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• W3112974370 abstract "•A comprehensive single-cell transcriptomic landscape of human MKs is constructed•MKs show cellular heterogeneity with distinct metabolic and cell cycle signatures•CD14+ MKs with immune characteristics are generated along a distinct trajectory•THBS1 is identified as an early marker for MK-biased endothelial cells from hESCs 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. 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. Megakaryocytes (MKs) are large (50–100 μm in diameter), rare (0.05%–0.1%), polyploid hematopoietic cells that, in adults, predominantly reside in the bone marrow (BM), where they are responsible for platelet production (Ebaugh and Bird, 1951Ebaugh Jr., F.G. Bird R.M. The normal megakaryocyte concentration in aspirated human bone marrow.Blood. 1951; 6: 75-80Crossref PubMed Google Scholar). MKs are also involved in coagulation, homeostasis, inflammation, angiogenesis, and innate immunity (Cunin and Nigrovic, 2019Cunin P. Nigrovic P.A. Megakaryocytes as immune cells.J. Leukoc. Biol. 2019; 105: 1111-1121Crossref PubMed Scopus (21) Google Scholar; Noetzli et al., 2019Noetzli L.J. French S.L. Machlus K.R. New Insights Into the Differentiation of Megakaryocytes From Hematopoietic Progenitors.Arterioscler. Thromb. Vasc. Biol. 2019; 39: 1288-1300Crossref PubMed Scopus (55) Google Scholar) as well as secreting inflammatory factors, cytokines, and chemokines that regulate the behavior of hematopoietic stem cells (HSCs) and other cell types in the BM (Bruns et al., 2014Bruns I. Lucas D. Pinho S. Ahmed J. Lambert M.P. Kunisaki Y. Scheiermann C. Schiff L. Poncz M. Bergman A. Frenette P.S. Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion.Nat. Med. 2014; 20: 1315-1320Crossref PubMed Scopus (347) Google Scholar; Capitano et al., 2018Capitano M. Zhao L. Cooper S. Thorsheim C. Suzuki A. Huang X. Dent A.L. Marks M.S. Abrams C.S. Broxmeyer H.E. Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-β1 secretion and hematopoiesis in mice.Blood. 2018; 132: 1027-1038Crossref PubMed Scopus (6) Google Scholar; Heazlewood et al., 2013Heazlewood S.Y. Neaves R.J. Williams B. Haylock D.N. Adams T.E. Nilsson S.K. Megakaryocytes co-localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation.Stem Cell Res. (Amst.). 2013; 11: 782-792Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar; Jiang et al., 1994Jiang S. Levine J.D. Fu Y. Deng B. London R. Groopman J.E. Avraham H. Cytokine production by primary bone marrow megakaryocytes.Blood. 1994; 84: 4151-4156Crossref PubMed Google Scholar, Jiang et al., 2017Jiang J. Kao C.Y. Papoutsakis E.T. How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells?.J. Control. Release. 2017; 247: 1-18Crossref PubMed Scopus (35) Google Scholar; Malara et al., 2014Malara A. Currao M. Gruppi C. Celesti G. Viarengo G. Buracchi C. Laghi L. Kaplan D.L. Balduini A. Megakaryocytes contribute to the bone marrow-matrix environment by expressing fibronectin, type IV collagen, and laminin.Stem Cells. 2014; 32: 926-937Crossref PubMed Scopus (85) Google Scholar; Nakamura-Ishizu et al., 2014Nakamura-Ishizu A. Takubo K. Fujioka M. Suda T. Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin.Biochem. Biophys. Res. Commun. 2014; 454: 353-357Crossref PubMed Scopus (87) Google Scholar; Zhao et al., 2014Zhao M. Perry J.M. Marshall H. Venkatraman A. Qian P. He X.C. Ahamed J. Li L. Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells.Nat. Med. 2014; 20: 1321-1326Crossref PubMed Scopus (333) Google Scholar). Along with their roles in the BM, recent studies have detected numerous MKs in the lungs that express multiple immune receptors and mediators, suggestive of important but undefined, functions (Lefrançais and Looney, 2019Lefrançais E. Looney M.R. Platelet Biogenesis in the Lung Circulation.Physiology (Bethesda). 