Identification of the Human Unipotent Megakaryocyte Progenitor, and Its Pathophysiological Roles in Human Thrombopoietic Disorders
Authors: Miyawaki Kohta , Iwasaki Hiromi , Akashi Koichi 
Published: October 2019

Abstract
In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP), which resides downstream of common myeloid progenitor (CMP). However, the definition of unipotent Meg progenitor (MegP) is still controversial.
To shed light on the heterogeneity of hematopoietic stem/progenitor cells (HSPCs), we analyzed the expression level of 96 lineage-specific genes in a single cell. While most of MEPs exclusively expressed erythroid- but not Meg-specific genes, a fraction of CMP cells (6.25%) showed distinct and exclusive expression pattern of platelet or Meg-specific genes. Among them were the cell surface molecules including CD41, the contents of alpha-granules of platelets and Meg-lineage specific transcription factors. Based on these data, we hypothesized that MegP might exist within the CD41+ CMP fraction.
In fact, a fraction (7.9 ± 3.2 %) of CMPs expressed significant level of CD41 in FACS analysis, but none of the other CD34+ cells including hematopoietic stem cells (HSCs), MEPs and granulocyte-macrophage progenitors (GMPs) expressed CD41. Purified CD41+CMPs exhibited myeloblast-like immature morphological characteristics. Interestingly, cells with 4N-nuclei were exclusively found in CD41+CMPs (1%), indicating the initiation of endomitosis and polyploidization process has already taken place at this early stage of hematopoiesis.
A conventional in vitro culture condition revealed that CD41+CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41-CMPs generated all types of myeloid colonies. To analyze Meg-lineage potential, we cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. The majority of single CD41+CMPs gave rise to pure megakaryocyte colonies.
In addition, the novel immunodeficient mouse strain, which was recently developed by us and could support human Meg-lineage hematopoiesis much more than any strains available today, enabled us to evaluate in vivo differentiation potential of lineage-restricted progenitors. As a result, CD41+CMPs showed unipotent and robust Meg-lineage differentiation potential compared to MEPs.
In order to elucidate gene expression profiling of CD41+CMPs more precisely, we performed cDNA microarray experiment. GSEA (Gene Set Enrichment Analysis) revealed that CD41+CMPs up-regulated Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulated erythroid and GM lineage-affiliated genes, clearly reflecting their lineage potentials.
We also evaluated lineal relationship of these populations in vitro. While MEP did not give rise to CD41+CMPs, CD41-CMPs successfully generated MegPs, suggesting that CD41+CMPs could be the progeny of CD41-CMPs.
Therefore, we defined the CD41+CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty times as strong as that of MEPs on per-cell-basis, indicating that this population is the major source of Megs rather than MEP.
Finally, to evaluate the pathophysiological significance of MegPs in human diseases, we analyzed population frequencies of each stem/progenitor population in essential thrombocythemia (ET) patients' bone marrow. The MegP pool dramatically expanded in ET BM as compared to that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We also evaluated JAK2 mutation (V617F) burden in each stem/progenitor population by utilizing the digital PCR method with allele specific probes and the amplicon sequencing-based detection. As a result, JAK2 mutation is highly concentrated to MegP population in accordance with expansion of MegP, suggesting that this population should play an important role in pathogenesis of ET.
In summary, we have prospectively identified the unipotent MegP population, which was endowed with robust Meg potential and emerged directly from CMPs bypassing MEPs. The newly-identified MegPs robustly contribute to physiological and pathological human megakaryopoiesis including ET.

Disclosures
No relevant conflicts of interest to declare.