9bf37bbd-2d4d-4f6c-a144-53466ea5127b - samples

The data provided here was critical in establishing that human long-term hematopoietic stem cells (LT-HSC), previously described as the most primitive HSC population, is actually composed of distinct subsets that can be prospectively isolated. Via mechanistic studies centering around the Rho-GTPase effector kinase PAK4 and its inhibitor INKA1, we identified the immune checkpoint ligand CD112 as a marker for hematopoietic stem and progenitor cells, that is highest expressed on LT-HSC. More importantly, CD112 can be used to stratify functionally distinct subsets within LT-HSC: In response to regeneration-mediated stress, the CD112low subset exhibits a transient restraint (termed latency) before contributing to hematopoietic reconstitution, while the CD112high subset is primed to respond rapidly. High resolution RNA-seq of the CD112 surface expression spectrum within rare LT-HSC subsets (human umbilical cord blood) demonstrated that more genes are differentially upregulated in the deeper quiescent and less metabolic active subset. Genes enriched in this subset centre around cell adhesion and Rho-GTPase signaling. This is in agreement with the scRNAseq data from human G-CSF mobilized peripheral blood (mPB) generated here that was used as an model of in vivo activation/priming revealing via RNA-velocity and pseudo-time analysis that INKA1high versus PAK4high, CDK6high and CD112high enrichment are either detected early or late in diffusion pseudotime indicative of quiescent versus primed cell status, respectively. RNAseq following INKA1 overexpression in LT-HSC and ST-HSC revealed by GSEA an overall stemness preserving phenotype and particularly in LT-HSC, but not in short-term HSC (ST-HSC), suppression of transcriptional programs linked to activation. Collectively, our data decipher the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to preserve a stem cell reservoir.EGA dataset EGAD00001006541

本数据集提供的数据对于确立一项关键结论至关重要：此前被认定为最原始造血干细胞（hematopoietic stem cells, HSC）群体的人类长期造血干细胞（long-term hematopoietic stem cells, LT-HSC），实则由多个可被前瞻性分离的独特亚群组成。本研究以Rho GTP酶（Rho-GTPase）效应激酶PAK4及其抑制剂INKA1为核心开展机制研究，鉴定出免疫检查点配体CD112可作为造血干祖细胞（hematopoietic stem and progenitor cells）的标志物，且该标志物在LT-HSC上的表达水平最高。更为重要的是，CD112可用于区分LT-HSC内部功能迥异的亚群：在再生介导的应激反应中，CD112低表达亚群在参与造血重建前会呈现短暂的蛰伏状态（又称潜伏期），而CD112高表达亚群则已做好快速响应的准备。对人类脐带血（umbilical cord blood）中稀有LT-HSC亚群的CD112表面表达谱进行高分辨率RNA测序（high resolution RNA-seq）结果显示，在深度静息且代谢活性更低的亚群中，差异上调的基因数量更多。该亚群中富集的基因主要集中于细胞黏附与Rho GTP酶信号通路。这与本研究生成的人类粒细胞集落刺激因子（granulocyte colony-stimulating factor, G-CSF）动员外周血（mobilized peripheral blood, mPB）单细胞RNA测序（single-cell RNA sequencing, scRNAseq）数据一致——该数据被用作体内激活/致敏的模型，通过RNA速率分析（RNA-velocity）与拟时间分析（pseudo-time analysis）发现，INKA1高表达与PAK4、CDK6、CD112高表达分别在扩散拟时间（diffusion pseudotime）的早期与晚期被检测到，分别对应静息细胞与致敏细胞的状态。对LT-HSC与短期造血干细胞（short-term hematopoietic stem cells, ST-HSC）进行INKA1过表达后的RNA测序结果经基因集富集分析（Gene Set Enrichment Analysis, GSEA）显示，整体呈现维持干细胞干性（stemness）的表型，尤其在LT-HSC中更为显著；而在ST-HSC中则未观察到该现象，且INKA1过表达会抑制与细胞激活相关的转录程序。综上，本研究数据解析了造血干细胞异质性与自我更新调控背后的分子机制，并揭示蛰伏状态是一种协调的生理反应：该反应整合了静息状态调控与造血干细胞命运抉择，从而维持干细胞储备库的稳定。EGA数据集EGAD00001006541