Exploring the regulation of megakaryocytic differentiation and platelet production by mechanical forces and cell-derived microparticles

Platelets, small anuclear blood cells, are life-saving products in clinical use but expensive due to limited source supply (from human blood donation) and short shelf life (3-5 days). Ex vivo production of platelets from mature megakaryocytes (Mks) differentiated from hematopoietic stem cells (HSCs) can provide a safe and unlimited supply of platelets for transfusions. However, the efficiencies currently associated with megakaryocytic (Mk) differentiation of HSCs, and platelet generation from Mks in vitro, are much lower compared to the corresponding in vivo processes. As a result, ex vivo platelet production is not practical. The low efficiencies are caused by the fact that some critical parameters, which control and regulate stem-cell fate and platelet biogenesis, are missing. Possible critical parameters would include stromal cells, extracellular matrix, soluble factors, and physicochemical properties of the culture environment, such as temperature, O 2 level, pH, stiffness (matrix elasticity) and shear forces. In this study, we investigated the effects of the rarely explored matrix elasticity and shear forces, as well as the previously unexplored Mk-derived microparticles (MkMPs), on Mk differentiation and platelet generation. Using a well-established human Mk cell line, matrix elasticity was shown to regulate Mk morphology and nuclear spreading on polyacrylamide hydrogels. Projection of proplatelets (PPTs), the Mk cytoplasmic extensions through which platelets are formed, and phosphatidylserine (PS) exposure, were enhanced on hydrogels with elasticities above 5.6 kPa. Nonmuscle myosin IIA, a major protein that generates contractile force in cells, was also found to be involved in matrix elasticity sensing by Mks. Fluid-flow application experiments demonstrated that shear forces are an important cues that regulates Mk differentiation and platelet generation in multifaceted ways. DNA synthesis of Mks at immature but not at mature stage was enhanced by shear force with optimal shear stress at 2.5 dyn/cm 2 within the 0-4.0 dyn/cm2 range, and Mks responded to shear stress very quickly (within 15 minutes) and maximally at 30 minutes. It has been shown that Mks undergo an apoptosis-like process to extend PPTs and release platelets. PS exposure and caspase-3 activation, two signature cellular events of cell apoptosis, were also enhanced by shear flow. Importantly, the generation of PPTs and platelet-like particles (PLPs) from mature Mks was increased by shear force by 4.1-10.8 fold, depending on the magnitude of the shear stress and exposure time, and flow-derived PLPs demonstrated better platelet functionality than those generated under static conditions. In addition, we found that flow application increased the generation of MkMPs, a type of membrane vesicles with diameter from 100-1000 nm, dramatically by 24-47 fold. The biological function of MkMPs, not previously explored in the literature, was explored in detail in this study. Coculture with MkMPs induced the Mk differentiation of HSCs without additional stimulation by thrombopoietin, the primary regulator of Mk differentiation. The resulting Mks from coculture were functional in that they were capable of projecting PPTs and synthesizing both alpha- and dense-granules, which are required for platelet function. In addition, the underlying mechanisms by which MkMPs exert their effect on HSCs were examined, and the results show that HSCs could take up MkMPs by two processes: direct membrane fusion and endocytosis. As shown by scanned electron microscopy, MkMP fusion into cells was characterized by four consecutive stages. RNase treatment of MkMPs reduced their inducing effect on HSCs, suggesting that RNA transfer from MkMPs to cells is essential for their biological function. RNA sequencing was used to explore the repertoire of RNAs carried by MkMPs and gain insight into the mechanisms underlying the biological effects of MkMPs, as well as into Mk differentiation. The results in this study support the concept that matrix elasticity, shear force and MkMPs regulate Mk differentiation and platelet biogenesis, and contribute to the understanding of the thrombopoiesis processes in vivo. They could also contribute to the design of bioreactors for producing platelets ex vivo.