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1.
Curr Opin Hematol ; 28(1): 28-35, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33186151

RESUMO

PURPOSE OF REVIEW: This review focuses on our current understanding of fate decisions in bipotent megakaryocyte-erythroid progenitors (MEPs). Although extensive research has been carried out over decades, our understanding of how MEP commit to the erythroid versus megakaryocyte fate remains unclear. RECENT FINDINGS: We discuss the isolation of primary human MEP, and focus on gene expression patterns, epigenetics, transcription factors and extrinsic factors that have been implicated in MEP fate determination. We conclude with an overview of the open debates in the field of MEP biology. SUMMARY: Understanding MEP fate is important because defects in megakaryocyte and erythrocyte development lead to disease states such as anaemia, thrombocytopenia and leukaemia. MEP also represent a model system for studying fundamental principles underlying cell fate decisions of bipotent and pluripotent progenitors, such that discoveries in MEP are broadly applicable to stem/progenitor cell biology.


Assuntos
Hematopoese , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Animais , Linhagem da Célula , Células Eritroides/citologia , Células Eritroides/metabolismo , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Transcriptoma
2.
Blood ; 136(9): 1044-1054, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32548608

RESUMO

Erythropoietin (EPO) provides the major survival signal to maturing erythroid precursors (EPs) and is essential for terminal erythropoiesis. Nonetheless, progenitor cells can irreversibly commit to an erythroid fate well before EPO acts, risking inefficiency if these progenitors are unneeded to maintain red blood cell (RBC) counts. We identified a new modular organization of erythropoiesis and, for the first time, demonstrate that the pre-EPO module is coupled to late EPO-dependent erythropoiesis by megakaryocyte (Mk) signals. Disrupting megakaryocytic transforming growth factor ß1 (Tgfb1) disorganized hematopoiesis by expanding the pre-EPO pool of progenitor cells and consequently triggering significant apoptosis of EPO-dependent EPs. Similarly, pharmacologic blockade of TGFß signaling in normal mice boosted the pre-EPO module, leading to apoptosis of EPO-sensitive EPs. Subsequent treatment with low-dose EPO triggered robust RBC production in both models. This work reveals modular regulation of erythropoiesis and offers a new strategy for overcoming chronic anemias.


Assuntos
Células Precursoras Eritroides/citologia , Eritropoese/fisiologia , Megacariócitos/citologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Células Precursoras Eritroides/metabolismo , Eritropoetina/farmacologia , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quimera por Radiação , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia
3.
Biochem Biophys Res Commun ; 528(1): 46-53, 2020 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-32456797

RESUMO

GATA1 is a master transcription factor of megakaryopoiesis and erythropoiesis, and loss-of-function mutation can induce accumulation of megakaryocyte-erythroid progenitors (MEPs) in mice and humans. Accordingly, the murine MEP cell line (termed G1ME2 cells) encoding doxycycline (dox)-inducible anti-Gata1 shRNA on Hprt locus has been developed. The cells were CD41+CD71+KIT+, expand under dox, stem cell factor, and thrombopoietin (TPO), and terminally differentiate into erythroid cells or megakaryocytes upon removal of dox. Surprisingly, in this study, these Gata1low murine MEPs displayed accelerated growth from around 90-100 days after cell culture, impeded megakaryocytic potential, and maintained erythropoiesis. We specified them as late G1ME2 cells and discovered that increased CD41-KIT+ population during long-term culture was the main reason for the delayed megakaryopoiesis. The CD41 expression level was partially de-repressed by PI3K/AKT inhibitors, suggesting that TPO-mediated cell survival signaling pathway might have impacted on CD41 in the late G1ME2 cells. Nevertheless, among the late cells, the CD41+KIT+ cells could still generate megakaryocytes on dox withdrawal. Taken together, G1ME2 cells could provide a good model to study molecular mechanism of hematopoiesis because of their ability to expand excessively without artificial immortalization.