2019; 34: 392-401Crossref PubMed Scopus (21) Google Scholar; Lefrançais et al., 2017Lefrançais E. Ortiz-Muñoz G. Caudrillier A. Mallavia B. Liu F. Sayah D.M. Thornton E.E. Headley M.B. David T. Coughlin S.R. et al.The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.Nature. 2017; 544: 105-109Crossref PubMed Scopus (486) Google Scholar). Thus, the varied and critical roles of MKs call for a thorough understanding of their development, biology, and contribution to health and disease. Development of the human hematopoietic system begins early, from 3–4 weeks post-conception (WPC), and occurs at multiple sites in the embryo, including the yolk sac (YS), aorta-gonad-mesonephros (AGM) region, and the fetal liver (FL) (Ivanovs et al., 2017Ivanovs A. Rybtsov S. Ng E.S. Stanley E.G. Elefanty A.G. Medvinsky A. Human haematopoietic stem cell development: from the embryo to the dish.Development. 2017; 144: 2323-2337Crossref PubMed Scopus (100) Google Scholar; Kelemen et al., 1979Kelemen E. Calvo W. Fliedner T.M. Atlas of human hemopoietic development. Springer, 1979Crossref Google Scholar; Moore, 1982Moore K.L. The developing human: clinically oriented embryology.Third Edition. Saunders, 1982Google Scholar; Petti et al., 1985Petti S. Testa U. Migliaccio A.R. Mavilio F. Marinucci M. Lazzaro D. Russo G. Mastroberardino G. Peschle C. Embryonic hemopoiesis in human liver: morphologic aspects at sequential stages of ontogenic development.Prog. Clin. Biol. Res. 1985; 193: 57-71PubMed Google Scholar; Tavian and Péault, 2005Tavian M. Péault B. Embryonic development of the human hematopoietic system.Int. J. Dev. Biol. 2005; 49: 243-250Crossref PubMed Scopus (184) Google Scholar). So far, most of the studies of human hematopoietic development in vivo have focused on erythrocytes and progenitor cells (Emerson et al., 1989Emerson S.G. Thomas S. Ferrara J.L. Greenstein J.L. Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver.Blood. 1989; 74: 49-55Crossref PubMed Google Scholar; Migliaccio et al., 1986Migliaccio G. Migliaccio A.R. Petti S. Mavilio F. Russo G. Lazzaro D. Testa U. Marinucci M. Peschle C. Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac----liver transition.J. Clin. Invest. 1986; 78: 51-60Crossref PubMed Scopus (213) Google Scholar; Peschle et al., 1981Peschle C. Migliaccio A.R. Migliaccio G. Ciccariello R. Lettieri F. Quattrin S. Russo G. Mastroberardino G. Identification and characterization of three classes of erythroid progenitors in human fetal liver.Blood. 1981; 58: 565-572Crossref PubMed Google Scholar; Stamatoyannopoulos et al., 1987Stamatoyannopoulos G. Constantoulakis P. Brice M. Kurachi S. Papayannopoulou T. Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching.Dev. Biol. 1987; 123: 191-197Crossref PubMed Scopus (25) Google Scholar; Tavian and Péault, 2005Tavian M. Péault B. Embryonic development of the human hematopoietic system.Int. J. Dev. Biol. 2005; 49: 243-250Crossref PubMed Scopus (184) Google Scholar). With the advance of single-cell technologies, we and others have recently defined the transcriptomic features of multiple hematopoietic cells in human embryos, including HSCs, lymphoid progenitors, and macrophages (Bian et al., 2020Bian Z. Gong Y. Huang T. Lee C.Z.W. Bian L. Bai Z. Shi H. Zeng Y. Liu C. He J. et al.Deciphering human macrophage development at single-cell resolution.Nature. 2020; 582: 571-576Crossref PubMed Scopus (104) Google Scholar; Park et al., 2020Park J.E. Botting R.A. Dominguez Conde C. Popescu D.M. Lavaert M. Kunz D.J. Goh I. Stephenson E. Ragazzini R. Tuck E. et al.A cell atlas of human thymic development defines T cell repertoire formation.Science. 