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Journal ArticleDOI: 10.1016/J.STEM.2020.11.006

Decoding Human Megakaryocyte Development.

04 Mar 2021-Cell Stem Cell (Cell Press)-Vol. 28, Iss: 3
Abstract: Summary Despite our growing understanding of embryonic immune development, rare early megakaryocytes (MKs) remain relatively understudied. Here we used single-cell RNA sequencing of human MKs from embryonic yolk sac (YS) and fetal liver (FL) to characterize the transcriptome, cellular heterogeneity, and developmental trajectories of early megakaryopoiesis. In the YS and FL, we found heterogeneous MK subpopulations with distinct developmental routes and patterns of gene expression that could reflect early functional specialization. Intriguingly, we identified a subpopulation of CD42b+CD14+ MKs in vivo that exhibit high expression of genes associated with immune responses and can also be derived from human embryonic stem cells (hESCs) in vitro. Furthermore, we identified THBS1 as an early marker for MK-biased embryonic endothelial cells. Overall, we provide important insights and invaluable resources for dissection of the molecular and cellular programs underlying early human megakaryopoiesis.

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Topics: Megakaryopoiesis (55%), Transcriptome (51%), Embryonic stem cell (51%)
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13 results found


Open access
01 Apr 2016-
Abstract: Single-cell expression profiles of melanoma Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs. Science, this issue p. 189 Melanoma cells show transcriptional heterogeneity. To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

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57 Citations


Journal ArticleDOI: 10.1182/BLOOD-2018-99-109544
Mark R. Looney1Institutions (1)
21 Nov 2018-Blood
Abstract: Platelets are indispensable in hemostasis, thrombosis, and immune responses. In humans, billions of platelets are produced each day from megakaryocytes, however the mechanisms of mature platelet production are incompletely understood. Megakaryocytes are produced in the bone marrow and have been visualized to communicate with the bone marrow sinusoids to release proplatelet fragments. Megakaryocytes have also been found in other tissues, including the lung, but the function of megakaryocytes in these locations is unclear. Historical data indicate that the lung may be a site of platelet biogenesis. The concentration of megakaryocytes in the blood exiting the lung is much lower than the blood entering the lung (implying filtering) and conversely, platelet counts are higher in blood draining from the lungs. Additionally, when the lung circulation in entirely bypassed, megakaryocytes accumulate in the blood and there is a high incidence of thrombocytopenia. However, direct proof of platelet biogenesis in the lung is lacking. We used lung intravital microscopy combined with fluorescently labeled mouse strains and directly visualized intravascular megakaryocytes releasing platelets in the lung circulation. We also visualized megakaryocytes in the bone marrow and spleen releasing proplatelet fragments, and megakaryocyte migration in toto from the bone marrow, which are presumably the source material for lung platelet production. The megakaryocyte-releasing events in the lung were quantified and represent at least half of the total platelet production in mice, which can be increased by the application of thrombopoietin. We also observed a much larger extravascular pool of megakaryocytes in the lung that were not platelet generating as observed by lung intravital imaging. The function of these lung-resident megakaryocytes is unknown, but RNA-Seq data points to a potential role in lung immunity. Orthotopic, single-lung transplantation experiments into thrombocytopenic and hematopoietic progenitor-deficient animals ( c-mpl -/- ) revealed that peripheral blood platelet counts and bone marrow hematopoietic progenitors could be fully reconstituted by the lung transplant procedure implying the presence of hematopoietic progenitors in the mouse lung. Indeed, these progenitors were directly detected in the extravascular lung and purified populations of hematopoietic progenitors in the lung could correct thrombocytopenia in c-mpl -/- animals. Finally, the lung transplant procedure produced donor-derived chimerism of other hematopoietic lineages such as neutrophils and lymphocytes. We conclude that the lung has significant hematopoietic potential including being a major site of platelet biogenesis. Disclosures No relevant conflicts of interest to declare.

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Topics: Thrombopoietin (61%), Megakaryocyte (60%), Platelet (56%) ... show more

4 Citations


Journal ArticleDOI: 10.1182/BLOOD.2021010697
Shu Sun1, Shu Sun2, Chen Jin1, Chen Jin2  +21 moreInstitutions (7)
07 Oct 2021-Blood
Abstract: 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.

