Differentiation route determines the functional outputs of adult megakaryopoiesis.

Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical differentiation route through a series of restricted hematopoietic progenitors (stepwise route). This raises the question of the importance of two alternative routes for megakaryopoiesis. Here, we developed fate-mapping systems to distinguish the two routes, comparing their quantitative and functional outputs. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes, respectively, but contributed to the platelet-producing megakaryocytes together. Megakaryocytes derived from the two routes displayed different activities and were differentially regulated by chemotherapy and inflammation. Our work links differentiation route to the heterogeneity of megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.

Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation.

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.

Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets.

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

Megakaryocyte maturation involves activation of the adaptive unfolded protein response.

Endoplasmic reticulum stress triggers the unfolded protein response (UPR) to promote cell survival or apoptosis. Transient endoplasmic reticulum stress activation has been reported to trigger megakaryocyte production, and UPR activation has been reported as a feature of megakaryocytic cancers. However, the role of UPR signaling in megakaryocyte biology is not fully understood. We studied the involvement of UPR in human megakaryocytic differentiation using PMA (phorbol 12-myristate 13-acetate)-induced maturation of megakaryoblastic cell lines and thrombopoietin-induced differentiation of human peripheral blood-derived progenitors. Our results demonstrate that an adaptive UPR is a feature of megakaryocytic differentiation and that this response is not associated with ER stress-induced apoptosis. Differentiation did not alter the response to the canonical endoplasmic reticulum stressors DTT or thapsigargin. However, thapsigargin, but not DTT, inhibited differentiation, consistent with the involvement of Ca2+ signaling in megakaryocyte differentiation.

ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity.

The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

Aryl hydrocarbon receptor activation alters immune cell populations in the lung and bone marrow during coronavirus infection.

Respiratory viral infections are one of the major causes of illness and death worldwide. Symptoms associated with respiratory infections can range from mild to severe, and there is limited understanding of why there is large variation in severity. Environmental exposures are a potential causative factor. The aryl hydrocarbon receptor (AHR) is an environment-sensing molecule expressed in all immune cells. Although there is considerable evidence that AHR signaling influences immune responses to other immune challenges, including respiratory pathogens, less is known about the impact of AHR signaling on immune responses during coronavirus (CoV) infection. In this study, we report that AHR activation significantly altered immune cells in the lungs and bone marrow of mice infected with a mouse CoV. AHR activation transiently reduced the frequency of multiple cells in the mononuclear phagocyte system, including monocytes, interstitial macrophages, and dendritic cells in the lung. In the bone marrow, AHR activation altered myelopoiesis, as evidenced by a reduction in granulocyte-monocyte progenitor cells and an increased frequency of myeloid-biased progenitor cells. Moreover, AHR activation significantly affected multiple stages of the megakaryocyte lineage. Overall, these findings indicate that AHR activation modulates multiple aspects of the immune response to a CoV infection. Given the significant burden of respiratory viruses on human health, understanding how environmental exposures shape immune responses to infection advances our knowledge of factors that contribute to variability in disease severity and provides insight into novel approaches to prevent or treat disease.NEW & NOTEWORTHY Our study reveals a multifaceted role for aryl hydrocarbon receptor (AHR) signaling in the immune response to coronavirus (CoV) infection. Sustained AHR activation during in vivo mouse CoV infection altered the frequency of mature immune cells in the lung and modulated emergency hematopoiesis, specifically myelopoiesis and megakaryopoiesis, in bone marrow. This provides new insight into immunoregulation by the AHR and extends our understanding of how environmental exposures can impact host responses to respiratory viral infections.

Long non-coding RNA NORAD regulates megakaryocyte differentiation and proplatelet formation via the DUSP6/ERK signaling pathway.

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.

Local and Systemic Overexpression of COMP-Ang1 Induces Ang1/Tie2-Related Thrombocytopenia and SDF-1/CXCR4-Dependent Anemia.

