The Notch Delta-4 ligand helps to maintain the quiescence and the short-term reconstitutive potential of Haematopoietic Progenitor Cells through activation of a key gene network: Delta-4/Notch pathway retains the HPCs potential

International audienceUnderstanding the role of Notch and its ligands within the different bone marrow niches could shed light on the mechanisms regulating haematopoietic progenitor cells (HPCs) maintenance and self renewal. Here, we report that murine bone marrow HPCs activation by the vascular Notch Delta4 ligand maintains a significant proportion of cells specifically in the G0 state. Furthermore, Delta4/Notch pathway limits significantly the loss of the in vivo short-term reconstitutive potential upon transplantation of Delta-4 activated HPCs into lethally irradiated recipient mice. Both effects are directly correlated with the decrease of cell cycle genes transcription such as CYCLIN-D1, − D2, and -D3, and the upregulation of stemness related genes transcription such as BMI1, GATA2, HOXB4 and C-MYC. In addition, the transcriptional screening also highlights new downstream post-transcriptional factors, named PUMILIO1 and − 2, as part of the stem signature associated with the Delta4/Notch signalling pathway

Comparative In Vitro Study of Various α2-Adrenoreceptor Agonist Drugs for Ticagrelor Reversal

Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y12 receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor through stimulation of α2A-adrenoreceptors. It subsequently inhibits cyclic adenosine monophosphate (cAMP) pathway and PI3K signaling. However, since epinephrine may expose a patient to deleterious hemodynamic effects, we hypothesized that other α2-adrenoreceptor agonist drugs used in clinical practice with fewer side effects could reverse the antiplatelet effects of ticagrelor. We compared in vitro the efficacy of clonidine, dexmedetomidine, brimonidine, and norepinephrine with epinephrine to restore ADP- and PAR-1-AP-induced washed platelet aggregation inhibited by ticagrelor, as well as resulting platelet cAMP levels. In ticagrelor-free samples, none of the α2-adrenoreceptor agonists induced aggregation by itself but all of them potentiated ADP-induced aggregation. Compared with epinephrine, norepinephrine, and brimonidine partially restored ADP- and fully restored PAR-1-AP-induced aggregation inhibited by ticagrelor while clonidine and dexmedetomidine were ineffective. Indeed, this lack of effect resulted from a lower decrease in cAMP concentration elicited by these partial α2-adrenoreceptor agonists, clonidine, and dexmedetomidine, compared with full α2-agonists. Our results support the development of specific full and systemic α2-adrenoreceptor agonists for ticagrelor reversal

Terminal Platelet Production is Regulated by Von Willebrand Factor

<div><p>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, <i>Vwf ^−/−</i> 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 <i>Vwf ^−/−</i> and <i>Vwf^+/+</i> 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 <i>Vwf^−/−</i> mice than from <i>Vwf^+/+</i> mice, suggesting a role of VWF <i>in vivo</i>. 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 <i>Vwf^−/−</i> 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 <i>in vivo</i> as regulator of bloodstream thrombopoiesis.</p></div

Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics

International audienceWe 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. Platelets are small anucleate cells that circulate in blood and are responsible for the arrest of bleeding. Platelets are formed by fragmentation of larger cells called megakaryocytes (MKs). Thrombocytopenia (insufficient platelet count) is a major condition, often requiring platelet transfusions. High collection costs, donor shortage, immuno-genicity, risk of contamination and storage issues represent the main limits of this therapeutic approach. People have tried to make artificial platelets but these objects seem promising for drug targeting rather than for therap

The core PCP protein Prickle2 regulates axon number and AIS maturation by binding to AnkG and modulating microtubule bundling

International audienceCore planar cell polarity (PCP) genes, which are involved in various neurodevelopmental disorders such as neural tube closure, epilepsy, and autism spectrum disorder, have poorly defined molecular signatures in neurons, mostly synapse-centric. Here, we show that the core PCP protein Prickle-like protein 2 (Prickle2) controls neuronal polarity and is a previously unidentified member of the axonal initial segment (AIS) proteome. We found that Prickle2 is present and colocalizes with AnkG480, the AIS master organizer, in the earliest stages of axonal specification and AIS formation. Furthermore, by binding to and regulating AnkG480, Prickle2 modulates its ability to bundle microtubules, a crucial mechanism for establishing neuronal polarity and AIS formation. Prickle2 depletion alters cytoskeleton organization, and Prickle2 levels determine both axon number and AIS maturation. Last, early Prickle2 depletion produces impaired action potential firing

