Search

Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Thrombocytopenia, which is associated with thrombopoietin (TPO) deficiency, presents very limited treatment options and can lead to life-threatening complications. Discovering new therapeutic agents against thrombocytopenia has proven to be a challenging task using traditional screening approaches. Fortunately, machine learning (ML) techniques offer a rapid avenue for exploring chemical space, thereby increasing the likelihood of uncovering new drug candidates. In this study, we focused on computational modeling for drug-induced megakaryocyte differentiation and platelet production using ML methods, aiming to gain insights into the structural characteristics of hematopoietic activity. We developed 112 different classifiers by combining eight ML algorithms with 14 molecule features. The top-performing model achieved good results on both 5-fold cross-validation (with an accuracy of 81.6% and MCC value of 0.589) and external validation (with an accuracy of 83.1% and MCC value of 0.642). Additionally, by leveraging the Shapley additive explanations method, the best model provided quantitative assessments of molecular properties and structures that significantly contributed to the predictions. Furthermore, we employed an ensemble strategy to integrate predictions from multiple models and performed in silico predictions for new molecules with potential activity against thrombocytopenia, sourced from traditional Chinese medicine and the Drug Repurposing Hub. The findings of this study could offer valuable insights into the structural characteristics and computational prediction of thrombopoiesis inducers.

Objective: To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice., Methods: PF4-Cre and Rictor fl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout ( Rictor -KO) mice and wild-type mice ( n =65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor -KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbβ3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy., Results: Compared with the wild-type mice, Rictor -KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor -KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbβ3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor -KO mice., Conclusion: Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.

Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms., Competing Interests: J.E. Italiano has a financial interest in and is a founder of Stellular Bio and Spry Bio. The interests of J.E. Italiano are managed by Boston Children’s Hospital. The other authors report no conflicts.

Abstract: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41 + MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

Platelet homeostasis is essential for vascular integrity and immune defence 1,2 . Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear 3,4 . Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection 5 . We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage., (© 2024. The Author(s).)

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Megakaryopoiesis and platelet production is a complex process that is underpotential regulation at multiple stages. Many long non-coding RNAs (lncRNAs) are distributed in hematopoietic stem cells and platelets. lncRNAs may play important roles as key epigenetic regulators in megakaryocyte differentiation and proplatelet formation. lncRNA NORAD can affect cell ploidy by sequestering PUMILIO proteins, although its direct effect on megakaryocyte differentiation and thrombopoiesis is still unknown. In this study, we demonstrate NORAD RNA is highly expressed in the cytoplasm during megakaryocyte differentiation. Interestingly, we identified for the first time that NORAD has a strong inhibitory effect on megakaryocyte differentiation and proplatelet formation from cultured megakaryocytes. DUSP6/ERK1/2 pathway is activated in response to NORAD knockdown during megakaryocytopoiesis, which is achieved by sequestering PUM2 proteins. Finally, compared with the wild-type control mice, NORAD knockout mice show a faster platelet recovery after severe thrombocytopenia induced by 6 Gy total body irradiation. These findings demonstrate lncRNA NORAD has a key role in regulating megakaryocyte differentiation and thrombopoiesis, which provides a promising molecular target for the treatment of platelet-related diseases such as severe thrombocytopenia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G 1 of interphase. Forced cell cycle arrest in G 1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.

Abstract: Lysyl oxidase (LOX) is a facilitator of extracellular matrix cross-linking. Using newly developed megakaryocyte-specific LOX knockout mice, we show that LOX expressed in these scarce bone marrow cells affects bone volume and collagen architecture in a sex-dependent manner., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP‑like cell line HEL western blotting, RT‑qPCR, lentivirus‑mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation‑specific (ETS) transcription factor friend leukemia integration factor 1 (Fli‑1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1‑mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation‑specific‑related gene ( ERG ) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA‑mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

