stem cells (HSCs) are a rare pool of self-renewing, multipotent cells residing in the bone marrow that support lifelong blood production. HSCs can be transplanted into ablated recipients, where they can reconstitute all hematopoietic cell types, making them highly useful as a curative therapy for patients with hematopoietic diseases via HSC transplantation. To sustain lifelong blood output and meet emergency demands, HSCs tightly regulate a quiescent versus cycling state to protect from exhaustion. A deeply quiescent HSC subset has been identified that rarely contributes to steady-state hematopoiesis, preserving the long-term fidelity of the HSC pool. Upon transplantation, deeply quiescent HSCs display increased self-renewal and stemness, suggesting these HSCs may be highly relevant to transplant outcomes. However, little is known about the relationship between HSC quiescence and cell fate and few molecular markers of deeply quiescent HSCs exist. My goal is to identify novel molecular markers of deeply quiescent HSCs for mechanistic interrogation. We recently identified GPRASP2 (G-protein Coupled Receptor-associated (GPCR) Sorting Protein 2), an HSC- enriched protein involved in GPCR trafficking, as a regulator of HSC transplantation. Pilot single HSC immunofluorescence studies reveal heterogeneous HSC Gprasp2 expression and single-cell RNA sequencing (RNAseq) data show low Gprasp2-expressing HSCs are enriched in lineage-specific differentiation and cell cycle programs relative to Gprasp2high HSCs. We generated Gprasp2 reporter mice to interrogate HSC subsets based on Gprasp2 expression (Gprasp2low/Gprasp2high). Preliminary transplant data reveal Gprasp2high HSCs display slow blood repopulation kinetics and robust, lineage-balanced reconstitution compared to faster, less robust, and lymphoid-biased reconstitution by Gprasp2low HSCs. Bulk RNAseq and pathway analysis reveal Gprasp2high HSCs downregulate cell cycle and cellular responses to stimuli gene expression. Consistently, more Gprasp2high HSCs occupy G0 than Gprasp2low HSCs and display greater self-renewal during ex vivo culture. I hypothesize Gprasp2 is a novel marker and regulator of a deeply quiescent HSC subset that preferentially self- renews. In Aim 1, I will test if Gprasp2 marks a subset of deeply quiescent HSCs by assaying in vivo division history of Gprasp2low/high HSCs, quantifying exit from quiescence upon stimulation, and interrogating reconstitution kinetics post-transplant. In Aim 2, I will use serial transplantation and single-cell assays of self- renewal and cell fate to test if Gprasp2 predicts cell fate. In Aim 3, I will investigate if Gprasp2 is a driver of HSC self-renewal by modifying Gprasp2 expression and identify GPCRs and cell-surface receptors with known roles in hematopoiesis regulated by Gprasp2 via proximity labeling assays. Completion of these aims will produce data for peer-reviewed publication and give mechanistic targets as preliminary results for a K99/K00 application. The McKinney-Freeman laboratory at St. Jude is the optimal training environment for gold-standard HSC assays, proteomics approaches, and bioinformatics required for my career as an independent HSC investigator.

项目摘要 造血干细胞（HSC）是一种罕见的自我更新，多能细胞，位于骨髓中 这支持终生的血液产生。 HSC可以将其移植到消融的接收者中，可以在其中 重建所有造血细胞类型，使其可作为治疗疗法非常有用 造血疾病通过HSC移植。为了维持终生的血输出并满足紧急需求， HSC严格调节静止状态与骑自行车状态，以防止疲惫。深度静止的HSC 已经确定了很少有助于稳态造血的子集，从而保留了长期保真度 HSC池。移植后，深度静止的HSC表现出增加的自我更新和茎， 建议这些HSC可能与移植结果高度相关。但是，关于 HSC静止与细胞命运之间的关系以及很少有深度静止的HSC的分子标记。 我的目标是确定深度静止的HSC的新型分子标记，以进行机理审讯。 我们最近确定了GPRASP2（G蛋白偶联受体相关（GPCR）排序蛋白2），HSC- 富含GPCR运输的蛋白质作为HSC移植的调节剂。飞行员单HSC 免疫荧光研究揭示了异质HSC GPRASP2表达和单细胞RNA测序 （RNASEQ）数据显示，低表达GPRASP2的HSC在谱系特异性分化和细胞周期中富集 相对于GPRASP2高HSC的程序。我们生成了GPRASP2记者小鼠以询问基于HSC子集 在GPRASP2表达（gprasp2low/gprasp2high）上。初步移植数据揭示了GPRASP2High HSC显示 与更快，较不健壮的速度相比 GPRASP2LOW HSCS淋巴偏见的重建。批量RNASEQ和途径分析揭示了GPRASP2High HSC下调细胞周期和对刺激基因表达的细胞反应。一贯，更多的gprasp2high HSC比GPRASP2LOW HSC占据G0，在离体培养过程中表现出更大的自我更新。我假设 GPRASP2是一个新颖的标记和调节剂，这是一个高度静止的HSC子集，优先自我 更新。在AIM 1中，我将测试GPRASP2是否通过在体内分析中测定深度静止的HSC的子集 GPRASP2LOW/HIGH HSC的历史，刺激时量化静止的退出和审问 移植后重建动力学。在AIM 2中，我将使用串行移植和自我的单细胞测定 更新和细胞命运测试GPRASP2是否预测细胞命运。在AIM 3中，我将调查GPRASP2是否是HSC的驱动程序 通过修改GPRASP2表达并识别具有已知作用的GPCR和细胞表面受体的自我更新 在由GPRASP2调节的造血中，通过接近标记测定法。这些目标的完成将产生 同行评审出版物的数据，并将机械目标作为K99/K00应用程序的初步结果。 圣裘德的麦金尼 - 弗里曼实验室是金色标准HSC分析的最佳培训环境， 我作为独立HSC研究员的职业所需的蛋白质组学方法和生物信息学。