Three-Dimensional Environment Sustains Hematopoietic Stem Cell Differentiation into Platelet-Producing Megakaryocytes

<div><p>Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve <i>in vitro</i> 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.</p></div

The core PCP protein Prickle2 regulates axon number and AIS maturation by binding to AnkG and modulating microtubule bundling

Core planar cell polarity (PCP) genes, which are involved in various neurodevelopmental disorders such as neural tube closure, epilepsy, and autism spectrum disorder, have poorly defined molecular signatures in neurons, mostly synapse-centric. Here, we show that the core PCP protein Prickle-like protein 2 (Prickle2) controls neuronal polarity and is a previously unidentified member of the axonal initial segment (AIS) proteome. We found that Prickle2 is present and colocalizes with AnkG480, the AIS master organizer, in the earliest stages of axonal specification and AIS formation. Furthermore, by binding to and regulating AnkG480, Prickle2 modulates its ability to bundle microtubules, a crucial mechanism for establishing neuronal polarity and AIS formation. Prickle2 depletion alters cytoskeleton organization, and Prickle2 levels determine both axon number and AIS maturation. Last, early Prickle2 depletion produces impaired action potential firing

Cell morphology in 3D and in liquid culture.

<p><b>(A)</b> Cell proliferation 12 days after cell seeding, relative to the initial cell number/scaffold. <b>(B)</b> Cell proliferation and differentiation in 3D between day 2 and day 36. <b>(C)</b> Cell proliferation and differentiation in liquid culture on days 2, 12 and 16. All images were acquired using the Axiovert 135 transmission optical microscope with 20X Plasdic magnification. Scale bar = 20 μm.</p

In situ characterization of differentiation markers and ploidy of mature MK.

<p><b>(A)</b> Immunofluorescence staining of CD41 (green) and CD42b (red) cells growing inside pores of 3D (I) and in liquid culture (II). Nuclear staining of cell grown in 3D (III) and liquid culture (IV) with YOYO-1 marker (white). All images were acquired 12 days after seeding using the Leica 510 confocal microscope with 40X Plan-NeoFluar objective lens. Scale bar = 10 μm. <b>(B)</b> Representative flow cytometry ploidy analysis of CD41⁺/CD42b⁺ UCB cells from 3D and liquid culture, 11 days after seeding. <b>(C)</b> Ploidy analysis of CD41⁺/CD42b⁺ UCB cells in 3D (black bars) compared to liquid culture (white bars), 11 days after seeding. Data are means ± SEM of 3 independent experiments. *p<0.05. Abbreviation: UCB, umbilical cord blood.</p

Characterization of MK differentiation as a function of time.

<p><b>(A, C, E)</b> Frequency of non-megakaryocytic cells (CD41^-/CD42b^-), MK precursors (CD41⁺/CD42b^-) and mature MK (CD41⁺/CD42b⁺) in 3D (closed circles, dotted lines) and liquid culture (open squares, full lines) between day 6 and day 36. Data are means ± SEM of 3 independent experiments.*p<0.05. In 3D, late time points (D23 and D36) were compared to day 6. <b>(B)</b> Histogram representation of total CD41^-/CD42b^- cell number in 3D (black bars) and liquid culture (white bars) at different days of culture. <b>(D)</b> Histogram representation of total CD41⁺/CD42b^- cell number in 3D (black bars) and liquid culture (white bars) at different days of culture. <b>(F)</b> Histogram representation of total CD41⁺/CD42b⁺ cell number in 3D (black bars) and liquid culture (white bars) at different days of culture. Total cell number of each MK population was determined by multiplying the total cell number by the frequency of each MK population. Data are means ± SEM of 3 independent experiments. In 3D, late time points (D23 and D36) were compared to day 7. *p<0.05. <b>(G)</b> CD41/CD34 dot plots of one representative experiment of 3 independent experiments in 3D and liquid culture on days 9, 16 and 23. Abbreviation: TCN, total cell number.</p