While supplemental angiopoietin-1 (Ang1) improves hematopoiesis, excessive Ang1 induces bone marrow (BM) impairment, hematopoietic stem cell (HSC) senescence, and erythropoietic defect. Here, we examined how excessive Ang1 disturbs hematopoiesis and explored whether hematopoietic defects were related to its level using K14-Cre;c-Ang1 and Col2.3-Cre;c-Ang1 transgenic mice that systemically and locally overexpress cartilage oligomeric matrix protein-Ang1, respectively. We also investigated the impacts of Tie2 inhibitor and AMD3100 on hematopoietic development. Transgenic mice exhibited excessive angiogenic phenotypes, but K14-Cre;c-Ang1 mice showed more severe defects in growth and life span with higher presence of Ang1 compared with Col2.3-Cre;c-Ang1 mice. Dissimilar to K14-Cre;c-Ang1 mice, Col2.3-Cre;c-Ang1 mice did not show impaired BM retention or senescence of HSCs, erythropoietic defect, or disruption of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis. However, these mice exhibited a defect in platelet production depending on the expression of Tie2 and globin transcription factor 1 (GATA-1), but not GATA-2, in megakaryocyte progenitor (MP) cells. Treatment with Tie2 inhibitor recovered GATA-1 expression in MP cells and platelet production without changes in circulating RBC in transgenic mice. Consecutive AMD3100 administration not only induced irrecoverable senescence of HSCs but also suppressed formation of RBC, but not platelets, via correlated decreases in number of erythroblasts and their GATA-1 expression in B6 mice. Our results indicate that genetic overexpression of Ang1 impairs hematopoietic development depending on its level, in which megakaryopoiesis is preferentially impaired via activation of Ang1/Tie2 signaling, whereas erythropoietic defect is orchestrated by HSC senescence, inflammation, and disruption of the SDF-1/CXCR4 axis.

GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development.

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.

Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors.

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

Dachshund Homolog 1: Unveiling Its Potential Role in Megakaryopoiesis and Bacillus anthracis Lethal Toxin-Induced Thrombocytopenia.

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Decreased cyclooxygenase-2 associated with impaired megakaryopoiesis and thrombopoiesis in primary immune thrombocytopenia.

BACKGROUND: Cyclooxygenase (COX)-2 is a rate-limiting enzyme in the biosynthesis of prostanoids, which is mostly inducible by inflammatory cytokines. The participation of COX-2 in the maturation of megakaryocytes has been reported but barely studied in primary immune thrombocytopenia (ITP). METHODS: The expressions of COX-2 and Caspase-1, Caspase-3 and Caspase-3 p17 subunit in platelets from ITP patients and healthy controls (HC), and the expressions of COX-2 and CD41 in bone marrow (BM) of ITP patients were measured and analyzed for correlations. The effects of COX-2 inhibitor on megakaryopoiesis and thrombopoiesis were assessed by in vitro culture of Meg01 cells and murine BM-derived megakaryocytes and in vivo experiments of passive ITP mice. RESULTS: The expression of COX-2 was decreased and Caspase-1 and Caspase-3 p17 were increased in platelets from ITP patients compared to HC. In platelets from ITP patients, the COX-2 expression was positively correlated with platelet count and negatively correlated to the expression of Caspase-1. In ITP patients BM, the expression of CD41 was positively correlated with the expression of COX-2. COX-2 inhibitor inhibited the count of megakaryocytes and impaired the maturation and platelet production in Meg01 cells and bone marrow-derived megakaryocytes. COX-2 inhibitor aggravated thrombocytopenia and damaged megakaryopoiesis in ITP murine model. CONCLUSION: COX-2 plays a vital role in the physiologic and pathologic conditions of ITP by intervening the survival of platelets and impairing the megakaryopoiesis and thrombopoiesis of megakaryocytes.

Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia.

