We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.

IntroductionThe Notch signaling pathway guides cell-fate decisions in multiple developmental processes. 1,2 Intercellular communications that control the developmental fate of multipotent cells are mediated by the Notch family of transmembrane receptors in several invertebrate and vertebrate developmental systems. The Notch proteins are single-pass receptors that are activated by the Delta (or Delta-like) and Jagged/Serrate families of membrane-bound ligands. 3 To date, 4 human Notch genes have been identified and all are expressed on hematopoietic cells. 2 Also, 5 human Notch ligands, Delta-like1/3/4, and Jagged1/2 were identified, and all were shown to bind to Notch-1, Notch-2, and Notch-4. 4,5 Notch receptors are matured in the secretory pathway and presented at the cell surface as heterodimeric molecules. Interaction with ligands leads to 2 additional proteolytic cleavages that liberate the Notch intracellular domain (NICD) from the plasma membrane. NICD enters the nucleus, where it interacts with the DNA-binding protein, CSL (C-promoter binding factor [CBF]-1, Suppressor of Hairless, LAG-1; also known as recombination signal-binding protein Jk [RBP-Jk]). 6 In the absence of NICD, CSL represses transcription through interactions with a corepressor complex, containing a histone deacetylase. Upon entering the nucleus, NICD displaces the corepressor complex from CSL and replaces it with a transcriptional activation complex that includes NICD, Mastermind (MAML-1), the histone acetyltransferase p300, and, possibly, PCAF p300/CBP (cyclic AMP response element-binding protein [CREB]-binding protein)-associated factor. Notch signaling, thus, converts CSL from a repressor to an activator, leading to the transcription of target genes. The target genes include members of the Hes and HRT/ HERP/Hey families of transcriptional repressors; therefore, Notch signaling is often viewed as a transcription cascade.The classical view holds that Notch signaling controls the balance between the progenitor pool and its differentiating progeny and thus is involved in the maintenance of stem cell fate. 1,2 In fact, a number of studies have provided evidence that ligand-induced Notch signaling favored hematopoietic stem cell (HSC) selfrenewal, increased the numbers of progenitors, and promoted HSC survival. Moreover, Notch signaling may be instructive for differentiation toward a particular fate. It plays a crucial role in the hematopoietic system, especially in the regulation of the T-cell lymphoid lineage commitment 7-9 and in late stages of B-cell development. 10 Because of the key role of Notch signaling in supporting early T-cell differentiation, it was generally established that Notch concomitantly negatively regulates myeloid lineage development. The megakaryocytic and erythroid lineages are extremely linked, because they share a common bipotent progenitor called the MEP (MK/erythroid progenitor). The role of Notch in megakaryocytic and erythroid development remains a matter of debate. While some data report that Notch signaling...

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng +/−) mice compared to Eng +/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-017-2694-7) contains supplementary material, which is available to authorized users.

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Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.

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It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s−1) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf −/− mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf −/− and Vwf +/+ blood, exposed to VWF at 1800 s−1 in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf−/− mice than from Vwf+/+ mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf−/− mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s−1 on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.