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Topics: Hematopoietic stem cell (53%), Progenitor cell (52%), Population (52%) ... show more

3 Citations


Open accessJournal ArticleDOI: 10.1002/SCTM.21-0264
Abstract: Platelets, the chief effector of hemostasis, are small anucleate blood cells generated from megakaryocytes (MKs), and the defects in platelet production or function lead to a variety of bleeding complications. Emerging evidence indicates that MKs and platelets are much more diverse than previously appreciated and involved in many physiological and pathological processes besides hemostasis, such as innate and adaptive immune responses, angiogenesis, and tumor metastasis, while the ontogenic variations in MK and platelet function have also become a focus in the field. However, whether MKs and platelets fulfill these distinct functions by utilizing distinct subpopulations remains poorly understood. New studies aimed at deciphering the MK transcriptome at the single-cell level have provided some key insights into the functional heterogeneity of MKs. In this review, we will discuss some of the recent discoveries of functional and developmental heterogeneity of MKs and its potential link to the heterogeneity of platelets. We will also discuss the implications of these findings while focusing on the ex vivo generation of platelets from human pluripotent stem cells. The improved understanding of the heterogeneity underlying human MK development and platelet production should open new avenues for future platelet regeneration and clinical treatment of related diseases.

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1 Citations


Journal ArticleDOI: 10.1182/BLOOD.2020008118
08 Jul 2021-Blood
Abstract: Mutations in the transcription factors GATA binding factor 1 (GATA1), growth factor independence 1B (GFI1B), and Runt-related transcription factor 1 (RUNX1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction resulting in a grayish appearance in blood smears. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from 11 individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1Q154Rfs, or RUNX1TD2-6 and 28 healthy controls were examined by label-free quantitative mass spectrometry. In total, 2875 platelet proteins were reliably quantified. Clustering analysis of more than 300 differentially expressed proteins revealed profound differences between cases and controls. Among cases, 44 of 143 significantly downregulated proteins were assigned to platelet function, hemostasis, and granule biology, in line with platelet dysfunction and bleedings. Remarkably, none of these proteins were significantly diminished in all affected cases. Similarly, no proteins were commonly overrepresented in all affected cases compared with controls. These data indicate that the studied transcription factor mutations alter platelet proteomes in distinct largely nonoverlapping manners. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders.

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Topics: Blood Platelet Disorders (64%), Platelet (54%), GATA1 (53%) ... show more

1 Citations


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103 results found


Open accessJournal ArticleDOI: 10.1073/PNAS.0506580102
Abstract: Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

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26,320 Citations


Open accessJournal ArticleDOI: 10.1101/GR.1239303
Paul Shannon1, Andrew Markiel, Owen Ozier, Nitin S. Baliga  +5 moreInstitutions (1)
01 Nov 2003-Genome Research
Abstract: Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.

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Topics: Interaction network (60%), ConsensusPathDB (55%), Human interactome (53%) ... show more

23,868 Citations


Open accessJournal ArticleDOI: 10.1093/BIOINFORMATICS/BTS635
01 Jan 2013-Bioinformatics
Abstract: Motivation Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. Results To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. Availability and implementation STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from http://code.google.com/p/rna-star/.

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Topics: MRNA Sequencing (57%)

20,172 Citations


Open accessJournal ArticleDOI: 10.1089/OMI.2011.0118
Abstract: Increasing quantitative data generated from transcriptomics and proteomics require integrative strategies for analysis Here, we present an R package, clusterProfiler that automates the process of biological-term classification and the enrichment analysis of gene clusters The analysis module and visualization module were combined into a reusable workflow Currently, clusterProfiler supports three species, including humans, mice, and yeast Methods provided in this package can be easily extended to other species and ontologies The clusterProfiler package is released under Artistic-20 License within Bioconductor project The source code and vignette are freely available at http://bioconductororg/packages/release/bioc/html/clusterProfilerhtml

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Topics: Bioconductor (61%)

8,173 Citations


Open accessJournal ArticleDOI: 10.1038/NBT.4096
Abstract: Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are present across multiple data sets remains challenging. Here, we introduce an analytical strategy for integrating scRNA-seq data sets based on common sources of variation, enabling the identification of shared populations across data sets and downstream comparative analysis. We apply this approach, implemented in our R toolkit Seurat (http://satijalab.org/seurat/), to align scRNA-seq data sets of peripheral blood mononuclear cells under resting and stimulated conditions, hematopoietic progenitors sequenced using two profiling technologies, and pancreatic cell 'atlases' generated from human and mouse islets. In each case, we learn distinct or transitional cell states jointly across data sets, while boosting statistical power through integrated analysis. Our approach facilitates general comparisons of scRNA-seq data sets, potentially deepening our understanding of how distinct cell states respond to perturbation, disease, and evolution.

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4,666 Citations


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