BACKGROUND: Thrombocytopenia is a common complication in cancer patients undergoing chemotherapy. Chemotherapy-induced thrombocytopenia (CIT) leads to dose reduction and treatment delays, lowering chemotherapy efficacy and survival rate. Thus, rapid recovery and continuous maintenance of platelet count during chemotherapy cycles are crucial in patients with CIT. Thrombopoietin (TPO) and its receptor, myeloid proliferative leukemia (MPL) protein, play a major role in platelet production. Although several MPL agonists have been developed to regulate thrombopoiesis, none have been approved for the management of CIT due to concerns regarding efficacy or safety. Therefore, the development of effective MPL agonists for treating CIT needs to be further expanded. METHODS: Anti-MPL antibodies were selected from the human combinatorial antibody phage libraries using phage display. We identified 2R13 as the most active clone among the binding antibodies via cell proliferation assay using BaF3/MPL cells. The effect of 2R13 on megakaryocyte differentiation was evaluated in peripheral blood CD34+ cells by analyzing megakaryocyte-specific differentiation markers (CD41a+ and CD42b+) and DNA ploidy using flow cytometry. The 2R13-induced platelet production was examined in 8- to 10-week-old wild-type BALB/c female mice and a thrombocytopenia mouse model established by intraperitoneal injection of 5-fluorouracil (150 mg/kg). The platelet counts were monitored twice a week over 14 days post-initiation of treatment with a single injection of 2R13, or recombinant human TPO (rhTPO) for seven consecutive days. RESULTS: We found that 2R13 specifically interacted with MPL and activated its signaling pathways. 2R13 stimulated megakaryocyte differentiation, evidenced by increasing the proportion of high-ploidy (≥ 8N) megakaryocytes in peripheral blood-CD34+ cells. The platelet count was increased by a single injection of 2R13 for up to 14 days. Injection of 5-fluorouracil considerably reduced the platelet count by day 4, which was recovered by 2R13. The platelets produced by 2R13 sustained a higher count than that achieved using seven consecutive injections of rhTPO. CONCLUSIONS: Our findings suggest that 2R13 is a promising therapeutic agent for CIT treatment.

Current insights into the role of Fli-1 in hematopoiesis and malignant transformation.

Fli-1, a member of the ETS family of transcription factors, was discovered in 1991 through retroviral insertional mutagenesis as a driver of mouse erythroleukemias. In the past 30 years, nearly 2000 papers have defined its biology and impact on normal development and cancer. In the hematopoietic system, Fli-1 controls self-renewal of stem cells and their differentiation into diverse mature blood cells. Fli-1 also controls endothelial survival and vasculogenesis, and high and low levels of Fli-1 are implicated in the auto-immune diseases systemic lupus erythematosus and systemic sclerosis, respectively. In addition, aberrant Fli-1 expression is observed in, and is essential for, the growth of multiple hematological malignancies and solid cancers. Here, we review the historical context and latest research on Fli-1, focusing on its role in hematopoiesis, immune response, and malignant transformation. The importance of identifying Fli-1 modulators (both agonists and antagonists) and their potential clinical applications is discussed.

SARS-CoV-2 Lysate Stimulation Impairs the Release of Platelet-like Particles and Megakaryopoiesis in the MEG-01 Cell Line.

