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1.
Oncogene ; 42(10): 771-781, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36646904

RESUMEN

Chemotherapy remains one of the main treatment modalities for cancer. While chemotherapy is mainly known for its ability to kill tumor cells directly, accumulating evidence indicates that it also acts indirectly by enhancing T cell-mediated anti-tumor immunity sometimes through immunogenic cell death. However, the role of immature immune cells in chemotherapy-induced immunomodulation has not been studied. Here, we utilized a mouse pancreatic cancer model to characterize the effects of gemcitabine chemotherapy on immature bone marrow cells in the context of tumor immunogenicity. Single cell RNA sequencing of hematopoietic stem and progenitor cells revealed a 3-fold increase in megakaryocyte-erythroid progenitors (MEPs) in the bone marrow of gemcitabine-treated mice in comparison to untreated control mice. Notably, adoptive transfer of MEPs to pancreatic tumor-bearing mice significantly reduced tumor growth and increased the levels of anti-tumor immune cells in tumors and peripheral blood. Furthermore, MEPs increased the tumor cell killing activity of CD8 + T cells and NK cells, an effect that was dependent on MEP-secreted CCL5 and CXCL16. Collectively, our findings demonstrate that chemotherapy-induced enrichment of MEPs in the bone marrow compartment contributes to anti-tumor immunity.


Asunto(s)
Antineoplásicos , Neoplasias Pancreáticas , Ratones , Animales , Células de la Médula Ósea , Médula Ósea , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/patología , Gemcitabina , Neoplasias Pancreáticas/patología , Antineoplásicos/farmacología
2.
Blood ; 139(25): 3630-3646, 2022 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-35421216

RESUMEN

Leukemic transformation (LT) of myeloproliferative neoplasm (MPN) has a dismal prognosis and is largely fatal. Mutational inactivation of TP53 is the most common somatic event in LT; however, the mechanisms by which TP53 mutations promote LT remain unresolved. Using an allelic series of mouse models of Jak2/Trp53 mutant MPN, we identify that only biallelic inactivation of Trp53 results in LT (to a pure erythroleukemia [PEL]). This PEL arises from the megakaryocyte-erythroid progenitor population. Importantly, the bone morphogenetic protein 2/SMAD pathway is aberrantly activated during LT and results in abnormal self-renewal of megakaryocyte-erythroid progenitors. Finally, we identify that Jak2/Trp53 mutant PEL is characterized by recurrent copy number alterations and DNA damage. Using a synthetic lethality strategy, by targeting active DNA repair pathways, we show that this PEL is highly sensitive to combination WEE1 and poly(ADP-ribose) polymerase inhibition. These observations yield new mechanistic insights into the process of p53 mutant LT and offer new, clinically translatable therapeutic approaches.


Asunto(s)
Trastornos Mieloproliferativos , Proteína p53 Supresora de Tumor , Animales , Proteína Morfogenética Ósea 2/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/metabolismo , Ratones , Mutación , Trastornos Mieloproliferativos/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
3.
Curr Opin Hematol ; 28(1): 28-35, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33186151

RESUMEN

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.


Asunto(s)
Hematopoyesis , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Animales , Linaje de la Célula , Células Eritroides/citología , Células Eritroides/metabolismo , Humanos , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Transcriptoma
4.
Blood ; 136(9): 1044-1054, 2020 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-32548608

RESUMEN

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.


Asunto(s)
Células Precursoras Eritroides/citología , Eritropoyesis/fisiología , Megacariocitos/citología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/patología , Células Precursoras Eritroides/metabolismo , Eritropoyetina/farmacología , Técnicas de Inactivación de Genes , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Quimera por Radiación , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/farmacología
5.
Biochem Biophys Res Commun ; 528(1): 46-53, 2020 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-32456797

RESUMEN

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.


Asunto(s)
Diferenciación Celular , Factor de Transcripción GATA1/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Animales , Ciclo Celular , Proliferación Celular , Células Cultivadas , Ratones , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Transducción de Señal
6.
Transfusion ; 60(3): 561-574, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32086956

RESUMEN

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.


Asunto(s)
Antígenos CD34/metabolismo , Plaquetas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Infección por el Virus Zika/metabolismo , Virus Zika/patogenicidad , Diferenciación Celular/fisiología , Línea Celular , Eritrocitos/citología , Humanos , ARN Viral/genética
7.
Blood ; 134(7): 614-625, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31270104

RESUMEN

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.


Asunto(s)
Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/patología , Células Progenitoras de Megacariocitos y Eritrocitos/patología , Factores de Transcripción/genética , Animales , Transformación Celular Neoplásica/genética , Regulación Leucémica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Leucemia Mieloide Aguda/genética , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Translocación Genética
8.
Cell Rep ; 25(8): 2083-2093.e4, 2018 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-30463007

RESUMEN

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.