2020; 367: eaay3224Crossref PubMed Scopus (134) Google Scholar; Popescu et al., 2019Popescu D.M. Botting R.A. Stephenson E. Green K. Webb S. Jardine L. Calderbank E.F. Polanski K. Goh I. Efremova M. et al.Decoding human fetal liver haematopoiesis.Nature. 2019; 574: 365-371Crossref PubMed Scopus (148) Google Scholar; Zeng et al., 2019aZeng Y. He J. Bai Z. Li Z. Gong Y. Liu C. Ni Y. Du J. Ma C. Bian L. et al.Tracing the first hematopoietic stem cell generation in human embryo by single-cell RNA sequencing.Cell Res. 2019; 29: 881-894Crossref PubMed Scopus (53) Google Scholar, Zeng et al., 2019bZeng Y. Liu C. Gong Y. Bai Z. Hou S. He J. Bian Z. Li Z. Ni Y. Yan J. et al.Single-Cell RNA Sequencing Resolves Spatiotemporal Development of Pre-thymic Lymphoid Progenitors and Thymus Organogenesis in Human Embryos.Immunity. 2019; 51: 930-948.e6Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, because of the extreme rarity of MKs in early development and the limited accessibility of human embryonic and fetal tissues, the process of megakaryopoiesis is relatively understudied. Early observations noted the presence of cells with MK morphology at multiple sites in developing human embryos (Allen Graeve and de Alarcon, 1989Allen Graeve J.L. de Alarcon P.A. Megakaryocytopoiesis in the human fetus.Arch. Dis. Child. 1989; 64: 481-484Crossref PubMed Scopus (43) Google Scholar; Fukuda, 1973Fukuda T. Fetal hemopoiesis. I. Electron microscopic studies on human yolk sac hemopoiesis.Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1973; 14: 197-213Crossref Scopus (75) Google Scholar; Kelemen et al., 1979Kelemen E. Calvo W. Fliedner T.M. Atlas of human hemopoietic development. Springer, 1979Crossref Google Scholar; Ma et al., 1996Ma D.C. Sun Y.H. Chang K.Z. Zuo W. Developmental change of megakaryocyte maturation and DNA ploidy in human fetus.Eur. J. Haematol. 1996; 57: 121-127Crossref PubMed Scopus (37) Google Scholar; Nogales Ortiz, 1993Nogales Ortiz F. The Human yolk sac and yolk sac tumors. Springer, 1993Crossref Google Scholar). However, these cells differed markedly from their adult counterparts; for example, human FL MKs are smaller and less mature and have lower ploidy than adult BM MKs (Allen Graeve and de Alarcon, 1989Allen Graeve J.L. de Alarcon P.A. Megakaryocytopoiesis in the human fetus.Arch. Dis. Child. 1989; 64: 481-484Crossref PubMed Scopus (43) Google Scholar; Ma et al., 1996Ma D.C. Sun Y.H. Chang K.Z. Zuo W. Developmental change of megakaryocyte maturation and DNA ploidy in human fetus.Eur. J. Haematol. 1996; 57: 121-127Crossref PubMed Scopus (37) Google Scholar). At present, the molecular and cellular features of early MKs from human embryos remain unknown. Because of the scarcity of fetal tissues and the difficulty of isolating sufficient cells for analysis, human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have become an important tool for the study of early human development (Rossant and Tam, 2017Rossant J. Tam P.P.L. New Insights into Early Human Development: Lessons for Stem Cell Derivation and Differentiation.Cell Stem Cell. 2017; 20: 18-28Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar; Yiangou et al., 2018Yiangou L. Ross A.D.B. Goh K.J. Vallier L. Human Pluripotent Stem Cell-Derived Endoderm for Modeling Development and Clinical Applications.Cell Stem Cell. 2018; 22: 485-499Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). A variety of MK differentiation protocols have been established from hPSCs and are used to generate functional platelets (Borst et al., 2017Borst S. Sim X. Poncz M. French D.L. Gadue P. Induced Pluripotent Stem Cell-Derived Megakaryocytes and Platelets for Disease Modeling and Future Clinical Applications.Arterioscler. Thromb. Vasc. Biol. 2017; 37: 2007-2013Crossref PubMed Scopus (16) Google Scholar; Sugimoto and Eto, 2017Sugimoto N. Eto K. Platelet production from induced pluripotent stem cells.J. Thromb. Haemost. 