Exposure of human megakaryocytes to high shear rates accelerates platelet production

Abstract

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation. (Blood. 2009;114:1875-1883) IntroductionMegakaryocyte (MK) differentiation is a continuous process characterized by sequential steps. MK ploidy increases through endomitosis, with parallel increase in cell size. Synthesis of storage organelles is enhanced and plasma membrane invaginates, resulting in the formation of demarcation membranes. Finally, mature MKs undergo complete cytoskeleton reorganization with microtubule involvement, to induce pseudopodial elongations called proplatelets. 1,2 Platelets are released from the tips of proplatelets that contain the organelles. 3 In static conditions, platelets are formed from mature MKs cultured in the presence of thrombopoietin (TPO). 3 MK fragmentation into platelets does not occur in the extravascular, hematopoietic space in normal bone marrow. To achieve platelet formation, MKs need to migrate into the sinusoid capillaries and mature MKs are generally located in the vicinity of sinusoids, more precisely on their abluminal side. 4 MK fragmentation occurs only in the intravascular space, and mature MKs have been identified in the blood circulation where they may interact via their surface receptors with proteins expressed on endothelial cells. [4][5][6] Adhesion of MKs to endothelial cells is essential for robust platelet production in vitro, a process that is believed to mimic what occurs in vivo when MKs interact with bone marrow endothelial cells within the vascular niche. 7 Recently, the process of proplatelet formation was examined in mouse skull bone marrow in vivo: the authors observed large MK fragments resembling imma...

Platelet bioreactor-on-a-chip

“…A major limitation of twodimensional liquid cultures has been their inability to account for three-dimensional BM composition and stiffness, directionality of proPLT extension, and proximity to venous endothelium. 15,24,25 Alternatively, intravital microscopy studies have provided physiologically accurate examples of proPLT production, however, poor resolution and limited control of the microenvironment has prohibited detailed study of how the BM microenvironment contributes to PLT release. 11,15 Nakagawa et al 50 recently published 3 bioreactor designs to produce human PLTs at scale from hiPSC-and ESC-derived MKs.…”

“…Mounting evidence that cell-cell contacts, 22,23 ECM composition 19,20 and stiffness, 21 vascular shear stress, 24,25 pO2/pH, 51,52 soluble factor interactions, 22,53 and temperature 54 contribute to proPLT formation and PLT release have suggested that recapitulating key components of BM and blood vessel microenvironments within a three-dimensional microfluidic culture system is necessary to achieve clinically significant numbers of functional human PLTs. Our microfluidic bioreactor design expands control and resolution of the microenvironment, and has allowed us to drastically improve the time to PLT release (from 18 hours down to 2 hours) and more For personal use only.…”

“…This results in yields of 10 122 PLTs per CD34 1 cord blood-derived or embryonic stem cell-derived MK, 18 which are themselves of limited availability, constituting a significant bottleneck in the ex vivo production of a PLT transfusion unit. Although second-generation cell culture approaches have provided further insight into the physiological drivers of PLT release, they have been unable to recreate the entire BM microenvironment, exhibiting limited individual control of extracellular matrix (ECM) composition, 19,20 BM stiffness, 21 endothelial cell contacts, 22,23 and vascular shear stresses, 24,25 and have been unsuccessful in synchronizing proPLT production, resulting in nonuniform PLT release over a period of 6 to 8 days. 26 Moreover, the inability to reproduce the BM microenvironment ex vivo, and resolve physiological proPLT extension and release by high-resolution live-cell microscopy, has significantly hampered efforts to study the cytoskeletal and signaling mechanics of PLT production.…”

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

“…The CD41 1 subpopulation is short lived, preactivated, weakly reactive, and underincorporated into thrombi. EV-PLPs generated in bioreactors [51][52][53][54][55] have been described and such high-throughput devices are clearly needed to achieve largescale EV-platelet production. Like infused EV-megakaryocytes, they would have the advantage of producing mostly young platelets.…”

A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis

“…Blood shear stress contributes to the shedding of PPs Dunois-Lardé et al, 2009;Thon et al, 2010); however, whether additional signals are required for efficient PP shedding was completely unknown. In this study, we show that hydrodynamic forces alone are not sufficient to allow the release of new platelets from MK PP extensions.…”