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/farmacologiaRESUMO
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 , TranscriptomaRESUMO
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/citologiaRESUMO
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 SinaisRESUMO
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 , HumanosRESUMO
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/citologiaRESUMO
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/metabolismoRESUMO
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 HerdávelRESUMO
Megakaryocyte and erythroid development are tightly controlled by a repertoire of cytokines, but it is not clear how cytokine-activated signaling pathways are controlled during development of these two lineages. Here, we identify that expression of DLX4, a transcription factor encoded by a homeobox gene, increases during megakaryopoiesis but decreases during erythropoiesis. Enforced expression of DLX4 in CD34(+) stem and progenitor cells and in bipotent K562 cells induced lineage markers and morphologic features of megakaryocytes and repressed erythroid marker expression and hemoglobin levels. Converse results were obtained when DLX4 was knocked down. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression revealed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. DLX4 also induced gene signatures that are associated with nuclear factor κB (NF-κB) signaling. The ability of DLX4 to promote megakaryocyte development at the expense of erythroid generation was diminished by blocking NF-κB activity or by repressing IL1B, a transcriptional target of DLX4. Collectively, our findings indicate that DLX4 exerts opposing effects on the megakaryocytic and erythroid lineages in part by inducing IL-1ß and NF-κB signaling.
Assuntos
Diferenciação Celular/genética , Eritropoese/genética , Proteínas de Homeodomínio/biossíntese , Interleucina-1beta/genética , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Fatores de Transcrição/biossíntese , Linhagem da Célula/genética , Eritrócitos/citologia , Eritrócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Interleucina-1beta/antagonistas & inibidores , Células K562 , Megacariócitos/citologia , Megacariócitos/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genéticaRESUMO
The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk- and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.
Assuntos
Eritropoese/genética , Fator de Transcrição GATA1/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Proteínas Nucleares/fisiologia , Trombopoese/genética , Fatores de Transcrição/fisiologia , Animais , Células da Medula Óssea/citologia , Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/deficiência , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem da Célula , Células Cultivadas/citologia , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/citologia , Fator de Transcrição GATA1/genética , Células Progenitoras de Megacariócitos/citologia , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta-gonad-mesonephros (AGM) region. The co-culture of Sox17-transduced hematopoietic progenitor cells (CD45(low) c-Kit(high) cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17-transduction in CD45(low) c-Kit(high) cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17-transduced cells were cultured with OP9 stromal cells, Sox17-transduced GMPs (Sox17-GMPs), Sox17-transduced CMPs (Sox17-CMPs), and Sox17-transduced MEPs (Sox17-MEPs) were generated. Sox17-GMPs and Sox17-CMPs maintained their self-renewal capacity and the hematopoietic ability upon co-culture with the OP9 stromal cells for some passages. Moreover, Sox17-GMPs exhibited the increase in expression of c-Mpl and GATA-2 in comparison to GMPs of BM and Sox17-CMPs showed the increase in expression of c-Mpl, NF-E2, and ß-globin genes in comparison to CMPs of BM. Furthermore, when Sox17-transduced cells were cultured in methylcellulose to examine the colony-forming ability, Sox17-GMPs and Sox17-CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.
Assuntos
Proteínas HMGB/metabolismo , Células Mieloides/citologia , Fatores de Transcrição SOXF/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Técnicas de Cocultura , Proteínas HMGB/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXF/genéticaRESUMO
MEIS1 is a transcription factor expressed in hematopoietic stem and progenitor cells and in mature megakaryocytes. This biphasic expression of MEIS1 suggests that the function of MEIS1 in stem cells is distinct from its function in lineage committed cells. Mouse models show that Meis1 is required for renewal of stem cells, but the function of MEIS1 in human hematopoietic progenitor cells has not been investigated. We show that two MEIS1 splice variants are expressed in hematopoietic progenitor cells. Constitutive expression of both variants directed human hematopoietic progenitors towards a megakaryocyte-erythrocyte progenitor fate. Ectopic expression of either MEIS1 splice variant in common myeloid progenitor cells, and even in granulocyte-monocyte progenitors, resulted in increased erythroid differentiation at the expense of granulocyte and macrophage differentiation. Conversely, silencing MEIS1 expression in progenitor cells induced a block in erythroid expansion and decreased megakaryocytic colony formation capacity. Gene expression profiling revealed that both MEIS1 splice variants induce a transcriptional program enriched for erythroid and megakaryocytic genes. Our results indicate that MEIS1 expression induces lineage commitment towards a megakaryocyte-erythroid progenitor cell fate in common myeloid progenitor cells through activation of genes that define a megakaryocyte-erythroid-specific gene expression program.
