Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia. Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia

Purpose and Methods: Immune thrombocytopenia (ITP), an autoimmune disorder, is mainly caused by megakaryocyte dysfunction, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow (BM) CD34+ hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Results: Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in natural killer/T cell progenitors and a pre-B cell subset, highlighting the selective immune aberratixon associated with defective megakaryopoiesis in ITP. CD9 can be used to enrich megakaryocyte-biased HSPCs, and CD9+Lin−CD34+CD45RA− HSPCs have the potential to be novel diagnostic and therapeutic targets in ITP. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. Conclusions: This study comprehensively dissects defective hematopoiesis and provides novel therapeutic targets for ITP. Overall design: We performed scRNA-seq of BM CD34+ HSPCs from four newly diagnosed treatment-naïve ITP patients and four healthy donors on the 10X Chromium platform to determine the megakaryopoiesis defects in ITP.

### 研究目的与方法
免疫性血小板减少症（immune thrombocytopenia, ITP）是一类自身免疫性疾病，其发病主要与巨核细胞功能异常相关，但具体潜在机制尚未阐明。本研究针对骨髓（bone marrow, BM）CD34+造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）开展单细胞转录组测序，以解析ITP患者巨核细胞生成过程中的缺陷。

### 研究结果
ITP患者的造血干祖细胞中，基因表达模式、细胞间相互作用及转录调控网络均存在异常，尤其在自然杀伤/T细胞祖细胞与前B细胞亚群中表现更为显著，这提示ITP患者的巨核细胞生成缺陷与选择性免疫异常紧密相关。研究发现CD9可用于富集偏向巨核细胞的造血干祖细胞，且CD9+Lin−CD34+CD45RA−造血干祖细胞有望成为ITP全新的诊断与治疗靶点。进一步分析显示，巨核细胞祖细胞（megakaryocytic progenitors, MkP）可分为7个具有不同基因表达谱及功能的亚群；与ITP相关的差异表达基因（differentially expressed genes, DEGs）具有MkP亚群特异性，且多数差异表达基因集中于同时具备免疫调节与血小板生成双重功能的亚群中。

### 研究结论
本研究全面解析了ITP患者的造血功能缺陷，为ITP提供了全新的治疗靶点。

### 实验整体设计
本研究借助10X Chromium平台，对4名初诊未接受治疗的ITP患者及4名健康供者的骨髓CD34+造血干祖细胞开展单细胞RNA测序（scRNA-seq），以此明确ITP患者的巨核细胞生成缺陷。