Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis

Abstract

Megakaryocytes (MKs), the platelet progenitor cell, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually-isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N), performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within three distinct subpopulations possessing gene signatures related to platelet-generation, HSC niche interaction, and inflammatory responses, respectively. In situ immunostaining of mouse BM demonstrated that platelet-generation and HSC-niche related MKs were physically in close proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under the blood shear forces, were predominantly formed on platelet-generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented approximately 5% of total MKs in the BM. These MKs could specifically respond to pathogen infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a pre-existing PU.1 and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance, as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable to engulf and digest bacteria and to stimulate T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.

The Heterogeneity of Megakaryocytes and Platelets and Implications for Ex Vivo Platelet Generation

“…In comparison with embryonic MKs, adult MKs in the BM display more striking hemostatic features, probably indicative of the high potential of platelet generation based on our observations and those from others (Figure 1 , top). 7 , 18 , 19 Our findings are in keeping with recent reports that mouse lung MKs exhibit unique immune characteristics that are distinct from mouse FL and BM MKs and skew them toward a role in microbial surveillance and antigen presentation in an MHC II‐dependent manner. 22 Together, these results are in support of the emerging hypothesis that MKs exert organ‐specific functions.…”

“…Recently, we and others developed new methodology to isolate MKs in vivo, enabling us to unveil distinct MK subpopulations and thereby providing new mechanistic insights into the functional heterogeneity of human MKs. 7 , 18 , 19 This review summarizes the current findings of functional diversity of MKs and platelets, the cellular heterogeneity of MKs, and ontogenic features of MKs and platelets. We also discuss the implications of cellular heterogeneity in platelet production from human pluripotent stem cells (hPSCs) and the future clinical applications.…”

hGATA1 Under the Control of a μLCR/β-Globin Promoter Rescues the Erythroid but Not the Megakaryocytic Phenotype Induced by the Gata1low Mutation in Mice

“…In addition, megakaryocyte-restricted expression of the driver mutations is necessary and sufficient to induce myelofibrosis even if the hematopoietic stem cells are normal ( Jeremy Wen et al, 2015 ; Zhan et al, 2016 ). This apparent contradiction has been recently clarified by new exciting single cell profiling data indicating that the megakaryocytes in the bone marrow are a mixture of three populations, each one exerting a different function ( Psaila et al, 2020 ; Yeung et al, 2020 ; Migliaccio and Hoffman, 2021 ; Sun et al, 2021 ; Wang et al, 2021 ). In fact, in addition to megakaryocytes poised to form platelets, the hematopoietic stem cells also generate megakaryocytes poised to exert immune-functions in the lungs ( Pariser et al, 2021 ) or niche-functions locally, by secreting collagen and other extracellular matrix proteins ( Malara et al, 2014 ).…”

A Novel Megakaryocyte Subpopulation Poised to Exert the Function of HSC Niche as Possible Driver of Myelofibrosis

“…Several laboratories are currently using single cell profiling and lineage tracking of megakaryocytes purified for primary sources to generate a comprehensive model for the progression and routes of megakaryocyte maturation in vivo. These studies have identified that in addition to cells poised to generate platelets (Plt, Plt-poised megakaryocytes), the hematopoietic stem cells (HSC) in the bone marrow generate megakaryocytes that are poised to exert immune (immune-poised) function in the lung and niche (niche-poised) functions in organs in need of tissue repair [ 18 , 19 , 20 , 21 ] ( Figure 1 ). The maturation of both Plt-poised and immune-poised megakaryocytes is intrinsically controlled by the balance between the transcription factors GATA1 (high expression) and GATA2 (low expression) [ 18 , 19 ] while that of niche poised cells is driven by low levels of GATA1 and high levels of GATA2 .…”

“…Based on distinctive morphological markers, megakaryocyte precursors are divided into four classes of progressively more mature cells: the pro-megakaryoblast (Stage 0), the megakaryoblast (Stage I), the promegakaryocyte (Stage II), and finally, the mature megakaryocyte (Stage III), which is capable of releasing platelets (Plt) [ 31 ]. Recent single cell RNAsequencing (RNAseq) data and lineage tracking analyses indicate that Stage I megakaryocytes may generate, in addition to platelet-poised megakaryocytes, cells that are poised to exert either immune or niche functions [ 18 , 19 , 20 , 21 ]. Since the morphology of the megakaryocytes poised to exert the different functions is the same, morphological analyses alone may not determine the relative frequency of the three populations in various tissues.…”