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

项目摘要 造血干细胞(Hscs)是一种罕见的存在于骨髓中的自我更新的多潜能细胞。 以支持终生血液生产。造血干细胞可以移植到消融受者体内，在那里他们可以 重建所有类型的造血细胞，使其成为治疗慢性粒细胞白血病患者的有效疗法 造血干细胞移植治疗血液病。为了维持终生输血和满足紧急需求， 肝星状细胞严格调节静止状态和循环状态，以防止精疲力竭。一个极度静止的HSC 已确定的子集很少对稳定状态的造血起作用，保持长期保真度 在HSC泳池里。在移植后，深度静止的HSC表现出更多的自我更新和茎。 提示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静止期与细胞命运的关系以及深静止期HSC的分子标志物很少。 我的目标是为机械审讯寻找深度静止的造血干细胞的新型分子标记。 我们最近发现了GPRASP2(G蛋白偶联受体相关(GPCR)分选蛋白2)，它是一种HSC- 参与GPCR转运的浓缩蛋白，作为造血干细胞移植的调节因子。试点单一HSC 免疫荧光研究显示HSC Gprasp2异质性表达和单细胞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丰富 与Gprasp2高HSC相关的程序。我们产生了Gprasp2报告小鼠来询问HSC亚群 Gprasp2的表达(Gprasp2low/Gprasp2High)。初步移植数据显示Gprasp2高HSCs显示 缓慢的血液再循环动力学和稳健的、谱系平衡的重建，与更快、不那么稳健和 Gprasp2低表达的造血干细胞偏向淋巴重建。Bulk RNAseq和通路分析显示Gprasp2高表达 HSCs下调细胞周期和细胞对刺激基因表达的反应。始终如一地，更高的Gprasp2 在体外培养中，HSCs占据G0的比例高于Gprasp2low HSCs，且具有更强的自我更新能力。我假设 Gprasp2是一种高度静止的HSC亚集的新标记和调节因子，它优先于 续订。在目标1中，我将测试Gprasp2是否通过分析体内分裂来标记深度静止的HSCs的子集 Gprasp2低/高HSC的病史，量化刺激后从静止状态退出，并询问 移植后重建动力学。在目标2中，我将使用系列移植和单细胞自体细胞分析 更新和细胞命运来测试Gprasp2是否可以预测细胞命运。在目标3中，我将调查Gprasp2是否是HSC的驱动因素 通过改变Gprasp2的表达进行自我更新并鉴定具有已知作用的GPCRs和细胞表面受体 Gprasp2通过邻近标记实验调节造血功能。完成这些目标将产生 为同行评议的出版物提供数据，并给出机械性目标作为K99/K00申请的初步结果。 圣裘德的麦金尼-弗里曼实验室是金标HSC分析的最佳培训环境， 作为一名独立的HSC调查员，我需要蛋白质组学和生物信息学。

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.