Identification of New Megakaryocytic Subpopulations in Mouse Bone Marrow.

Platelets (PLT) are produced from megakaryocytes (Mks) via proplatelet formation (PPF). However, the molecular mechanisms from Mks to PPF are not clearly elucidated, because the maturational steps of the Mks in bone marrow (BM) are not analyzed in detail. Until now, mouse Mks have been only isolated as acetylcholinesterase (AchE) positive cells and they are understood as well maturated population. In this study, we found the presence of different megakaryocytic subpopulations in BM by flowcytometry. To isolate the Mks, first we depleted lineage marker (CD4, CD8a, CD11b, B220, CD71, CD90, TER119, Gr-1, F4/80, 7/4) positive cells from BM cells of BALB/c mice. The analysis of the expression-pattern of CD41, CD45 and CD61 in the lineage negative (Lin⁻) cells showed the presence of two types of megakaryocytic subpopulations. By sorting, they were identified as Lin⁻CD41⁺/45⁺/61⁺ cells (AchE negative) and Lin⁻CD41⁺⁺/45⁺/61⁺⁺ cells (partially AchE positive), respectively. To assess the maturational stages of the subpopulations, each population was cultured with 10ng/mL of TPO followed by counting of PPF and PLT production. Both PPF and PLT production were observed in Lin⁻CD41⁺/45⁺/61⁺ cells later than those in Lin⁻CD41⁺⁺/45⁺/61⁺⁺ cells. On the other hand, CFU-Mk was scarcely detected in each subpopulation. The results indicate that both populations are the committed megakaryocytes and Lin⁻CD41⁺/45⁺/61⁺ cells are more immature population than Lin⁻CD41⁺⁺/45⁺/61⁺⁺ cells. Then to characterize these subpopulations in detail, gene expression profiling was performed against four-megakaryocytic lineage-populations, Lin⁻CD41⁻Thy1^lowc-kit⁺ cells as stem/progenitor, Lin⁻CD41⁺/45⁺/61⁺ cells, Lin⁻CD41⁺⁺/45⁺/61⁺⁺ cells and PLT using GeneChipU74 or RT-PCR. These analyses revealed that many PLT-specific genes including gpIb/IX, P-selectin, thrombin-R and ADP-R were already expressed on Lin⁻CD41⁺/45⁺/61⁺ cells but less than Lin⁻CD41⁺⁺/45⁺/61⁺⁺ cells. Especially, beta-1 tubulin that is necessary for PPF was only expressed on Lin⁻CD41⁺⁺/45⁺/61⁺⁺ cells. On the contrary, the expression of c-kit gene was gradually decreasing from stem/progenitor fraction to PLT. In conclusion, we succeeded in the isolation of new subpopulations distinguishable between immature Mks and more matured Mks beginning to prepare PLT. The present finding can contribute to elucidate the molecular mechanisms during terminal maturation.