Background: Megakaryocytes (MKs) are polyploid cells responsible for producing ∼10 11 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the promise for the production of clinical-grade platelets from stem cells, overcoming significant current limitations in platelet transfusion medicine. Previous work identified that loss of the epigenetic regulator SET domain containing 2 (SETD2) was associated with an increased platelet count in mice. However, the role of SETD2 in megakaryopoiesis remains unknown., Objectives: Here, we examined how SETD2 regulated MK development and platelet production using complementary murine and human systems., Methods: We manipulated the expression of SETD2 in multiple in vitro and ex vivo models to assess the ploidy of MKs and the function of platelets., Results: The genetic ablation of Setd2 increased the number of high-ploidy bone marrow MKs. Peripheral platelet counts in Setd2 knockout mice were significantly increased ∼2-fold, and platelets exhibited normal size, morphology, and function. By knocking down and overexpressing SETD2 in ex vivo human cell systems, we demonstrated that SETD2 negatively regulated MK polyploidization by controlling methylation of α-tubulin, microtubule polymerization, and MK nuclear division. Small-molecule inactivation of SETD2 significantly increased the production of high-ploidy MKs and platelets from human-induced pluripotent stem cells and cord blood CD34 + cells., Conclusion: These findings identify a previously unrecognized role for SETD2 in regulating megakaryopoiesis and highlight the potential of targeting SETD2 to increase platelet production from human cells for transfusion practices., Competing Interests: Declaration of competing interests All authors declare they have no competing interests., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of peripheral blood CD34+ or CD45+ cells from subjects with the G42S CYCS variant showed an acceleration in megakaryopoiesis compared to wild-type (WT) cells. To determine whether this result reflects a common feature of the CYCS variants, the c.145T>C mutation (Y49H variant) was introduced into the endogenous CYCS locus in K-562 cells, which undergo megakaryocytic maturation in response to treatment with a phorbol ester. The c.145T>C (Y49H) variant enhanced the megakaryocyte maturation of the K-562 cells, and this effect was seen when the cells were cultured at both 18 % and 5 % oxygen. Thus, alteration of megakaryopoiesis is common to both the G42S and Y49H CYCS variants and may contribute to the low platelet phenotype. The Y49H CYCS variant has previously been reported to impair mitochondrial respiratory chain function in vitro, however using extracellular flux analysis the c.145T>C (Y49H) variant does not alter mitochondrial bioenergetics of the K-562 cells, consistent with the lack of a phenotype characteristic of mitochondrial diseases in CYCS variant families. The Y49H variant has also been reported to enhance the ability of cytochrome c to trigger caspase activation in the intrinsic apoptosis pathway. However, as seen in peripheral blood cells from G42S CYCS variant carriers, the presence of Y49H cytochrome c in K-562 cells did not significantly change their response to an apoptotic stimulus., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge., (© 2024. The Author(s).)

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato + cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis., (© 2024. The Author(s).)

Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.

Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was cloned in 1994. Since its initial cloning and characterization, the hormone has revealed many surprises. For example, instead of acting as the postulated differentiation factor for platelet precursors, megakaryocytes, it is the most potent stimulator of megakaryocyte progenitor expansion known. Moreover, it also stimulates the survival, and in combination with stem cell factor leads to the expansion of hematopoietic stem cells. All of these growth-promoting activities have resulted in its clinical use in patients with thrombocytopenia and aplastic anemia, although the clinical development of the native molecule illustrated that "it's not wise to mess with mother nature", as a highly engineered version of the native hormone led to autoantibody formation and severe thrombocytopenia. Finally, another unexpected finding was the role of the thrombopoietin receptor in stem cell biology, including the development of myeloproliferative neoplasms, an important disorder of hematopoietic stem cells. Overall, the past 30 years of clinical and basic research has yielded many important insights, which are reviewed in this paper.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis 1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton., (© 2024. The Author(s).)

