Single-Cell RNA-Seq Analysis of Native Murine Megakaryocytes from Young and Old Mice Reveals Significant Metabolic and Mitochondrial Differences throughout Megakaryocyte Development

Traditional transcriptome analysis of megakaryocytes relies on ex-vivo culture and expansion of hematopoietic cells in thrombopoietin-rich media for several days prior to RNA isolation for bulk RNA-seq analysis. This approach has been widely used, however, it is not clear as to what extend this ex vivo expansion of megakaryocytes represents the transcriptome landscape of megakaryocytes at the time of harvest from the bone marrow. In order to get a more detailed megakaryocytic transcriptome landscape immediately after bone marrow harvest, we have optimized a method to perform single cell RNA-seq analysis of native freshly isolated bone marrow megakaryocytes. This was accomplished throughout a series of steps to enrich murine bone marrow for megakaryocytes followed by cell capture using the 10X Genomics platform. In order to assess the transcriptome of native murine megakaryocytes and the effect of age on transcriptional signatures we performed single cell RNA seq analysis of 3 young mice (age 2-3 months old) and 3 old mice (>18 months old). Bioinformatics analyses identified seven transcriptionally different clusters of cells that represent the megakaryocyte ploidy status (Figures 1 and 2). Within these seven regions, 3 of them appear to represent late states of maturation (regions 5, 6 and 7) and possibly, the pro-platelet forming groups of cells due to the elevated expression of megakaryocyte transcripts such as Vwf, Pf4, Itga2b, Itgb3, Gata-1 and Mpl. Ingenuity pathway analysis (IPA) shows that the top three differentially regulated pathways between megakaryocytes from young and old mice are: a) protein ubiquitination; b) mitochondrial dysfunction and; c) oxidative phosphorylation. Furthermore, we validated the RNA-seq analysis at the protein level by measuring ALDHA1 from platelet lysates, the top differentially expressed transcript between groups. Finally, platelets from old mice have a distinctive mitochondrial phenotype characterized by elevated mitochondrial mass and significantly elevated oxygen consumption upon activation by thrombin, features that might be directly contributing to platelet hyperreactivity of aging. In summary, we present a novel methodology to study the transcriptional profile of native megakaryocytes. This initial approach highlights that metabolic and mitochondrial pathways appear to be important modulators of megakaryocyte and platelet development and function.

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