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.

项目概要 造血干细胞 (HSC) 是存在于骨髓中的一种罕见的自我更新、多能细胞库 支持终生血液生产。造血干细胞可以被移植到被切除的受者体内，在那里它们可以 重建所有造血细胞类型，使其成为治疗患有以下疾病的患者的有效疗法 通过 HSC 移植治疗造血系统疾病。为了维持终生血液输出并满足紧急需求， HSC 严格调节静态与循环状态，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 HSC 池的成员。移植后，深度静止的 HSC 表现出增强的自我更新和干性， 表明这些造血干细胞可能与移植结果高度相关。然而，人们对此知之甚少 HSC 静止与细胞命运之间的关系以及深度静止 HSC 的分子标记很少。 我的目标是识别深度静止 HSC 的新分子标记，以进行机械分析。 我们最近发现了 GPRASP2（G 蛋白偶联受体相关 (GPCR) 分选蛋白 2），一种 HSC- 丰富的蛋白质参与 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 HSC Gprasp2 表达和单细胞 RNA 测序 (RNAseq) 数据显示低 Gprasp2 表达的 HSC 在谱系特异性分化和细胞周期中富集 与 Gprasp2high HSC 相关的程序。我们生成了 Gprasp2 报告小鼠来询问基于 HSC 子集的 Gprasp2 表达（Gprasp2low/Gprasp2high）。初步移植数据显示 Gprasp2high HSCs 显示 与更快、更不稳健和更快速的血液重建动力学相比，血液再增殖动力学更慢，并且重建稳健、谱系平衡。 Gprasp2low HSC 进行淋巴偏向重建。批量 RNAseq 和通路分析揭示 Gprasp2high HSC 下调细胞周期和细胞对刺激基因表达的反应。始终如一，更多 Gprasp2high HSC 比 Gprasp2low HSC 占据 G0，并且在离体培养期间表现出更强的自我更新能力。我假设 Gprasp2 是深度静止 HSC 亚群的新型标记物和调节因子，优先自我调节 续订。在目标 1 中，我将通过检测体内分裂来测试 Gprasp2 是否标记深度静止 HSC 的子集 Gprasp2low/high HSC 的历史，量化刺激后从静止状态的退出，并询问 移植后的重建动力学。在目标 2 中，我将使用连续移植和单细胞检测来检测自身 更新和细胞命运来测试 Gprasp2 是否预测细胞命运。在目标 3 中，我将调查 Gprasp2 是否是 HSC 的驱动程序 通过修改 Gprasp2 表达进行自我更新，并识别具有已知作用的 GPCR 和细胞表面受体 通过邻近标记测定，Gprasp2 调节造血作用。完成这些目标将产生 同行评审出版物的数据，并给出机械目标作为 K99/K00 应用的初步结果。 圣裘德的 McKinney-Freeman 实验室是黄金标准 HSC 检测的最佳培训环境， 作为一名独立的 HSC 研究者，我的职业生涯需要蛋白质组学方法和生物信息学。