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Lipids contribute to hematopoiesis and membrane properties and dynamics; however, little is known about the role of lipids in megakaryopoiesis. Here we show that megakaryocyte progenitors, megakaryocytes and platelets present a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake as well as de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in low platelets. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production and that changes in dietary fatty acids may be a viable target to modulate platelet counts., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Diamond-Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is associated with anemia, congenital anomalies, and cancer predisposition. It is categorized as a ribosomopathy, because more than 80% or patients have haploinsufficiency of either a small or large subunit-associated ribosomal protein (RP). The erythroid pathology is due predominantly to a block and delay in early committed erythropoiesis with reduced megakaryocyte/erythroid progenitors (MEPs). To understand the molecular pathways leading to pathogenesis of DBA, we performed RNA sequencing on mRNA and miRNA from RPS19-deficient human hematopoietic stem and progenitor cells (HSPCs) and compared existing database documenting transcript fluctuations across stages of early normal erythropoiesis. We determined the chromatin regulator, SATB1 was prematurely downregulated through the coordinated action of upregulated miR-34 and miR-30 during differentiation in ribosomal insufficiency. Restoration of SATB1 rescued MEP expansion, leading to a modest improvement in erythroid and megakaryocyte expansion in RPS19 insufficiency. However, SATB1 expression did not affect expansion of committed erythroid progenitors, indicating ribosomal insufficiency affects multiple stages during erythroid differentiation., Competing Interests: Conflict of interest disclosure The authors have no competing interests to declare., (Copyright © 2022 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Transforming growth factor β1 (TGF-β1) regulates a wide variety of events in adult bone marrow (BM), including quiescence of hematopoietic stem cells, via undefined mechanisms. Because megakaryocytes (MKs)/platelets are a rich source of TGF-β1, we assessed whether TGF-β1 might inhibit its own production by comparing mice with conditional inactivation of Tgfb1 in MKs (PF4Cre;Tgfb1flox/flox) and control mice. PF4Cre;Tgfb1flox/flox mice had ∼30% more MKs in BM and ∼15% more circulating platelets than control mice (P < .001). Thrombopoietin (TPO) levels in plasma and TPO expression in liver were approximately twofold higher in PF4Cre;Tgfb1flox/flox than in control mice (P < .01), whereas TPO expression in BM cells was similar between these mice. In BM cell culture, TPO treatment increased the number of MKs from wild-type mice by approximately threefold, which increased approximately twofold further in the presence of a TGF-β1-neutralizing antibody and increased the number of MKs from PF4Cre;Tgfb1flox/flox mice approximately fourfold. Our data reveal a new role for TGF-β1 produced by MKs/platelets in regulating its own production in BM via increased TPO production in the liver. Additional studies are required to determine the mechanism., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abivertinib, a third-generation tyrosine kinase inhibitor, is originally designed to target epidermal growth factor receptor (EGFR)-activating mutations. Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer. However, abivertinib also exhibited high inhibitory activity against Bruton's tyrosine kinase and Janus kinase 3. Given that these kinases play some roles in the progression of megakaryopoiesis, we speculate that abivertinib can affect megakaryocyte (MK) differentiation and platelet biogenesis. We treated cord blood CD34 + hematopoietic stem cells, Meg-01 cells, and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis. Our in vitro results showed that abivertinib impaired the CFU-MK formation, proliferation of CD34 + HSC-derived MK progenitor cells, and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation. These results suggested that megakaryopoiesis was inhibited by abivertinib. We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice, which suggested that thrombopoiesis was also inhibited. Thus, these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia., (© 2021. Higher Education Press.)

Translation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains unknown. In this study, we found that MK/platelet-specific lactate dehydrogenase A (LdhA) knockout mice exhibited an increased number of platelets with remarkably accelerated MK maturation and proplatelet formation. Interestingly, the role of LDHA in MK maturation and platelet formation did not depend on lactate content, which was the major product of LDHA. Mechanism studies revealed that LDHA interacted with eukaryotic elongation factor 2 (eEF2) in the cytoplasm, controlling the participation of eEF2 in translation at the ribosome. Furthermore, the interaction of LDHA and eEF2 was dependent on nicotinamide adenine dinucleotide (NADH), a coenzyme of LDHA. NADH-competitive inhibitors of LDHA could release eEF2 from the LDHA pool, upregulate translation, and enhance MK maturation in vitro. Among LDHA inhibitors, stiripentol significantly promoted the production of platelets in vivo under a physiological state and in the immune thrombocytopenia model. Moreover, stiripentol could promote platelet production from human cord blood mononuclear cell-derived MKs and also have a superposed effect with romiplostim. In short, this study shows a novel nonclassical function of LDHA in translation that may serve as a potential target for thrombocytopenia therapy., (© 2022 by The American Society of Hematology.)