Assuntos
Células Eritroides/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Megacariócitos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Processamento Alternativo , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Análise por Conglomerados , Células Eritroides/citologia , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/citologia , Proteína Meis1 , Trombopoese/genéticaRESUMO
Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. The cofactor FOG1 interacts with transcription factors GATA1 and GATA2 to control erythroid and megakaryocyte (MK) differentiation. In contrast, FOG1 antagonizes the ability of GATA factors to promote mast cell (MC) development. Normal FOG1 function in late-stage erythroid cells and MK requires interaction with the chromatin remodeling complex NuRD. Here, we report that mice in which the FOG1/NuRD interaction is disrupted (Fog(ki/ki)) produce MK-erythroid progenitors that give rise to significantly fewer and less mature MK and erythroid colonies in vitro while retaining multilineage capacity, capable of generating MCs and other myeloid lineage cells. Gene expression profiling of Fog(ki/ki) MK-erythroid progenitors revealed inappropriate expression of several MC-specific genes. Strikingly, aberrant MC gene expression persisted in mature Fog(ki/ki) MK and erythroid progeny. Using a GATA1-dependent committed erythroid cell line, select MC genes were found to be occupied by NuRD, suggesting a direct mechanism of repression. Together, these observations suggest that a simple heritable silencing mechanism is insufficient to permanently repress MC genes. Instead, the continuous presence of GATA1, FOG1, and NuRD is required to maintain lineage fidelity throughout MK-erythroid ontogeny.
Assuntos
Compartimento Celular , Linhagem da Célula , Células Eritroides/citologia , Hematopoese , Megacariócitos/citologia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Células Eritroides/enzimologia , Eritropoese , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Mastócitos/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/enzimologia , Camundongos , Proteínas Nucleares/genética , Especificidade de Órgãos , Proteínas Repressoras/metabolismo , Baço/citologia , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Growth factor independence-1B (Gfi-1B) is a transcriptional repressor essential for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI1B in mice leads to embryonic lethality resulting from failure to produce definitive erythrocytes, hindering the study of Gfi-1B function in adult hematopoiesis. We here show that, in humans, Gfi-1B controls the development of erythrocytes and megakaryocytes by regulating the proliferation and differentiation of bipotent erythro-megakaryocytic progenitors. We further identify in this cell population the type III transforming growth factor-beta receptor gene, TGFBR3, as a direct target of Gfi-1B. Knockdown of Gfi-1B results in altered transforming growth factor-beta (TGF-beta) signaling as shown by the increase in Smad2 phosphorylation and its inability to associate to the transcription intermediary factor 1-gamma (TIF1-gamma). Because the Smad2/TIF1-gamma complex is known to specifically regulate erythroid differentiation, we propose that, by repressing TGF-beta type III receptor (TbetaRIotaII) expression, Gfi-1B favors the Smad2/TIF1-gamma interaction downstream of TGF-beta signaling, allowing immature progenitors to differentiate toward the erythroid lineage.
Assuntos
Diferenciação Celular/fisiologia , Eritrócitos/metabolismo , Megacariócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Proteínas Reguladoras de Apoptose/metabolismo , Proliferação de Células , Eritrócitos/citologia , Eritropoese/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Células K562 , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/citologia , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação/fisiologia , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Trombopoese/fisiologiaRESUMO
Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis, but its mechanism of action had not been addressed. Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) has been shown to mediate endothelial cell responses to PF4 and so we tested this receptor's importance in PF4's role in megakaryopoiesis. We found that LRP1 is absent from megakaryocyte-erythrocyte progenitor cells, is maximally present on large, polyploidy megakaryocytes, and near absent on platelets. Blocking LRP1 with either receptor-associated protein (RAP), an antagonist of LDL family member receptors, or specific anti-LRP1 antibodies reversed the inhibition of megakaryocyte colony growth by PF4. In addition, using shRNA to reduce LRP1 expression was able to restore megakaryocyte colony formation in bone marrow isolated from human PF4-overexpressing mice (hPF4(High)). Further, shRNA knockdown of LRP1 expression was able to limit the effects of PF4 on megakaryopoiesis. Finally, infusion of RAP into hPF4(High) mice was able to increase baseline platelet counts without affecting other lineages, suggesting that this mechanism is important in vivo. These studies extend our understanding of PF4's negative paracrine effect in megakaryopoiesis and its potential clinical implications as well as provide insights into the biology of LRP1, which is transiently expressed during megakaryopoiesis.
Assuntos
Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Comunicação Parácrina/fisiologia , Fator Plaquetário 4/metabolismo , Receptores de LDL/metabolismo , Trombopoese/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Anticorpos/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/genética , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Comunicação Parácrina/efeitos dos fármacos , Fator Plaquetário 4/genética , Receptores de LDL/antagonistas & inibidores , Receptores de LDL/genética , Trombopoese/efeitos dos fármacos , Proteínas Supressoras de Tumor/genéticaRESUMO
PURPOSE: In order to address cell dose limitations associated with the use of cord blood hematopoietic stem cell (HSC) transplantation, we explored the effect of bone marrow stroma-derived heparan sulfate (HS) on the ex vivo expansion of HSCs. METHODS: Heparan sulfate was isolated and purified from the conditioned media of human bone marrow stromal cells and used for the expansion of cord blood-derived CD34(+) cells in the presence of a cocktail of cytokines. RESULTS: The number of myeloid lineage-committed progenitor cells was increased at low dosage of HS as illustrated by an increase in the total number of colony-forming cells (CFC) and colonies of erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) precursors. Notably, the stroma-derived HS did not alter the growth of CD34(+) HSCs or negatively affect the levels of various HSC phenotypic markers after expansion. CONCLUSIONS: This study shows that HS secreted into solution by stromal cells has the capacity to support hematopoietic cytokines in the maintenance and expansion of HSCs. The incorporation of stroma-derived HS as a reagent may improve the efficacy of cord blood HSC transplantation by enhancing the number of committed cells and accelerating the rate of engraftment.