SARS-CoV-2 infection causes a considerable inflammatory response coupled with impaired platelet reactivity, which can lead to platelet disorders recognized as negative prognostic factors in COVID-19 patients. The virus may cause thrombocytopenia or thrombocytosis during the different disease stages by destroying or activating platelets and influencing platelet production. While it is known that several viruses can impair megakaryopoiesis by generating an improper production and activation of platelets, the potential involvement of SARS-CoV-2 in affecting megakaryopoiesis is poorly understood. To this purpose, we explored, in vitro, the impact of SARS-CoV-2 stimulation in the MEG-01 cell line, a human megakaryoblastic leukemia cell line, considering its spontaneous capacity of releasing platelet-like particles (PLPs). We interrogated the effect of heat-inactivated SARS-CoV-2 lysate in the release of PLPs and activation from MEG-01, the signaling pathway influenced by SARS-CoV-2, and the functional effect on macrophagic skewing. The results highlight the potential influence of SARS-CoV-2 in the early stages of megakaryopoiesis by enhancing the production and activation of platelets, very likely due to the impairment of STATs signaling and AMPK activity. Overall, these findings provide new insight into the role of SARS-CoV-2 in affecting megakaryocyte-platelet compartment, possibly unlocking another avenue by which SARS-CoV-2 moves.

Metabolic sensor O-GlcNAcylation regulates megakaryopoiesis and thrombopoiesis through c-Myc stabilization and integrin perturbation.

Metabolic state of hematopoietic stem cells (HSCs) is an important regulator of self-renewal and lineage-specific differentiation. Posttranslational modification of proteins via O-GlcNAcylation is an ideal metabolic sensor, but how it contributes to megakaryopoiesis and thrombopoiesis remains unknown. Here, we reveal for the first time that cellular O-GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human-derived hematopoietic stem and progenitor cells (HSPCs). Inhibition of O-GlcNAc transferase (OGT) that catalyzes O-GlcNAcylation prolongedly decreases O-GlcNAcylation and induces the acquisition of CD34+ CD41a+ MK-like progenitors and its progeny CD34- CD41a+ /CD42b+ megakaryoblasts (MBs)/MKs from HSPCs, consequently resulting in increased CD41a+ and CD42b+ platelets. Using correlation and co-immunoprecipitation analyses, we further identify c-Myc as a direct downstream target of O-GlcNAcylation in MBs/MKs and provide compelling evidence on the regulation of platelets by novel O-GlcNAc/c-Myc axis. Our data indicate that O-GlcNAcylation posttranslationally regulates c-Myc stability by interfering with its ubiquitin-mediated proteasomal degradation. Depletion of c-Myc upon inhibition of OGT promotes platelet formation in part through the perturbation of cell adhesion molecules, that is, integrin-α4 and integrin-ß7, as advised by gene ontology and enrichment analysis for RNA sequencing and validated herein. Together, our findings provide a novel basic knowledge on the regulatory role of O-GlcNAcylation in megakaryopoiesis and thrombopoiesis that could be important in understanding hematologic disorders whose etiology are related to impaired platelet production and may have clinical applications toward an ex vivo platelet production for transfusion.

miRNA 146b mediates the regulation of nucleolar size and activity in polyploid megakaryocytes.

BACKGROUND INFORMATION: Megakaryocytes (MKs) follow a unique cell cycle duplication process, called endomitosis, resulting in polyploidisation of cells. It is hypothesised that polyploidy, as well as an increment in cytoplasm volume, allow more efficient platelets generation from MKs. Although polyploidy leads to an increase in the DNA amount, which impacts gene expression, little is known about ribosomal biogenesis in these polylobulated polyploid cells. RESULTS: The nucleolus acts as a hub for ribosomal biogenesis, which in turn governs the protein synthesis rate of the cells. We therefore estimated the size and activity of the nucleolus in polyploid cells during megakaryopoiesis in vitro. Polyploid megakaryocytic cell lines and in vitro cultured MKs, which were obtained from human cord blood-derived CD 34+ cells, revealed that miRNA 146b regulated the activity of nucleolar and coiled-body phosphoprotein 1, which plays an integral role in determining nucleolar size and activity. Additionally, miRNA-146b was up-regulated during endomitosis and was found to promote megakaryopoiesis. CONCLUSION: We propose that miRNA 146b regulates not only nucleolar size and activity, but also megakaryopoiesis. SIGNIFICANCE: This study highlights the importance of nucleolar activity and miRNA in the progression of megakaryopoiesis and thrombopoiesis.

