OC 27.3 - Single-cell transcriptomics reveal critical roles of G6b-B in regulating the megakaryocyte maturation

Background: Platelets are produced by megakaryocytes (MKs), which differentiate from hematopoietic stem cells in the bone marrow. Although thrombopoietin receptor signaling constitutes a key pathway regulating MK differentiation, it remains unclear how other receptors regulate this process. G6b-B (encoded by Mpig6b) is an inhibitory receptor with an MK lineage-specific expression pattern. Human patients with pathogenic variants in MPIG6B, or transgenic mice lacking the receptor develop severe macrothrombocytopenia and myelofibrosis. Using bulk RNA-sequencing on MKs from Mpig6bmut mice, we recently found reduced mRNA levels suggesting that G6b-B is required for MK maturation, however the molecular mechanisms remain unknown.

Aims: To identify the role of G6b-B in regulating the MK transcriptional program.

Methods: Native bone marrow MKs of wild-type and Mpig6bmut mice were enriched using magnetic cell separation and processed for single-cell RNA sequencing (scRNA-seq) utilizing the 10x Genomics Chromium system. Data was analysed using the Seurat package. Proplatelet formation, MK maturation and different MK subsets were assessed using flow cytometry and immunofluorescence microscopy.

Results: Our scRNA-seq experiments corroborated the recently reported transcriptomic heterogeneity of MKs but the higher number of sequenced cells (~40’000 cells/genotype) yielded higher resolution. Our data show that the loss of functional G6b-B leads to an increased number of MK progenitors and a striking, 4-fold expansion of the immune-modulatory MK subtype. Considering the influence of MKs on surrounding cells, these expanded subsets could contribute to the rapid development of myelofibrosis in these mice. Furthermore, we observed a drastic reduction in the proportion of platelet-generating mature MKs, which showed pronouncedly reduced expression of MK-specific transcripts, likely contributing to the macrothrombocytopenia.

Conclusion(s): Our results highlight G6b-B as the second central receptor in MK development and provide unprecedented insights into its role in coordinating the MK transcriptional program. Loss of G6b-B results in aberrant differentiation of MK subtypes, potentially explaining both the macrothrombocytopenia and myelofibrosis phenotypes.