Asunto(s)
Ciclo Celular , Linaje de la Célula , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo
9.
DNA Cell Biol ; 37(10): 831-838, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30124330

RESUMEN

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.


Asunto(s)
Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/metabolismo , Síndromes Mielodisplásicos/genética , Proteínas/genética , Animales , Diferenciación Celular , Femenino , Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/patología , Péptidos y Proteínas de Señalización Intracelular , Células Progenitoras de Megacariocitos y Eritrocitos/patología , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Proteínas/metabolismo , Transgenes , Irradiación Corporal Total
10.
Br J Haematol ; 180(4): 526-533, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29377069

RESUMEN

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.


Asunto(s)
Antineoplásicos/uso terapéutico , Deleción Cromosómica , Cromosomas Humanos Par 5 , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Megacariocitos/metabolismo , Talidomida/análogos & derivados , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Médula Ósea/patología , Evolución Clonal , Análisis Citogenético , Neoplasias Hematológicas/diagnóstico , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Lenalidomida , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/patología , Megacariocitos/patología , Inducción de Remisión , Talidomida/administración & dosificación , Talidomida/efectos adversos , Talidomida/uso terapéutico , Resultado del Tratamiento
11.
Curr Opin Hematol ; 24(3): 173-182, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28212192

RESUMEN

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.


Asunto(s)
Eritropoyesis/genética , Regulación de la Expresión Génica , Variación Genética , Animales , Biomarcadores , Diferenciación Celular/genética , Estudio de Asociación del Genoma Completo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hemoglobinas/genética , Humanos , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Sitios de Carácter Cuantitativo , Carácter Cuantitativo Heredable
12.
Blood ; 128(11): 1503-15, 2016 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-27443289

RESUMEN

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.


Asunto(s)
Diferenciación Celular , Transformación Celular Neoplásica/patología , Leucemia Experimental/patología , Células Progenitoras de Megacariocitos y Eritrocitos/patología , Proteínas de Fusión Oncogénica/metabolismo , Animales , Western Blotting , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Femenino , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Masculino , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Fusión Oncogénica/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
14.
Blood ; 128(7): 923-33, 2016 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-27268089

RESUMEN

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.


Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Antígenos CD34/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Adulto , Linaje de la Célula , Separación Celular , Ensayo de Unidades Formadoras de Colonias , Células Eritroides/citología , Células Eritroides/metabolismo , Humanos , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/citología , Fenotipo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores de Trombopoyetina/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo
15.
Genome Biol ; 17: 83, 2016 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-27142433

RESUMEN

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.


Asunto(s)
Hematopoyesis , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Análisis de la Célula Individual/métodos , Adulto , Anciano , Linaje de la Célula , Separación Celular/métodos , Células Eritroides/citología , Células Eritroides/metabolismo , Femenino , Humanos , Masculino , Células Progenitoras de Megacariocitos y Eritrocitos/clasificación , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Persona de Mediana Edad
16.
Blood ; 127(10): 1242-8, 2016 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-26787736

RESUMEN

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.


Asunto(s)
Diferenciación Celular/fisiología , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/metabolismo , Mielopoyesis/fisiología , Trombopoyesis/fisiología , Animales , Humanos , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Linfopoyesis/fisiología , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Megacariocitos/citología
18.
J Clin Invest ; 125(6): 2261-3, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25961451

RESUMEN

The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megakaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications.


Asunto(s)
Antibacterianos/farmacología , Diferenciación Celular/efectos de los fármacos , Doxiciclina/farmacología , Células Madre Embrionarias/metabolismo , Factor de Transcripción GATA1/biosíntesis , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Trombopoyetina/farmacología , Animales , Humanos
19.
J Clin Invest ; 125(6): 2369-74, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25961454

RESUMEN

Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10¹³-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.


Asunto(s)
Antibacterianos/farmacología , Diferenciación Celular/efectos de los fármacos , Doxiciclina/farmacología , Células Madre Embrionarias/metabolismo , Factor de Transcripción GATA1/biosíntesis , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Trombopoyetina/farmacología , Animales , Diferenciación Celular/genética , Células Madre Embrionarias/citología , Factor de Transcripción GATA1/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones
20.
PLoS One ; 9(11): e112671, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25401736

RESUMEN

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.


Asunto(s)
Hematopoyesis/genética , Leucemia/genética , Leucemia/patología , Fenotipo , Proteínas Supresoras de Tumor/genética , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Análisis por Conglomerados , Perfilación de la Expresión Génica , Humanos , Leucemia/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/patología , Ratones , Mutación , Células Mieloides/metabolismo , Células Mieloides/patología , Dominios y Motivos de Interacción de Proteínas/genética , Linfocitos T/metabolismo , Linfocitos T/patología , Transactivadores , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo
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