胶质母细胞瘤干细胞（GSCs）对胶质母细胞瘤（GBM）的进展、治疗抵抗和复发起了贡献。然而，目前对GSCs的研究通常是在人体肿瘤微环境之外进行的，忽视了原发性GSCs的细胞状态的重要性。在本研究中，我们利用来自公共数据库的6个独立的GBM队列的单细胞转录组测序数据，结合谱系和干性特征，识别了原发性GSCs。我们分解了GSCs的细胞状态，并将它们与患者的临床结果进行了相关性分析。结果，我们构建了一个GSCs位于中心的细胞层次结构。此外，我们还确定和表征了两个不同且反复出现的GSCs亚群：增殖性GSCs（pGSCs）和静止性GSCs（qGSCs）。pGSCs显示出高细胞周期活性，表明快速细胞分裂，而qGSCs则呈静止状态。然后，我们通过假时间分析和肿瘤谱系追踪了肿瘤发展的过程，并发现GSCs在整个肿瘤发展期间累积。在此过程中，pGSCs主要对早期阶段起作用，而qGSCs则在后期富集。最后，我们构建了一个反映pGSCs活性的8基因预后标志，并发现肿瘤富含pGSCs标志的患者临床结果不佳。我们的研究突出了原发性GSCs的异质性及其与肿瘤发展和临床结果的相关性，为GBM治疗提供了潜在的靶点。© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Glioblastoma stem cells (GSCs) contributed to the progression, treatment resistance and relapse of glioblastoma (GBM). However, current researches on GSCs were performed usually outside the human tumor microenvironment, ignoring the importance of the cellular states of primary GSCs. In this study, we leveraged single-cell transcriptome sequencing data of six independent GBM cohorts from public databases, and combined lineage and stemness features to identify primary GSCs. We dissected the cell states of GSCs and correlated them with the clinical outcomes of patients. As a result, we constructed a cellular hierarchy where GSCs resided at the center. In addition, we identified and characterized two different and recurrent GSCs subpopulations: proliferative GSCs (pGSCs) and quiescent GSCs (qGSCs). The pGSCs showed high cell cycle activity, indicating rapid cell division, while qGSCs showed a quiescent state. Then we traced the processes of tumor development by pseudo-time analysis and tumor phylogeny, and found that GSCs accumulated throughout the whole tumor development period. During the process, pGSCs mainly contributed to the early stage and qGSCs were enriched in the later stage. Finally, we constructed an 8-gene prognostic signature reflecting pGSCs activity and found that patients whose tumors were enriched for the pGSC signature had poor clinical outcomes. Our study highlights the primary GSCs heterogeneity and its correlation to tumor development and clinical outcomes, providing the potential targets for GBM treatment.© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.