Assuntos
Diferenciação Celular , Fator de Transcrição GATA1/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Células Cultivadas , Camundongos , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Transdução de Sinais
4.
Transfusion ; 60(3): 561-574, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32086956

RESUMO

BACKGROUND: To date, several cases of transfusion-transmitted ZIKV infections have been confirmed. Multiple studies detected prolonged occurrence of ZIKV viral RNA in whole blood as compared to plasma samples indicating potential ZIKV interaction with hematopoietic cells. Also, infection of cells from the granulocyte/macrophage lineage has been demonstrated. Patients may develop severe thrombocytopenia, microcytic anemia, and a fatal course of disease occurred in a patient with sickle cell anemia suggesting additional interference of ZIKV with erythroid and megakaryocytic cells. Therefore, we analyzed whether ZIKV propagates in or compartmentalizes with hematopoietic progenitor, erythroid, and megakaryocytic cells. METHODS: ZIKV RNA replication, protein translation and infectious particle formation in hematopoietic cell lines as well as primary CD34+ HSPCs and ex vivo differentiated erythroid and megakaryocytic cells was monitored using qRT-PCR, FACS, immunofluorescence analysis and infectivity assays. Distribution of ZIKV RNA and infectious particles in spiked red blood cell (RBC) units or platelet concentrates (PCs) was evaluated. RESULTS: While subsets of K562 and KU812Ep6EPO cells supported ZIKV propagation, primary CD34+ HSPCs, MEP cells, RBCs, and platelets were non-permissive for ZIKV infection. In spiking studies, ZIKV RNA was detectable for 7 days in all fractions of RBC units and PCs, however, ZIKV infectious particles were not associated with erythrocytes or platelets. CONCLUSION: Viral particles from plasma or contaminating leukocytes, rather than purified CD34+ HSPCs or the cellular component of RBC units or PCs, present the greatest risk for transfusion-transmitted ZIKV infections.


Assuntos
Antígenos CD34/metabolismo , Plaquetas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Infecção por Zika virus/metabolismo , Zika virus/patogenicidade , Diferenciação Celular/fisiologia , Linhagem Celular , Eritrócitos/citologia , Humanos , RNA Viral/genética
5.
Blood ; 134(18): 1484-1485, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31698443
6.
Blood ; 134(7): 614-625, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31270104

RESUMO

Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16 s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.


Assuntos
Transformação Celular Neoplásica/patologia , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/patologia , Células Progenitoras de Megacariócitos e Eritrócitos/patologia , Fatores de Transcrição/genética , Animais , Transformação Celular Neoplásica/genética , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Leucemia Mieloide Aguda/genética , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Translocação Genética
7.
Blood ; 133(13): 1427-1435, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30728145

RESUMO

The classical model of hematopoiesis proposes a hierarchy in which a small number of multipotent hematopoietic stem cells (HSCs) maintain all blood lineages by giving rise to progeny that pass through discrete progenitor stages. At each stage, lineage differentiation potential is restricted, coupled with the loss of ability to self-renew. Recently, single-cell approaches have been used to test certain assumptions made by this model, in particular relating to megakaryocyte (Mk) and erythroid (E) development. An alternative model has emerged in which substantial heterogeneity and lineage-priming exists within the HSC compartment, including the existence of multipotent but megakaryocyte/platelet-biased HSCs. Hematopoietic differentiation follows a hierarchical continuum, passing through cellular nodes and branch points. Megakaryocytes are produced via a shared pathway with the erythroid lineage, also shared in its early stages with mast cells, eosinophils, and basophils, but separate from other myeloid and lymphoid lineages. In addition, distinct pathways for direct differentiation of Mk from HSCs may coexist and could be important in situations of increased physiological requirements or in malignancies. Further work at single-cell resolution using multiomic approaches and examining Mk-E biased subsets within their physiological context will undoubtedly improve our understanding of normal hematopoiesis and ability to manipulate this in pathology.


Assuntos
Células Eritroides/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Análise de Célula Única/métodos , Animais , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/citologia
8.
Cell Rep ; 25(11): 2935-2936, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30540928

RESUMO

Beige fat cells hold significant promise for reducing obesity and metabolic disease. Su et al. (2018) present a robust method to produce human beige adipocytes from pluripotent stem cells via the generation of an expandable intermediary population of mesenchymal progenitor cells.