2017; 15: 1717-1727Crossref PubMed Scopus (40) Google Scholar). By applying these models, we and others have identified several key regulators of human megakaryopoiesis in vitro, including c-MYC, RUNX1, MEIS1, TAL1, GATA1, and FLI1 (Iizuka et al., 2015Iizuka H. Kagoya Y. Kataoka K. Yoshimi A. Miyauchi M. Taoka K. Kumano K. Yamamoto T. Hotta A. Arai S. Kurokawa M. Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis.Exp. Hematol. 2015; 43: 849-857Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar; Li et al., 2018Li Y. Jin C. Bai H. Gao Y. Sun S. Chen L. Qin L. Liu P.P. Cheng L. Wang Q.F. Human NOTCH4 is a key target of RUNX1 in megakaryocytic differentiation.Blood. 2018; 131: 191-201Crossref PubMed Scopus (14) Google Scholar; Moreau et al., 2016Moreau T. Evans A.L. Vasquez L. Tijssen M.R. Yan Y. Trotter M.W. Howard D. Colzani M. Arumugam M. Wu W.H. et al.Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.Nat. Commun. 2016; 7: 11208Crossref PubMed Scopus (143) Google Scholar; Takayama et al., 2010Takayama N. Nishimura S. Nakamura S. Shimizu T. Ohnishi R. Endo H. Yamaguchi T. Otsu M. Nishimura K. Nakanishi M. et al.Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells.J. Exp. Med. 2010; 207: 2817-2830Crossref PubMed Scopus (232) Google Scholar; Toscano et al., 2015Toscano M.G. Navarro-Montero O. Ayllon V. Ramos-Mejia V. Guerrero-Carreno X. Bueno C. Romero T. Lamolda M. Cobo M. Martin F. et al.SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.Mol. Ther. 2015; 23: 158-170Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar; Wang et al., 2018Wang H. Liu C. Liu X. Wang M. Wu D. Gao J. Su P. Nakahata T. Zhou W. Xu Y. et al.MEIS1 Regulates Hemogenic Endothelial Generation, Megakaryopoiesis, and Thrombopoiesis in Human Pluripotent Stem Cells by Targeting TAL1 and FLI1.Stem Cell Reports. 2018; 10: 447-460Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). One aim of such studies is to facilitate generation of platelets from hPSC-derived MKs for clinical use. However, to date, producing platelets in this way is highly inefficient and results in poor-quality products, calling into question the extent to which the MK derivation protocols recapitulate key features of in vivo megakaryopoiesis. Without detailed knowledge of MK generation in human embryos, we cannot properly evaluate hPSC-based models. To fill these important gaps in our understanding of early human megakaryopoiesis, here we aimed to characterize the molecular characteristics, heterogeneity, and developmental trajectories of MKs in human embryos. Using single-cell RNA sequencing (scRNA-seq) of cells from embryonic YS and FL, we identified several subpopulations of MKs with distinct patterns of gene expression that could potentially reflect early functional specification. We also found that the hESC-based differentiation model can simulate early human megakaryopoiesis, including emergence of a subpopulation of MKs expressing immune-related genes. Finally, we identified THBS1 expression as an early marker that can be used to identify MK precursor endothelial cells. To characterize primary MKs from healthy human embryos at the transcriptomic level, we performed droplet-based scRNA-seq (10X Genomics) using cells collected from two YS specimens at 4 WPC and two FL specimens at 8 WPC using different enrichment methods (Figures 1A and S1A; Table S1). From the YS samples, we captured 11,021 cells in total, enabling us to identify nine cell clusters defined by their expression of established marker genes: MKs, erythrocytes, MK-erythroid progenitors, two subpopulations of YS-derived myeloid-biased progenitors (Bian et al., 2020Bian Z. Gong Y. Huang T. Lee C.Z.W. Bian L. Bai Z. Shi H. Zeng Y. Liu C. He J. et al.Deciphering human macrophage development at single-cell resolution.Nature. 