RESUMO
This corrects the article DOI: 10.1038/ncomms11208.
RESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Reprogramação Celular , Fator de Transcrição GATA1/genética , Megacariócitos/citologia , Células-Tronco Pluripotentes/citologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Plaquetas/citologia , Plaquetas/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Criopreservação/métodos , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus/genética , Megacariócitos/metabolismo , Análise em Microsséries , Células-Tronco Pluripotentes/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transdução Genética , TransgenesRESUMO
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.
Assuntos
Plaquetas/enzimologia , Janus Quinase 2/sangue , Janus Quinase 2/genética , Proteínas Mutantes/sangue , Proteínas Mutantes/genética , Mutação , Trombocitemia Essencial/sangue , Trombocitemia Essencial/genética , Animais , Plaquetas/patologia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Megacariócitos/enzimologia , Megacariócitos/patologia , Camundongos , Camundongos Transgênicos , Agregação Plaquetária/genética , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Trombocitemia Essencial/enzimologia , Trombopoese/genéticaRESUMO
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.