Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo.

Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

GPIbα-filamin A interaction regulates megakaryocyte localization and budding during platelet biogenesis.

ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.

Development of an efficient, ready to use, blood platelet-release device based on two new flow regime parameters: The periodic hydrodynamic loading and the shear stress accumulation.

In vitro production of blood platelets for transfusion purposes is gaining interest. While platelet production is now possible on a laboratory scale, the challenge is to move towards industrial production. Attaining this goal calls for the development of platelet release devices capable of producing large quantities of platelets. To this end, we have developed a continuous-flow platelet release device composed of five spherical chambers each containing two calibrated cones placed in a staggered configuration. Following perfusion of proplatelet-bearing cultured megakaryocytes, the device achieves a high yield of about 100 bona-fide platelets/megakaryocyte, at a flow rate of ∼80 mL/min. Performances and operating conditions comply with the requirements of large-scale platelet production. Moreover, this device enabled an in-depth analysis of the flow regimes through Computational Fluid Dynamics (CFD). This revealed two new universal parameters to be taken into account for an optimal platelet release: i.e. a periodic hydrodynamic load and a sufficient accumulation of shear stress. An efficient 16 Pa.s shear stress accumulation is obtained in our system at a flow rate of 80 mL/min.

Matrix stiffness controls megakaryocyte adhesion, fibronectin fibrillogenesis, and proplatelet formation through Itgß3.

Megakaryocytes (MKs) are the precursor cells of platelets, located in the bone marrow (BM). Once mature, they extend elongated projections named proplatelets through sinusoid vessels, emerging from the marrow stroma into the circulating blood. Not all signals from the microenvironment that regulate proplatelet formation are understood, particularly those from the BM biomechanics. We sought to investigate how MKs perceive and adapt to modifications of the stiffness of their environment. Although the BM is one of the softest tissue of the body, its rigidification results from excess fibronectin (FN), and other matrix protein deposition occur upon myelofibrosis. Here, we have shown that mouse MKs are able to detect the stiffness of a FN-coated substrate and adapt their morphology accordingly. Using a polydimethylsiloxane substrate with stiffness varying from physiological to pathological marrow, we found that a stiff matrix favors spreading, intracellular contractility, and FN fibrils assembly at the expense of proplatelet formation. Itgb3, but not Itgb1, is required for stiffness sensing, whereas both integrins are involved in fibrils assembly. In contrast, soft substrates promote proplatelet formation in an Itgb3-dependent manner, consistent with the ex vivo decrease in proplatelet formation and the in vivo decrease in platelet number in Itgb3-deficient mice. Our findings demonstrate the importance of environmental stiffness for MK functions with potential pathophysiological implications during pathologies that deregulate FN deposition and modulate stiffness in the marrow.

The tubulin code in platelet biogenesis.

Blood platelets are small non-nucleated cellular fragments that prevent and stop hemorrhages. They are produced in the bone marrow by megakaryocytes through megakaryopoiesis. This intricate process involves profound microtubule rearrangements culminating in the formation of a unique circular sub-membranous microtubule array, the marginal band, which supports the typical disc-shaped morphology of platelets. Mechanistically, these processes are thought to be controlled by a specific tubulin code. In this review, we summarize the current knowledge on the key isotypes, notably ß1-, α4A- and α8-tubulin, and putative post-translational modifications, involved in platelet and marginal band formation. Additionally, we provide a provisional list of microtubule-associated proteins (MAPs) involved in these processes and a survey of tubulin variants identified in patients presenting defective platelet production. A comprehensive characterization of the platelet tubulin code and the identification of essential MAPs may be expected in the near future to shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion.

Loss of α4A- and ß1-tubulins leads to severe platelet spherocytosis and strongly impairs hemostasis in mice.

Native circulating blood platelets present with a discoid flat morphology maintained by a submembranous peripheral ring of microtubules, named marginal band. The functional importance of this particular shape is still debated, but it was initially hypothesized to facilitate platelet interaction with the injured vessel wall and to contribute to hemostasis. The importance of the platelet discoid morphology has since been questioned on the absence of clear bleeding tendency in mice lacking the platelet-specific ß1-tubulin isotype, which exhibits platelets with a thinner marginal band and an ovoid shape. Here, we generated a mouse model inactivated for ß1-tubulin and α4A-tubulin, an α-tubulin isotype strongly enriched in platelets. These mice present with fully spherical platelets completely devoid of a marginal band. In contrast to the single knockouts, the double deletion resulted in a severe bleeding defect in a tail-clipping assay, which was not corrected by increasing the platelet count to normal values by the thrombopoietin-analog romiplostim. In vivo, thrombus formation was almost abolished in a ferric chloride-injury model, with only a thin layer of loosely packed platelets, and mice were protected against death in a model of thromboembolism. In vitro, platelets adhered less efficiently and formed smaller-sized and loosely assembled aggregates when perfused over von Willebrand factor and collagen matrices. In conclusion, this study shows that blood platelets require 2 unique α- and ß-tubulin isotypes to acquire their characteristic discoid morphology. Lack of these 2 isotypes has a deleterious effect on flow-dependent aggregate formation and stability, leading to a severe bleeding disorder.

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti-GPIbß antibody derivative.

BACKGROUND: Glycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb-IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R-IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb-IX-mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild-type mice. Previously, an anti-GPIbß antibody RAM.1 was developed. RAM.1 inhibits GPIbα-dependent platelet signaling and activation. Further, RAM.1 also inhibits anti-GPIbα antibody-mediated filopodia formation. OBJECTIVE: To investigate whether RAM.1 can ameliorate trigger sequence exposure-mediated platelet clearance. METHODS: Spontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody. RESULT: Transfected Chinese hamster ovary cells stably expressing the same chimeric IL4R-Ibα protein complex as in IL4R-IbαTg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R-IbαTg platelets. CONCLUSION: These results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb-IX-mediated thrombocytopenia.