Generation and manipulation of human iPSC-derived platelets.

The discovery of iPSCs has led to the ex vivo production of differentiated cells for regenerative medicine. In the case of transfusion products, the derivation of platelets from iPSCs is expected to complement our current blood-donor supplied transfusion system through donor-independent production with complete pathogen-free assurance. This derivation can also overcome alloimmune platelet transfusion refractoriness by resulting in autologous, HLA-homologous or HLA-deficient products. Several developments were necessary to produce a massive number of platelets required for a single transfusion. First, expandable megakaryocytes were established from iPSCs through transgene expression. Second, a turbulent-type bioreactor with improved platelet yield and quality was developed. Third, novel drugs that enabled efficient feeder cell-free conditions were developed. Fourth, the platelet-containing suspension was purified and resuspended in an appropriate buffer. Finally, the platelet product needed to be assured for competency and safety including non-tumorigenicity through in vitro and in vivo preclinical tests. Based on these advancements, a clinical trial has started. The generation of human iPSC-derived platelets could evolve transfusion medicine to the next stage and assure a ubiquitous, safe supply of platelet products. Further, considering the feasibility of gene manipulations in iPSCs, other platelet products may bring forth novel therapeutic measures.

miR-486-5p and miR-22-3p Enable Megakaryocytic Differentiation of Hematopoietic Stem and Progenitor Cells without Thrombopoietin.

Megakaryocytes release submicron size microparticles (MkMPs) in circulation. We have shown that MkMPs target CD34+ hematopoietic stem/progenitor cells (HSPCs) to induce megakaryocytic differentiation, and that small RNAs in MkMPs play an important role in the development of this phenotype. Here, using single-molecule real-time (SMRT) RNA sequencing (RNAseq), we identify the synergetic effect of two microRNAs (miRs), miR-486-5p and miR-22-3p (highly enriched in MkMPs), in driving the Mk differentiation of HSPCs in the absence of thrombopoietin (TPO). Separately, our data suggest that the MkMP-induced Mk differentiation of HSPCs is enabled through JNK and PI3K/Akt/mTOR signaling. The interaction between the two signaling pathways is likely mediated by a direct target of miR-486-5p and a negative regulator of PI3K/Akt signaling, the phosphatase and tensin homologue (PTEN) protein. Our data provide a possible mechanistic explanation of the biological effect of MkMPs in inducing megakaryocytic differentiation of HSPCs, a phenotype of potential physiological significance in stress megakaryopoiesis.

The Analysis of the Human Megakaryocyte and Platelet Coding Transcriptome in Healthy and Diseased Subjects.

Platelets are generated and released into the bloodstream from their precursor cells, megakaryocytes that reside in the bone marrow. Though platelets have no nucleus or DNA, they contain a full transcriptome that, during platelet formation, is transported from the megakaryocyte to the platelet. It has been described that transcripts in platelets can be translated into proteins that influence platelet response. The platelet transcriptome is highly dynamic and has been extensively studied using microarrays and, more recently, RNA sequencing (RNA-seq) in relation to diverse conditions (inflammation, obesity, cancer, pathogens and others). In this review, we focus on bulk and single-cell RNA-seq studies that have aimed to characterize the coding transcriptome of healthy megakaryocytes and platelets in humans. It has been noted that bulk RNA-seq has limitations when studying in vitro-generated megakaryocyte cultures that are highly heterogeneous, while single-cell RNA-seq has not yet been applied to platelets due to their very limited RNA content. Next, we illustrate how these methods can be applied in the field of inherited platelet disorders for gene discovery and for unraveling novel disease mechanisms using RNA from platelets and megakaryocytes and rare disease bioinformatics. Next, future perspectives are discussed on how this field of coding transcriptomics can be integrated with other next-generation technologies to decipher unexplained inherited platelet disorders in a multiomics approach.