Assuntos
Adipócitos Bege , Células-Tronco Mesenquimais , Ciclo Celular , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos , Obesidade
9.
Cell Rep ; 25(8): 2083-2093.e4, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30463007

RESUMO

Megakaryocytic-erythroid progenitors (MEPs) give rise to the cells that produce red blood cells and platelets. Although the mechanisms underlying megakaryocytic (MK) and erythroid (E) maturation have been described, those controlling their specification from MEPs are unknown. Single-cell RNA sequencing of primary human MEPs, common myeloid progenitors (CMPs), megakaryocyte progenitors, and E progenitors revealed a distinct transitional MEP signature. Inferred regulatory transcription factors (TFs) were associated with differential expression of cell cycle regulators. Genetic manipulation of selected TFs validated their role in lineage specification and demonstrated coincident modulation of the cell cycle. Genetic and pharmacologic modulation demonstrated that cell cycle activation is sufficient to promote E versus MK specification. These findings, obtained from healthy human cells, lay a foundation to study the mechanisms underlying benign and malignant disease states of the megakaryocytic and E lineages.


Assuntos
Ciclo Celular , Linhagem da Célula , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais , Transcrição Genética , Proteína Supressora de Tumor p53/metabolismo
10.
DNA Cell Biol ; 37(10): 831-838, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30124330

RESUMO

Nd1-S is the nuclear-localizing short variant form of Nd1 (Ivns1abp) encoding a Kelch family transcription factor. While the function of Nd1 has been investigated in the context of metastasis and doxorubicin-induced cardiotoxicity, little is known about its role in hematopoiesis. In this study, we investigated the function of Nd1-S in hematopoiesis by transplanting the Nd1-S-overexpressing murine hematopoietic stem and progenitor cells (HSPCs) into recipient mice (Nd1-S mice). Enforced expression of Nd1-S led to erythroid and megakaryocyte dysplasia, demonstrated by dramatically decreased red blood cells and platelets, and megakaryocytes in the peripheral blood and bone marrow of the Nd1-S mice. Moreover, phenotypic megakaryocyte-erythroid progenitors (MEPs) accumulated in these Nd1-S mice with aberrant morphology and defective colony-forming capability. Furthermore, these phenotypic MEPs showed impaired pathways regulating erythroid differentiation and megakaryocyte development. Therefore, our study provides de novo evidence that overexpression of Nd1-S in HSPCs leads to erythroid and megakaryocyte dysplasia in vivo by targeting MEPs.


Assuntos
Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Síndromes Mielodisplásicas/genética , Proteínas/genética , Animais , Diferenciação Celular , Feminino , Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Células Progenitoras de Megacariócitos e Eritrócitos/patologia , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteínas/metabolismo , Transgenes , Irradiação Corporal Total
11.
Stem Cells ; 36(8): 1138-1145, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29658164

RESUMO

Hematopoietic stem and progenitor cells maintain blood formation throughout our lifetime by undergoing long- and short-term self-renewal, respectively. As progenitor cells progress through the hematopoiesis process, their differentiation capabilities narrow, such that the precursors become committed to only one or two lineages. This Review focuses on recent advances in the identification and characterization of bipotent megakaryocytic-erythroid progenitors (MEP), the cells that can further produce two completely different functional outputs: platelets and red blood cells. The existence of MEP has sparked controversy as studies describing the requirement for this intermediate progenitor stage prior to commitment to the erythroid and megakaryocytic lineages have been potentially contradictory. Interpretation of these studies is complicated by the variety of species, cell sources, and analytical approaches used along with inherent challenges in the continuum of hematopoiesis, where hematopoietic progenitors do not stop at discrete steps on single paths as classically drawn in hematopoietic hierarchy models. With the goal of improving our understanding of human hematopoiesis, we discuss findings in both human and murine cells. Based on these data, MEP clearly represent a transitional stage of differentiation in at least one route to the generation of both megakaryocytes and erythroid cells. Stem Cells 2018;36:1138-1145.


Assuntos
Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Animais , Linhagem da Célula , Eritropoese , Humanos
12.
Br J Haematol ; 180(4): 526-533, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29377069

RESUMO

The mechanisms underlying lenalidomide-resistance of del(5q) MDS stem cells remain to be elucidated and may include cell-intrinsic as well as microenvironmental causes. Abnormal hypolobated megakaryocytes constitute one of the hallmarks of del(5q) MDS. We hypothesized that these cells have potential implications for the regulation of haematopoietic stem cells (HSC) similarly to what has recently been described for megakaryocytes in the murine system. Therefore, we conducted a study to determine the response of abnormal hypolobated megakaryocytes to lenalidomide therapy. We studied lenalidomide-treated patients in the MDS-004 trial as well as a cohort seen at our institution. Morphological evaluation at time of complete cytogenetic remission (CCyR) demonstrated the persistence of hypolobated megakaryocytes in all evaluable patients (n = 9). Furthermore, we provide evidence that the abnormal hypolobated morphology is restricted to del(5q) megakaryocytes, both at diagnosis and during CCyR. Using fluorescence in situ hybridisation analysis on flow-sorted stem- and progenitor populations, we observed a similar degree of clonal involvement in megakaryocyte-erythroid-progenitors as in HSC. Taken together, our findings suggest that megakaryocyte morphology might aid in the evaluation of patients where discontinuation of lenalidomide is considered and offers interesting hypotheses for further investigation of lenalidomide resistance.