Glycoprotein V : the unsolved GPV puzzle.

Glycoprotein V (GPV) is a highly expressed 82 KDa platelet surface transmembrane protein which is loosely attached to the GPIb-IX complex. Despite remaining questions concerning its function, GPV presents several unique features which have repercussions in hematology, atherothrombosis, immunology and transfusion. GPV is specifically expressed in platelets and megakaryocytes and is an ideal marker and reporter gene for the late stages of megakaryopoiesis. The ectodomain of GPV can be released by a number of proteases, namely thrombin, elastase and ADAM10 and 17. Although it was originally proposed as a thrombin receptor, this hypothesis was abandoned since thrombin activation was preserved after blockade of GPV cleavage and in Gp5 knockout mice. The combined potential of GPV to reflect the direct action of thrombin, platelet exposure to strong agonists and inflammatory conditions has led one to evaluate its utility as a marker in the context of atherothrombosis. Increased plasma levels of soluble GPV have notably been recorded in myocardial infarction, stroke and venous thromboembolism. It is also highly valued in transfusion to monitor platelet storage lesions. GPV presents several polymorphisms, which are a possible source of alloantibodies, while autoantibodies have been frequently detected in immune thrombocytopenia. The real biological function of this glycoprotein nevertheless remains an enigma, despite the respectively decreased and increased responses to low concentrations of collagen and thrombin observed in Gp5 knockout mice. Current studies are exploring its role in modulating general or VWF-induced platelet signaling, which could bear relevance in thrombosis and platelet clearance.

Mutations in the most divergent α-tubulin isotype, α8-tubulin, cause defective platelet biogenesis.

BACKGROUND: In the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia-causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring-like structure that supports the platelet typical disc-shaped morphology. This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation. OBJECTIVES: To assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. METHODS: We used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. RESULTS: We identified six distinct novel mutations in TUBA8, which encodes the most-divergent α-tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8-tubulin in the microtubule network. CONCLUSION: This study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy.

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement.

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing. Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

Isolation of Mouse Megakaryocyte Progenitors.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation.

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants.

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thiéry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 °C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Differential regulation of the platelet GPIb-IX complex by anti-GPIbß antibodies.

BACKGROUND: Platelets' initial recognition of endothelial damage proceeds through the interaction between collagen, plasma von Willebrand factor (VWF), and the platelet glycoprotein (GP)Ib-IX complex (CD42). The GPIb-IX complex consists of one GPIbα, one GPIX, and two GPIbß subunits. Once platelets are immobilized to the subendothelial matrix, shear generated by blood flow unfolds a membrane-proximal mechanosensory domain (MSD) in GPIbα, exposing a conserved trigger sequence and activating the receptor. Currently, GPIbα appears to solely facilitate ligand-induced activation because it contains both the MSD and the binding sites for all known ligands to GPIb-IX. Despite being positioned directly adjacent to the MSD, the roles of GPIbß and GPIX in signal transduction remain murky. OBJECTIVES: To characterize a novel rat monoclonal antibody 3G6 that binds GPIbß. METHODS: Effects of 3G6 on activation of GPIb-IX are characterized in platelets and Chinese hamster ovary cells expressing GPIb-IX (CHO-Ib-IX) and compared with those of an inhibitory anti-GPIbß antibody, RAM.1. RESULTS: Both RAM.1 and 3G6 bind to purified GPIbß and GPIb-IX with high affinity. 3G6 potentiates GPIb-IX-associated filopodia formation in platelets or CHO-Ib-IX when they adhere VWF or antibodies against the ligand-binding domain (LBD) of GPIbα. Pretreatment with 3G6 also increased anti-LBD antibody-induced GPIb-IX activation. Conversely, RAM.1 inhibits nearly all GPIb-IX-related signaling in platelets and CHO-Ib-IX cells. CONCLUSIONS: These data represent the first report of a positive modulator of GPIb-IX activation. The divergent modulatory effects of 3G6 and RAM.1, both targeting GPIbß, strongly suggest that changes in the conformation of GPIbß underlie outside-in activation via GPIb-IX.

Renin-angiotensin system is involved in embryonic emergence of hematopoietic stem/progenitor cells.

Angiotensin-converting enzyme (ACE), a key element of the renin-angiotensin system (RAS), has recently been identified as a new marker of both adult and embryonic human hematopoietic stem/progenitor cells (HSPCs). However, whether a full renin-angiotensin pathway is locally present during the hematopoietic emergence is still an open question. In the present study, we show that this enzyme is expressed by hematopoietic progenitors in the developing mouse embryo. Furthermore, ACE and the other elements of RAS-namely angiotensinogen, renin, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors-are expressed in the paraaortic splanchnopleura (P-Sp) and in its derivative, the aorta-gonad-mesonephros region, both in human and mouse embryos. Their localization is compatible with the existence of a local autocrine and/or paracrine RAS in these hemogenic sites. in vitro perturbation of the RAS by administration of a specific AT1 receptor antagonist inhibits almost totally the generation of blood CD45-positive cells from dissected P-Sp, implying that angiotensin II signaling is necessary for the emergence of hematopoietic cells. Conversely, addition of exogenous angiotensin II peptide stimulates hematopoiesis in culture, with an increase in the number of immature c-Kit+ CD41+ CD31+ CD45+ hematopoietic progenitors, compared to the control. These results highlight a novel role of local-RAS during embryogenesis, suggesting that angiotensin II, via activation of AT1 receptor, promotes the emergence of undifferentiated hematopoietic progenitors.