ABSTRACT
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.
Subject(s)
B-Lymphocytes/metabolism , CCAAT-Enhancer-Binding Proteins/genetics , Cellular Reprogramming/genetics , Fetal Blood/metabolism , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Base Sequence , CCAAT-Enhancer-Binding Proteins/immunology , Cell Differentiation , Cell Separation , Cellular Reprogramming/immunology , Fetal Blood/cytology , Fetal Blood/immunology , Gene Expression , Genetic Vectors , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/immunology , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/immunology , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Molecular Sequence Data , Myeloid Cells/cytology , Myeloid Cells/immunology , Myeloid Cells/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/immunology , Primary Cell Culture , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/immunology , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/immunology , Sendai virus/genetics , V(D)J Recombination/immunologyABSTRACT
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.