2020; 582: 571-576Crossref PubMed Scopus (104) Google Scholar), macrophages, endothelial cells, epithelial cells, and mesenchymal cells (Figures 1B and S1B–S1D). In the MK population, we observed specific expression of genes encoding platelet factor 4 (PF4) and pro-platelet basic protein (PPBP) (Figure 1C). Furthermore, we detected specific expression of KLF1, GYPA, and ALAS2 in erythrocytes (Erys); GATA1, GATA2, and CD34 in MK-erythroid progenitor (MEPs); CD34 and SPINK2 in YS-derived myeloid-biased progenitors (YSMPs); C1QA and CD14 in macrophages (Macs); CDH5 in endothelial cells (ECs); EPCAM and AFP in epithelial cells (Epis); and ACTC1 in mesenchymal cells (Mes) (Figures 1C and S1E; Tables S2A and S3A). We also validated the identity of these cell clusters using Gene Ontology (GO) enrichment analysis (Figure S1F). In MKs, the major biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched were related to “platelet activation” and “regulation of actin cytoskeleton,” consistent with their known functions (Figure 1D). Interestingly, within the MK population (defined by expression of the MK markers CD41a and CD42b), we observed two distinct morphologies: some MKs (16 of 29) possessed numerous cytoplasmic blebs, similar to the previously reported diploid platelet-forming cells in the mouse YS (Figures 1E and 1F, bottom; Potts et al., 2014Potts K.S. Sargeant T.J. Markham J.F. Shi W. Biben C. Josefsson E.C. Whitehead L.W. Rogers K.L. Liakhovitskaia A. Smyth G.K. et al.A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo.Blood. 2014; 124: 2725-2729Crossref PubMed Scopus (35) Google Scholar, Potts et al., 2015Potts K.S. Sargeant T.J. Dawson C.A. Josefsson E.C. Hilton D.J. Alexander W.S. Taoudi S. Mouse prenatal platelet-forming lineages share a core transcriptional program but divergent dependence on MPL.Blood. 2015; 126: 807-816Crossref PubMed Scopus (19) Google Scholar), implying that this type of MK might be in the process of producing platelets; in contrast, the remaining MKs (13 of 29) exhibited relatively “smooth” cell membranes (Figures 1E and 1F, top), perhaps indicative of a less mature or functionally distinct state. In addition, fluorescence-activated cell sorting (FACS)-isolated Erys and Macs exhibited the expected morphologies (Figures S1G–S1I). To closely examine the transcriptional landscape of the MK population from the YS, we next identified the top 10 most highly expressed transcription factors (TFs) and cell surface marker genes (Figure 1G). Among the top 10 TFs were several with established essential roles in megakaryopoiesis and thrombopoiesis, such as NFE2, GFI1B, LYL1, GATA1, and MYLK (Chiu et al., 2019Chiu S.K. Orive S.L. Moon M.J. Saw J. Ellis S. Kile B.T. Huang Y. Chacon D. Pimanda J.E. Beck D. et al.Shared roles for Scl and Lyl1 in murine platelet production and function.Blood. 2019; 134: 826-835Crossref PubMed Scopus (7) Google Scholar; Foudi et al., 2014Foudi A. Kramer D.J. Qin J. Ye D. Behlich A.S. Mordecai S. Preffer F.I. Amzallag A. Ramaswamy S. Hochedlinger K. et al.Distinct, strict requirements for Gfi-1b in adult bone marrow red cell and platelet generation.J. Exp. Med. 2014; 211: 909-927Crossref PubMed Scopus (45) Google Scholar; Meinders et al., 2015Meinders M. Kulu D.I. van de Werken H.J. Hoogenboezem M. Janssen H. Brouwer R.W. van Ijcken W.F. Rijkers E.J. Demmers J.A. Krüger I. et al.Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function.Blood. 2015; 125: 1957-1967Crossref PubMed Scopus (40) Google Scholar; Shivdasani et al., 1997Shivdasani R.A. Fujiwara Y. McDevitt M.A. Orkin S.H. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development.EMBO J. 1997; 16: 3965-3973Crossref PubMed Scopus (570) Google Scholar; Shivdasani et al., 1995Shivdasani R.A. Rosenblatt M.F. Zucker-Franklin D. Jackson C.W. Hunt P. Saris C.J. Orkin S.H. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development.Cell. 1995; 81: 695-704Abstract Full Text PDF PubMed Scopus (606) Google Scholar; Figure 1G, left). Furthermore, many well-known MK marker genes, such as SELP, GP1BA, CD9, and ITGA2B (Clay et al., 2001Clay D. Rubinstein E. Mishal Z. Anjo A. Prenant M. Jasmin C. Boucheix C. Le Bousse-Kerdilès M.C. CD9 and megakaryocyte differentiation.Blood. 2001; 97: 1982-1989Crossref PubMed Scopus (50) Google Scholar; Majka et al., 2001Majka M. Baj-Krzyworzeka M. Kijowski J. Reca R. Ratajczak J. Ratajczak M.Z. 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Thus, MKs from the human YS abundantly express several canonical MK-related genes and potential novel regulators of human megakaryopoiesis that warrants further study. Because early megakaryopoiesis in mammals occurs in the YS and FL (Palis, 2016Palis J. Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo.FEBS Lett. 2016; 590: 3965-3974Crossref PubMed Scopus (61) Google Scholar), we also collected cells from the FL at 8 WPC for comparative analysis (Figure 1A; Table S1). In total, we isolated 17,677 cells and identified 15 cell populations, including MKs, Erys, and Monocyte-Macs (Figures 2A and S2A–S2C), in keeping with a recent study of hematopoiesis in human FL (Popescu et al., 2019Popescu D.M. Botting R.A. Stephenson E. Green K. Webb S. Jardine L. Calderbank E.F. Polanski K. Goh I. Efremova M. et al.Decoding human fetal liver haematopoiesis.Nature. 2019; 574: 365-371Crossref PubMed Scopus (148) Google Scholar). The expression of established markers in each cell population was clear (Figure 2B; Tables S2B and S3B); in MKs, we again saw specific PF4 and PPBP expression as well as enrichment of “platelet activation” and “coagulation” (Figures 2B and S2D). Morphologically, FACS-isolated MKs, Erys, and Macs from FL exhibited the expected phenotypes (Figures 2C, 2D, and S2E–S2G). Interestingly, we found that FL contained a significantly smaller proportion of haploid MKs and a significantly greater proportion of 8N or more polyploid MKs (around 15%) than the YS (around 1.7%) (Figure 2E). These findings are consistent with previous report in mice (Potts et al., 2015Potts K.S. Sargeant T.J. Dawson C.A. Josefsson E.C. Hilton D.J. Alexander W.S. Taoudi S. Mouse prenatal platelet-forming lineages share a core transcriptional program but divergent dependence on MPL.Blood. 2015; 126: 807-816Crossref PubMed Scopus (19) Google Scholar). To explore the potential differences between megakaryopoiesis in the YS and FL, we examined significantly differentially expressed genes among MKs from these two sources. We found that several glycolysis-associated genes, such as ENO1 and PKM, were expressed more highly in MKs from the YS than in those from the FL (Figure 2F; Table S3C). Consistent with these observations, we saw enrichment of “glycolysis/gluconeogenesis” in MKs from the YS (Figure 2G). Gene set enrichment analysis (GSEA) also revealed enrichment of the “glycolysis” pathway in MKs from the YS (Figure 2H). In contrast, consistent with the high proliferative activity of cells from the FL (Calvanese et al., 2019Calvanese V. Nguyen A.T. Bolan T.J. Vavilina A. Su T. Lee L.K. Wang Y. Lay F.D. Magnusson M. Crooks G.M. et al.MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment.Nature. 