Assuntos
Antineoplásicos/uso terapêutico , Deleção Cromossômica , Cromossomos Humanos Par 5 , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Megacariócitos/metabolismo , Talidomida/análogos & derivados , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Medula Óssea/patologia , Evolução Clonal , Análise Citogenética , Neoplasias Hematológicas/diagnóstico , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Lenalidomida , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/patologia , Megacariócitos/patologia , Indução de Remissão , Talidomida/administração & dosagem , Talidomida/efeitos adversos , Talidomida/uso terapêutico , Resultado do Tratamento
13.
Curr Opin Hematol ; 24(3): 173-182, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28212192

RESUMO

PURPOSE OF REVIEW: The discovery of several genetic variants associated with erythroid traits and subsequent elucidation of their functional mechanisms are exemplars of the power of the new genetic and genomic technology. The present review highlights findings from recent genetic studies related to the control of erythropoiesis and dyserythropoiesis, and fetal hemoglobin, an erythroid-related trait. RECENT FINDINGS: Identification of the genetic modulators of erythropoiesis involved two approaches: genome-wide association studies (GWASs) using single nucleotide polymorphism (SNP) arrays that revealed the common genetic variants associated with erythroid phenotypes (hemoglobin, red cell count, MCV, MCH) and fetal hemoglobin; and massive parallel sequencing such as whole genome sequencing (WGS) and whole exome sequencing (WES) that led to the discovery of the rarer variants (GFI1B, SBDS, RPS19, PKLR, EPO, EPOR, KLF1, GATA1). Functional and genomic studies aided by computational approaches and gene editing technology refined the regions encompassing the putative causative SNPs and confirmed their regulatory role at different stages of erythropoiesis. SUMMARY: Five meta-analysis of GWASs identified 17 genetic loci associated with erythroid phenotypes, which are potential regulators of erythropoiesis. Some of these loci showed pleiotropy associated with multiple erythroid traits, suggesting undiscovered molecular mechanisms and challenges underlying erythroid biology. Other sequencing strategies (WGS and WES) further elucidated the role of rare variants in dyserythropoiesis. Integration of common and rare variant studies with functional assays involving latest genome-editing technologies will significantly improve our understanding of the genetics underlying erythropoiesis and erythroid disorders.


Assuntos
Eritropoese/genética , Regulação da Expressão Gênica , Variação Genética , Animais , Biomarcadores , Diferenciação Celular/genética , Estudo de Associação Genômica Ampla , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinas/genética , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Locos de Características Quantitativas , Característica Quantitativa Herdável
14.
Blood ; 129(3): 358-370, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-27815262

RESUMO

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53-/- mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53-/- bone marrow cells rapidly develop a highly penetrant AML. We find that p53-/- cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53-/- MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53-/- synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.


Assuntos
Transformação Celular Neoplásica/genética , GTP Fosfo-Hidrolases/genética , Leucemia Mieloide Aguda/patologia , Células Progenitoras de Megacariócitos e Eritrócitos/patologia , Proteínas de Membrana/genética , Proteína Supressora de Tumor p53/genética , Animais , Transplante de Medula Óssea , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Mutação , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiência
16.
Blood ; 128(11): 1503-15, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27443289

RESUMO

Acute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFß-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.


