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

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 造血干细胞是一种存在于骨髓中的具有自我更新能力的多能细胞 支持终生血液生产。HSC可以移植到消融的受体中，在那里它们可以 重建所有造血细胞类型，使其作为治疗性疗法非常有用， 造血干细胞移植治疗造血系统疾病为了维持终生的血液输出量并满足紧急需求， HSC严格调节静止与循环状态，以防止疲劳。深度静止的HSC 已确定很少有助于稳态造血的子集，从而保持长期保真度 的HSC池。在移植后，深度静止的HSC显示出增加的自我更新和干性， 提示这些HSC可能与移植结果高度相关。然而，人们对此知之甚少。 HSC静止和细胞命运之间的关系，并且存在很少的深度静止HSC的分子标记。 我的目标是确定新的分子标记物的深度静止HSC的机械审讯。 我们最近发现GPRASP 2（G蛋白偶联受体相关（GPCR）分选蛋白2），一种HSC- 富集的蛋白参与GPCR运输，作为HSC移植的调节剂。试点单HSC 免疫荧光研究揭示异质性HSC GPrasp 2表达和单细胞RNA测序 RNAseq数据显示低GPrasp 2表达的HSC在谱系特异性分化和细胞周期中富集。 Gprasp 2 high HSC相关的程序。我们产生了GPrasp 2报告小鼠来询问HSC亚群， 对GPrasp 2表达的影响（GPrasp 2low/GPrasp 2 high）。初步移植数据显示Gprasp 2 high HSC显示 缓慢的血液再生动力学和稳健的、谱系平衡的重建，相比之下， Gprasp 2low HSC的淋巴偏向性重建。批量RNAseq和途径分析揭示GPrasp 2 high HSC下调细胞周期和细胞对刺激基因表达的反应。一致，更Gprasp 2 high HSC比Gprasp 2low HSC占据G 0，并且在离体培养期间显示出更大的自我更新。我假设 GPrasp 2是一种新的标记物，也是一种高度静止的HSC亚群的调节因子， 更新。在目标1中，我将通过测定体内分裂来测试GPrasp 2是否标记深度静止的HSC的子集 Gprasp 2低/高HSC的历史，量化刺激后从静止期退出，并询问 移植后的重建动力学。在目标2中，我将使用系列移植和单细胞分析自体移植， 更新和细胞命运来测试GPrasp 2是否预测细胞命运。在目标3中，我将研究GPRAP 2是否是HSC的驱动程序 通过修饰Gprasp 2表达进行自我更新，并鉴定具有已知作用的GPCR和细胞表面受体 通过邻近标记分析，在由GPrasp 2调节的造血中。实现这些目标将产生 数据同行评审的出版物，并给出了机制的目标作为初步结果的K99/K 00应用。 圣裘德的麦金尼-弗里曼实验室是金标准HSC检测的最佳培训环境， 蛋白质组学方法，和生物信息学需要我的职业生涯作为一个独立的HSC研究者。