Identification of the Human Unipotent Megakaryocyte Progenitor, and Its Pathophysiological Roles in Human Thrombopoietic Disorders

In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP; CD34⁺ CD38⁺ IL3Ra^- CD45RA^-), which resides downstream of common myeloid progenitor (CMP; CD34⁺ CD38⁺ IL3Ra⁺ CD45RA^-). However, the definition of unipotent Meg progenitor (MegP) is still controversial and, in the hierarchical map, where Meg potential mainly resides remains unclear. Here, we report prospective isolation of unipotent MegP, which is endowed with much more robust Meg potential than MEP, and emerges directly from CMP bypassing MEP.

Because CD41 is known as a Meg lineage-specific marker, we first searched for the CD41-expressing cell population in the CD34 positive stem/progenitor fraction of healthy individuals. A fraction (7.9 ± 3.2 %) of CMPs expressed CD41, but none of the other CD34⁺ subpopulation including hematopoietic stem cells (HSCs; CD34⁺ CD38^- CD45RA^-), MEPs and granulocyte-macrophage progenitors (GMPs; CD34⁺ CD38⁺ IL3Ra⁺ CD45RA⁺) expressed significant levels of CD41. Purified CD41⁺ CMPs retained myeloblast-like immature morphological characteristics with large and round nucleus and basophilic cytoplasm, closely resembling those of CD41^- CMPs and more immature HSCs. Interestingly, cells with immature 4N-nuclei were exclusively found in CD41⁺ CMPs (about 1%), indicating that a minor fraction of this population is initiating endomitosis and polyploidization process, an important characteristic of megakaryoblasts. A conventional In vitro culture condition with fetal bovine serum and a myeloerythroid cytokine cocktail revealed that CD41⁺ CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41^- CMPs generated all types of myeloid colonies. These unexpected observations led us to hypothesize that CD41⁺ CMPs were committed to the Meg lineage. We then cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. In this condition, the majority of single CD41⁺ CMPs gave rise to pure megakaryocyte colonies, without forming any other myeloerythroid colonies. In addition, in cDNA microarray analysis, CD41⁺ CMPs possessed a distinct gene expression profile with up-regulation of Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulation of erythroid and GM lineage-affiliated genes, reflecting their lineage potential. 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 (672 ± 232 vs. 20 ± 6.2 colonies per 1000 cells) and MegP-derived Meg colonies accounted for more than ninety percent of those from whole CD34⁺ CD38⁺ progenitor populations, indicating that this population is the major source of megakaryocytes and platelets production rather than MEP. The MegP population was found in 7 day culture of CD41^- CMPs but not of MEPs, suggesting that MegPs are downstream of CMPs, but are independent of MEP. More importantly, MegP pool dramatically expanded in essential thrombocythemia (ET) patients’ bone marrow, constituting three times larger part of CMP than that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We are currently further analyzing ET patients’ bone marrow to obtain data regarding V617F JAK2 mutation frequency, for example. Thus, the newly-identified MegP robustly contributes to physiological and pathological human megakaryopoiesis, thus could be a therapeutic target in various thrombopoietic disorders.