Mature megakaryocytes, the platelet precursors, originate from hematopoietic stem cell progenitors, which, once committed to this lineage, undergo endomitosis leading to polyploidization. The process entails repeated rounds of DNA replication without cell division, yielding polyploid cells. Supporting the cell's developmental process and various cellular functions are integrin receptors, a conduit of communication between the extracellular environment and the cell actin cytoskeleton. Integrins are heterodimers of α and β subunits, where different combinations of the known 18 α and 8 β subunits confer specificity to the receptor. Integrin ligands range from extracellular matrices through soluble ligands, infectious agents, and counterreceptors, to cells. In this review, we describe the different integrins expressed on bone marrow megakaryocytes and their attributed roles in lineage development and cellular functions, including adhesion, spreading, proplatelet formation, and functional interaction with other cells. Pathologies associated with dysregulated megakaryocyte integrin expression are also reviewed., Competing Interests: Conflict of interest disclosure None of the authors have relevant conflicts of interest., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Megakaryocytes (MKs), the largest of the hematopoietic cells, are responsible for producing platelets by extending and depositing long proplatelet extensions into the bloodstream. The traditional view of megakaryopoiesis describes the cellular journey from hematopoietic stem cells (HSCs) along the myeloid branch of hematopoiesis. However, recent studies suggest that MKs can be generated from multiple pathways, some of which do not require transit through multipotent or bipotent MK-erythroid progenitor stages in steady-state and emergency conditions. Growing evidence suggests that these emergency conditions are due to stress-induced molecular changes in the bone marrow (BM) microenvironment, also called the BM niche. These changes can result from insults that affect the BM cellular composition, microenvironment, architecture, or a combination of these factors. In this review, we explore MK development, focusing on recent studies showing that MKs can be generated from multiple divergent pathways. We highlight how the BM niche may encourage and alter these processes using different mechanisms of communication, such as direct cell-to-cell contact, secreted molecules (autocrine and paracrine signaling), and the release of cellular components (eg, extracellular vesicles). We also explore how MKs can actively build and shape the surrounding BM niche., (© 2022 by The American Society of Hematology.)

Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.

Cells do not make fate decisions independently. Arguably, every cell-fate decision occurs in response to environmental signals. In many cases, cell-cell communication alters the dynamics of the internal gene regulatory network of a cell to initiate cell-fate transitions, yet models rarely take this into account. Here, we have developed a multiscale perspective to study the granulocyte-monocyte versus megakaryocyte-erythrocyte fate decisions. This transition is dictated by the GATA1-PU.1 network: a classical example of a bistable cell-fate system. We show that, for a wide range of cell communication topologies, even subtle changes in signaling can have pronounced effects on cell-fate decisions. We go on to show how cell-cell coupling through signaling can spontaneously break the symmetry of a homogenous cell population. Noise, both intrinsic and extrinsic, shapes the decision landscape profoundly, and affects the transcriptional dynamics underlying this important hematopoietic cell-fate decision-making system. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Hematopoietic cells differentiate through several progenitors in a hierarchical manner, and recent single-cell analyses have revealed substantial heterogeneity within each progenitor. Although common myeloid progenitors (CMPs) are defined as a multipotent cell population that can differentiate into granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs), and GMPs generate neutrophils and monocytes, these myeloid progenitors must contain some lineage-committed progenitors. Through gene expression analysis at single-cell levels, we identified CD62L as a marker to reveal the heterogeneity. We confirmed that CD62L-negative CMPs represent "bona fide" CMPs, whereas CD62L-high CMPs are mostly restricted to GMP potentials both in mice and humans. In addition, we identified CD62L-negative GMPs as the most immature subsets in GMPs and Ly6C + CD62L-intermediate and Ly6C + CD62L-high GMPs are skewed to neutrophil and monocyte differentiation in mice, respectively. Our findings contribute to more profound understanding about the mechanism of myeloid differentiation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The purpose of this research was to assess the effects of a microRNA (miRNA) cluster on platelet production. Human chromosome 19q13.41 harbors an evolutionarily conserved cluster of three miRNA genes (MIR99B, MIRLET7E, MIR125A) within 727 base-pairs. We now report that levels of miR-99b-5p, miR-let7e-5p and miR-125a-5p are strongly correlated in human platelets, and all are positively associated with platelet count, but not white blood count or hemoglobin level. Although the cluster regulates hematopoietic stem cell proliferation, the function of this genomic locus in megakaryocyte (MK) differentiation and platelet production is unknown. Furthermore, studies of individual miRNAs do not represent broader effects in the context of a cluster. To address this possibility, MK/platelet lineage-specific Mir-99b/let7e/125a knockout mice were generated. Compared to wild type littermates, cluster knockout mice had significantly lower platelet counts and reduced MK proplatelet formation, but no differences in MK numbers, ploidy, maturation or ultra-structural morphology, and no differences in platelet function. Compared to wild type littermates, knockout mice showed similar survival after pulmonary embolism. The major conclusions are that the effect of the Mir-99b/let7e/125a cluster is confined to a late stage of thrombopoiesis, and this effect on platelet number is uncoupled from platelet function., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. In light of recent advances in single-cell technologies, we sought to redefine the transcriptional and epigenetic landscape of mouse and human hematopoietic stem cells under telomere attrition, as induced by pathogenic germline variants in telomerase complex genes. Here, we show that telomere attrition maintains hematopoietic stem cells under persistent metabolic activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of the innate immune signaling response, which directly compromises hematopoietic stem cells' self-renewal capabilities and eventually leads to their exhaustion. Mechanistically, we demonstrate that targeting members of the Ifi20x/IFI16 family of cytosolic DNA sensors using the oligodeoxynucleotide A151, which comprises four repeats of the TTAGGG motif of the telomeric DNA, overcomes interferon signaling activation in telomere-dysfunctional hematopoietic stem cells and these cells' skewed differentiation towards the megakaryocytic lineage. This study challenges the historical hypothesis that telomere attrition limits the proliferative potential of hematopoietic stem cells by inducing apoptosis, autophagy, or senescence, and suggests that targeting IFI16 signaling axis might prevent hematopoietic stem cell functional decline in conditions affecting telomere maintenance., (© 2021. The Author(s).)