Assuntos
Diferenciação Celular , Transformação Celular Neoplásica/patologia , Leucemia Experimental/patologia , Células Progenitoras de Megacariócitos e Eritrócitos/patologia , Proteínas de Fusão Oncogênica/metabolismo , Animais , Western Blotting , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Feminino , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Masculino , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Blood ; 128(7): 923-33, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27268089

RESUMO

Bipotent megakaryocyte/erythroid progenitors (MEPs) give rise to progeny limited to the megakaryocyte (Mk) and erythroid (E) lineages. We developed a novel dual-detection functional in vitro colony-forming unit (CFU) assay for single cells that differentiates down both the Mk and E lineages (CFU-Mk/E), which allowed development and validation of a novel purification strategy for the identification and quantitation of primary functional human MEPs from granulocyte colony-stimulating factor-mobilized peripheral blood and bone marrow. Applying this assay to fluorescence-activated cell sorter-sorted cell populations, we found that the Lin(-)CD34(+)CD38(mid)CD45RA(-)FLT3(-)MPL(+)CD36(-)CD41(-) population is much more highly enriched for bipotent MEPs than any previously reported subpopulations. We also developed purification strategies for primary human lineage-committed Mk and E progenitors identified as CFU-Mk and burst forming unit-E. Comparative expression analyses in MEP, MkP, and ErP populations revealed differential expression of MYB We tested whether alterations in MYB concentration affect the Mk-E fate decision at the single cell level in MEPs and found that short hairpin RNA-mediated MYB knockdown promoted commitment of MEPs to the Mk lineage, further defining its role in MEP lineage fate. There are numerous applications for these novel enrichment strategies, including facilitating mechanistic studies of MEP lineage commitment, improving approaches for in vitro expansion of Mk and E cells, and developing improved therapies for benign and malignant hematologic disease.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD34/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Adulto , Linhagem da Célula , Separação Celular , Ensaio de Unidades Formadoras de Colônias , Células Eritroides/citologia , Células Eritroides/metabolismo , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/citologia , Fenótipo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Receptores de Trombopoetina/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo
18.
Genome Biol ; 17: 83, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27142433

RESUMO

BACKGROUND: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by "bulk" assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined. RESULTS: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: "Pre-MEP," enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; "E-MEP," strongly biased towards erythroid differentiation; and "MK-MEP," a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output. CONCLUSIONS: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias.


Assuntos
Hematopoese , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Análise de Célula Única/métodos , Adulto , Idoso , Linhagem da Célula , Separação Celular/métodos , Células Eritroides/citologia , Células Eritroides/metabolismo , Feminino , Humanos , Masculino , Células Progenitoras de Megacariócitos e Eritrócitos/classificação , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Pessoa de Meia-Idade
19.
Virology ; 493: 162-72, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27058763

RESUMO

Megakaryocyte-erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis.


Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Células Progenitoras de Megacariócitos e Eritrócitos/virologia , Replicação Viral , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Linhagem Celular , Chlorocebus aethiops , Dengue/virologia , Vírus da Dengue/imunologia , Vírus da Dengue/ultraestrutura , Humanos , Camundongos , Células Vero , Proteínas do Envelope Viral/imunologia
20.
Blood ; 127(10): 1242-8, 2016 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-26787736

RESUMO

The classical model of hematopoiesis has long held that hematopoietic stem cells (HSCs) sit at the apex of a developmental hierarchy in which HSCs undergo long-term self-renewal while giving rise to cells of all the blood lineages. In this model, self-renewing HSCs progressively lose the capacity for self-renewal as they transit into short-term self-renewing and multipotent progenitor states, with the first major lineage commitment occurring in multipotent progenitors, thus giving rise to progenitors that initiate the myeloid and lymphoid branches of hematopoiesis. Subsequently, within the myeloid lineage, bipotent megakaryocyte-erythrocyte and granulocyte-macrophage progenitors give rise to unipotent progenitors that ultimately give rise to all mature progeny. However, over the past several years, this developmental scheme has been challenged, with the origin of megakaryocyte precursors being one of the most debated subjects. Recent studies have suggested that megakaryocytes can be generated from multiple pathways and that some differentiation pathways do not require transit through a requisite multipotent or bipotent megakaryocyte-erythrocyte progenitor stage. Indeed, some investigators have argued that HSCs contain a subset of cells with biased megakaryocyte potential, with megakaryocytes directly arising from HSCs under steady-state and stress conditions. In this review, we discuss the evidence supporting these nonclassical megakaryocytic differentiation pathways and consider their relative strengths and weaknesses as well as the technical limitations and potential pitfalls in interpreting these studies. Ultimately, such pitfalls will need to be overcome to provide a comprehensive and definitive understanding of megakaryopoiesis.


Assuntos
Diferenciação Celular/fisiologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Mielopoese/fisiologia , Trombopoese/fisiologia , Animais , Humanos , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Linfopoese/fisiologia , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Megacariócitos/citologia
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