RESUMEN

This corrects the article DOI: 10.1038/ncomms11208.

RESUMEN

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Reprogramación Celular , Factor de Transcripción GATA1/genética , Megacariocitos/citología , Células Madre Pluripotentes/citología , Proteína Proto-Oncogénica c-fli-1/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Plaquetas/citología , Plaquetas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Criopreservación/métodos , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lentivirus/genética , Megacariocitos/metabolismo , Análisis por Micromatrices , Células Madre Pluripotentes/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteína 1 de la Leucemia Linfocítica T Aguda , Transducción Genética , Transgenes

RESUMEN

The principal morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia rubra vera (PV) stems from thrombotic events. Most patients with ET/PV harbor a JAK2V617F mutation, but its role in the thrombotic diathesis remains obscure. Platelet function studies in patients are difficult to interpret because of interindividual heterogeneity, reflecting variations in the proportion of platelets derived from the malignant clone, differences in the presence of additional mutations, and the effects of medical treatments. To circumvent these issues, we have studied a JAK2V617F knock-in mouse model of ET in which all megakaryocytes and platelets express JAK2V617F at a physiological level, equivalent to that present in human ET patients. We show that, in addition to increased differentiation, JAK2V617F-positive megakaryocytes display greater migratory ability and proplatelet formation. We demonstrate in a range of assays that platelet reactivity to agonists is enhanced, with a concomitant increase in platelet aggregation in vitro and a reduced duration of bleeding in vivo. These data suggest that JAK2V617F leads to intrinsic changes in both megakaryocyte and platelet biology beyond an increase in cell number. In support of this hypothesis, we identify multiple differentially expressed genes in JAK2V617F megakaryocytes that may underlie the observed biological differences.

Asunto(s)

Plaquetas/enzimología , Janus Quinasa 2/sangre , Janus Quinasa 2/genética , Proteínas Mutantes/sangre , Proteínas Mutantes/genética , Mutación , Trombocitemia Esencial/sangre , Trombocitemia Esencial/genética , Animales , Plaquetas/patología , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Megacariocitos/enzimología , Megacariocitos/patología , Ratones , Ratones Transgénicos , Agregación Plaquetaria/genética , Proteínas Recombinantes/sangre , Proteínas Recombinantes/genética , Trombocitemia Esencial/enzimología , Trombopoyesis/genética

RESUMEN

The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 × 10(-228)) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.

Asunto(s)

Predisposición Genética a la Enfermedad , Proteínas de Unión al ARN/genética , Trombocitopenia/genética , Deformidades Congénitas de las Extremidades Superiores/genética , Regiones no Traducidas 5'/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Niño , Preescolar , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Femenino , Variación Genética , Humanos , Lactante , Recién Nacido , Masculino , Mutación , Recuento de Plaquetas , Polimorfismo de Nucleótido Simple , Radio (Anatomía)/anomalías , Alineación de Secuencia , Análisis de Secuencia de ADN , Trombocitopenia/congénito , Adulto Joven , Pez Cebra/genética