Human ZNF648 is a novel poly C-terminal C2H2 zinc finger protein identified amongst the most dysregulated proteins in erythroid cells differentiated from iPSC. Its nuclear localisation and structure indicate it is likely a DNA-binding protein. Using a combination of ZNF648 overexpression in an iPSC line and primary adult erythroid cells, ZNF648 knockdown in primary adult erythroid cells and megakaryocytes, comparative proteomics and transcriptomics we show that ZNF648 is required for both erythroid and megakaryocyte differentiation. Orthologues of ZNF648 were detected across Mammals, Reptilia, Actinopterygii, in some Aves, Amphibia and Coelacanthiformes suggesting the gene originated in the common ancestor of Osteichthyes (Euteleostomi or bony fish). Conservation of the C-terminal zinc finger domain is higher, with some variation in zinc finger number but a core of at least six zinc fingers conserved across all groups, with the N-terminus recognisably similar within but not between major lineages. This suggests the N-terminus of ZNF648 evolves faster than the C-terminus, however this is not due to exon-shuffling as the entire coding region of ZNF648 is within a single exon. As for other such transcription factors, the N-terminus likely carries out regulatory functions, but showed no sequence similarity to any known domains. The greater functional constraint on the zinc finger domain suggests ZNF648 binds at least some similar regions of DNA in the different organisms. However, divergence of the N-terminal region may enable differential expression, allowing adaptation of function in the different organisms.

EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss-of-function mutations have been found in myeloproliferative neoplasms, particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and short hairpin RNA induces megakaryocyte (MK) commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and polyploidization and decreases proplatelet formation. EZH2 inhibitors similarly reduce MK polyploidization and proplatelet formation in vitro and platelet levels in vivo in a JAK2V617F background. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression of genes involved in DNA replication and repair and an upregulation of cyclin-dependent kinase inhibitors, particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and to a lesser extent CDKN2D could partially rescue the percentage of polyploid MKs. Moreover, H3K27me3 and EZH2 chromatin immunoprecipitation assays revealed that CDKN1A is a direct EZH2 target and CDKN2D expression is not directly regulated by EZH2, suggesting that EZH2 controls MK polyploidization directly through CDKN1A and indirectly through CDKN2D., (© 2021 by The American Society of Hematology.)

NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte-erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.

Megakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche-related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented ∼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell., (© 2021 by The American Society of Hematology.)

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Cova et al.)

The activity of hematopoietic factor GATA-1 is modulated through p300/CBP-mediated acetylation and FOG-1 mediated indirect interaction with HDAC1/2 containing NuRD complex. Although GATA-1 acetylation is implicated in GATA-1 activation, the role of deacetylation is not studied. Here, we found that the FOG-1/NuRD does not deacetylate GATA-1. However, HDAC1/2 can directly bind and deacetylate GATA-1. Two arginine residues within the GATA-1 linker region mediates direct interaction with HDAC1. The arginine to alanine mutation (2RA) blocks GATA-1 deacetylation and fails to induce erythroid differentiation. Gene expression profiling and ChIP-seq analysis further demonstrate the importance of GATA-1 deacetylation for gene activation and chromatin recruitment. GATA-12RA knock-in (KI) mice suffer mild anemia and thrombocytopenia with accumulation of immature erythrocytes and megakaryocytes in bone marrow and spleen. Single cell RNA-seq analysis of Lin- cKit+ (LK) cells further reveal a profound change in cell subpopulations and signature gene expression patterns in HSC, myeloid progenitors, and erythroid/megakaryocyte clusters in KI mice. Thus, GATA-1 deacetylation and its interaction with HDAC1 modulates GATA-1 chromatin binding and transcriptional activity that control erythroid/megakaryocyte commitment and differentiation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Competing Interests: None declared.

Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS., Competing Interests: Declaration of interests M.L. has served on the Scientific Advisory Board for Epi One. A.V. has received research funding from GlaxoSmithKline, Incyte, MedPacto, Novartis, Curis, and Eli Lilly and Company; has received compensation as a scientific advisor to Novartis, Stelexis Therapeutics, Acceleron Pharma, and Celgene; and has equity ownership in Stelexis Therapeutics. The remaining authors declare no competing interests. Array BioPharma provided the p38 inhibitor pexmetinib (ARRY614) and participated in its phase I study (ClinicalTrials.gov: NCT01496495)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocytes, granulocytes, mast cells and macrophages, the latter in a Myb transcription factor-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from Myb-dependent bipotent progenitors expressing Csf2rb and colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes. Understanding megakaryocyte development is crucial as they play key functions during vascular development, in particular in separating blood and lymphatic networks., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Liver X receptor β (LXRβ), a nuclear receptor involved in important cellular processes such as cholesterol, glucose and fatty acid metabolism, was suggested to be involved in platelet aggregation but its detailed roles are not clear. In the present study, we evaluated the contribution of LXRβ to platelet functions and production. In the systemic collagen-epinephrine thrombosis mouse model, LXRβ-deficient mice showed increased area of blood clots compared with control wide-type littermates. The aggregation of LXRβ-deficient platelets in response to ADP was stronger than that of control mice platelets. More importantly, the number of platelets in blood of LXRβ-deficient mice was significantly higher than that of wild-type mice, especially for female mice. Knockdown of LXRβ expression in human megakaryoblastic Dami cells also enhanced cell polyploidization, formation of proplatelets and production of platelet-like particles. Increase in expression levels of proteins related to oxidative phosphorylation such as NADH:ubiquinone oxidoreductase core subunit V1 (Ndufv1) was observed in LXRβ-knockdown Dami cells. The levels of Ndufv1 in LXRβ-deficient mice platelets were also higher than that of wild-type mice. Taken together, our findings suggested LXRβ might participate in control of platelet production from megakaryocytes by regulating mitochondrial metabolism., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This paper aims to emphasize the importance of applying international consensus guidelines to detect qualitative and quantitative abnormalities of megakaryocytes on smears of bone marrow aspirates (BMA) for a shared and harmonized diagnostic path between different laboratories. Careful evaluation of megakaryocytes on BMA smears represents a cornerstone in the diagnosis of most clonal and nonclonal hematological diseases. Images associated with the detailed morphologic description of normal, reactive, abnormal, and dysplastic megakaryocytes are also reported together with examples of similar cells that, if not promptly identified, can lead to a morphological misdiagnosis., (Copyright © 2021 John Wiley & Sons Ltd.)