Uncovering Transcriptome Characteristics in Bone Marrow Hematopoiesis in Immune Thrombocytopenia at Single-Cell Resolution

Abstract

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we hypothesized that bone marrow (BM) CD34+ hematopoietic stem and progenitor cells (HSPCs) participated in the defective megakaryogenesis in ITP patients, and we performed single-cell RNA-seq (scRNA-seq) of BM CD34+ HSPCs to reveal the overall transcriptome alterations in HSPCs in ITP. Dimensional reduction using uniform manifold approximation and projection (UMAP) showed the effective integration of the datasets from different samples. We manually annotated the cell clusters into 17 different cell types with distinct gene expression patterns. By performing differential expression analysis, enrichment analysis, trajectory analysis, transcriptional regulatory network analysis and cell-cell interaction analysis, we found that gene expression, transcriptional regulatory networks and cell-cell interactions varied in HSPCs of ITP, particularly in a pre-B cell population and natural killer/T cell progenitors, highlighting the selective immune aberration associated with defective megakaryopoiesis in ITP. Differentially expressed gene (DEG) analysis indicated that the top upregulated genes in a megakaryocytic progenitors (MkP) population of ITP were associated with erythropoiesis, suggesting a bias toward erythrocytes in megakaryopoiesis in ITP. Moreover, Flow cytometry confirmed that the number of CD9+ cells from CD34+ HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+ CD34+ HSPCs from healthy controls tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients. The percentage of megakaryocytes differentiated from CD9+CD34+ HSPCs was higher than that of the CD9- counterparts from healthy controls, whereas in ITP patients, the percentage decreased to only 1/4th of that in the healthy controls and was comparable to that from the CD9- HSPCs, indicating that differentiation of CD9+ HSPCs toward the megakaryopoietic lineage was impaired in ITP. Further analysis revealed the universality of their heterogeneity and provided an analytical basis for the study of megakaryocyte-related diseases, including ITP. Enrichment analysis confirmed the definition of MkP in our ITP integrated dataset. To further analyze the transcriptional difference between ITP and HC, we pooled MkP from all samples. The expression of several molecular features of MkP was visualized, and seven transcriptionally heterogeneous subpopulations of MkP were identified. We then assessed the expression of marker genes in each subpopulation and characterized the gene sets enriched in different clusters, which showed pronounced heterogeneity within MkP, consistent with the results of previous analyses of MkP/Mk from multi-hematopoietic tissues. We evaluated ITP-associated DEGs in each MkP subcluster, and we found that most of these DEGs were subtype-specific, indicative of the subtype-specific effects of the disorder. In summary, this study dissects the significant heterogeneity and disease characteristics of BM CD34+ HSPCs from ITP patients at single-cell resolution, revealing new insights regarding the pathophysiology of ITP.