RESUMEN
B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.
Asunto(s)
Linfocitos B/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Reprogramación Celular/genética , Sangre Fetal/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/inmunología , Diferenciación Celular , Separación Celular , Reprogramación Celular/inmunología , Sangre Fetal/citología , Sangre Fetal/inmunología , Expresión Génica , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/inmunología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Datos de Secuencia Molecular , Células Mieloides/citología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/inmunología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/inmunología , Virus Sendai/genética , Recombinación V(D)J/inmunologíaRESUMEN
Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34(+) cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22(phox)-dependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.