2019; 576: 281-286Crossref PubMed Scopus (27) Google Scholar; Ivanovs et al., 2017Ivanovs A. Rybtsov S. Ng E.S. Stanley E.G. Elefanty A.G. Medvinsky A. Human haematopoietic stem cell development: from the embryo to the dish.Development. 2017; 144: 2323-2337Crossref PubMed Scopus (100) Google Scholar; Oberlin et al., 2010Oberlin E. Fleury M. Clay D. Petit-Cocault L. Candelier J.J. Mennesson B. Jaffredo T. Souyri M. VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.Blood. 2010; 116: 4444-4455Crossref PubMed Scopus (33) Google Scholar), MKs from this site were enriched for gene sets associated with “cell cycle” (Figures 2G and 2H). In accordance with these observations, we detected higher expression of the cell cycle-related genes HMGB1, CDK1, and MKI67 in FL MKs compared with YS MKs (Figure 2F). Interestingly, although the β-globin-encoding gene HBB was specifically expressed in MKs from the FL, expression of HBE1, which encodes hemoglobin subunit epsilon 1, was higher in MKs from the YS (Figures 2F and S2H). “Leaky” expression of such typical erythroid genes has been reported previously in fetal MKs (Elagib et al., 2018Elagib K.E. Brock A.T. Goldfarb A.N. Megakaryocyte ontogeny: Clinical and molecular significance.Exp. Hematol. 2018; 61: 1-9Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar; Woo et al., 2013Woo A.J. Wieland K. Huang H. Akie T.E. Piers T. Kim J. Cantor A.B. Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation.J. Clin. Invest. 2013; 123: 3292-3304Crossref Scopus (23) Google Scholar). We found distinct features of MKs from human YS and from FL. We envision that the differentially expressed genes and distinct metabolic fingerprints and cell cycle signatures might be applied to distinguish megakaryopoiesis in these two sites and shed light on the functional development of MKs through embryogenesis. To further characterize cellular heterogeneity, we pooled all MKs from the YS and FL for detailed analysis. Six transcriptionally heterogeneous subpopulations of MKs were identified in the YS and FL (Figures 3A–3D and S3A; Table S2C). We then assessed the expression of marker genes in each subpopulation and characterized the gene sets enriched in the different MK clusters (Figures 3B and 3C; Table S3D). MK1 showed enrichment of GO terms related to glycolysis (Figure 3C), whereas MK2 exhibited enriched GO terms including “positive regulation of cell cycle” (Figures 3C and S3B). MK3 highly expressed genes associated with “blood coagulation” and might therefore represent a subset of MKs dedicated to early platelet production (Figure 3C). Indeed, the top 10 differentially expressed genes (DEGs) enriched in the MK3 subpopulation included CCL5, which reportedly increases MK ploidy and subsequent proplatelet formation in a CCR5-dependent manner (Figure 3B; Machlus et al., 2016Machlus K.R. Johnson K.E. Kulenthirarajan R. Forward J.A. Tippy M.D. Soussou T.S. El-Husayni S.H. Wu S.K. Wang S. Watnick R.S. et al.CCL5 derived from platelets increases megakaryocyte proplatelet formation.Blood. 2016; 127: 921-926Crossref PubMed Scopus (55) Google Scholar). In addition, many other thrombopoiesis-associated genes, such as TUBB1, MYL9, MYLK, GFI1B, NFE2, and GP1BA (Chang et al., 2007Chang Y. Blute" @default.
• W3112974370 created "2020-12-21" @default.
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• W3112974370 date "2021-03-01" @default.
• W3112974370 modified "2023-10-16" @default.
• W3112974370 title "Decoding Human Megakaryocyte Development" @default.
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