Assuntos
Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Heparitina Sulfato/farmacologia , Células da Medula Óssea/química , Contagem de Células , Técnicas de Cultura de Células/métodos , Células Cultivadas , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Meios de Cultivo Condicionados , Glicosaminoglicanos/química , Glicosaminoglicanos/isolamento & purificação , Glicosaminoglicanos/farmacologia , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/efeitos dos fármacos , Heparitina Sulfato/química , Heparitina Sulfato/isolamento & purificação , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/efeitos dos fármacos , Células-Tronco , Células Estromais/químicaRESUMO
Commitment towards megakaryocyte versus erythroid blood cell lineages occurs in the megakaryocyte-erythroid progenitor, where mutually exclusive expression of either EKLF (Klf1) or Fli1 defines alternative outcomes. Here we show there is a marked increase in the number of circulating platelets in mice lacking the erythroid transcription factor EKLF. In addition, committed erythroid cells retain key signatures of megakaryocytes both on the cell surface and at the mRNA level. We also show that the effect of EKLF on megakaryocyte-erythroid progenitor lineage decision and commitment is cell autonomous in bone marrow reconstitution assays where stem cells lacking EKLF favor the megakaryocyte differentiation pathway. We conclude the megakaryocyte program is aberrantly activated in EKLF null erythroid cells.
Assuntos
Linhagem da Célula/genética , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Fatores de Transcrição Kruppel-Like/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos TransgênicosRESUMO
These studies examined the effects of X radiation and interleukin 3 (IL-3), which is an effective cytokine for the generation of megakaryocytopoiesis from X-irradiated hematopoietic stem/progenitor cells, on the terminal process of human megakaryocytopoiesis and thrombopoiesis. Mature megakaryocytes were induced by culturing CD34(+) cells from normal human peripheral blood in a serum-free liquid culture stimulated with thrombopoietin. The experiments contained the following groups: control cultures with nonirradiated cells incubated for 15 days; cultures treated with IL-3 on day 7 or day 11, cultures irradiated with 2 Gy on day 7 or day 11, and cultures treated with IL-3 immediately after X irradiation. The nonirradiated control cultures produced megakaryocytes from day 7, and both the megakaryocyte and platelet generation reached a peak on day 12-13. When X irradiation was performed on day 7, both the megakaryocyte and platelet numbers decreased remarkably, while no significant effect was observed on those numbers when cultures were X-irradiated on day 11. IL-3 showed neither protective nor promoting effects on the terminal stages of megakaryocytic maturation and platelet production. The results demonstrated that mature megakaryocytes are radiosensitive but that the radiosensitivity decreased with the terminal stages of megakaryocytic maturation, especially for the megakaryocytes entering into proplatelet formation.
Assuntos
Plaquetas/citologia , Plaquetas/fisiologia , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/fisiologia , Tolerância a Radiação/fisiologia , Plaquetas/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/efeitos da radiação , Doses de Radiação , Tolerância a Radiação/efeitos da radiaçãoRESUMO
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 Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Peripheral blood (PB) of children with viral infection had remarkable increase in colony-forming unit-granulocyte/macrophage (CFU-GM)-derived colonies and clusters. The authors sought to investigate the hematopoietic changes in children with infection. METHODS: The CFU-GM-derived colonies and clusters, burst-forming unit-erythroid (BFU-E)-derived colonies, and colony-forming unit-megakaryocyte (CFU-MK)-derived colonies assays and methylcellulose and/or agarose culture of PB were performed in 25 controls and 42 infected patients. Hematopoietic cell assays and cell culture of bone marrow (BM) were performed in 28 controls and 12 infected patients. RESULTS: The PB controls had very few CFU-GM-derived colonies and clusters. The PB of infected children had many more CFU-GM-derived colonies and clusters. In addition, PB of infected children had abundant BFU-E-derived colonies and CFU-MK-derived colonies, whereas, from BM there were no statistical differences in the numbers of CFU-GM-derived colonies and clusters, BFU-E-derived colonies, and CFU-MK-derived colonies between infection group and control group. The numbers of CFU-GM-derived colonies and clusters were greater in PB of bacteria-infected patients than those in PB of non-bacteria-infected patients. CONCLUSIONS: PB of infected children had increased numbers of colonies, especially in the bacteria-infected group.