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.
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This work was supported by the European Research Council to P.M (ERC-2014-CoG-646903), the ISCIII/FEDER (E-Rare-2 Call PI12/03112 to PM, CP11/00131 to AFF and PI14/01191 to CB), MINECO (SAF2013-43065 to PM and SAF2012-37167 to TG), The Foundation TV3 Marato (120410 to TG), The Spanish Association Against Cancer to PM and MCC, and The Generalitat de Catalunya (SGR330 to PM and SGR1136 to TG). CB is supported by a Miguel Servet II contract (CPII13/00011). DRM and AM-L are supported by PFIS (FI11/0511) and FPI (BES-2014-067844) scholarships, respectively. PM also acknowledges the financial support from The Obra Social La Caixa-Fundaciò Josep Carreras. TG acknowledges the support by the Technology and Business Development Office of CRG within the framework of its proof-of-concept programs. CB, PM, JCS and OQ-B are investigators of the Spanish Cell Therapy cooperative network (TERCEL). Special thanks to Dr Lee Carpenter (University of Oxford, UK) and Dr Igor Slukvin (University of Wisconsin, MA) for providing MS5 and OP9 cells, and Dra. Dolors Comoler (IDIBAPS, Barcelona) for valuable help with the VDJH assays.
CB, JLS: conceived the study, designed and performed experiments, analyzed data and wrote the manuscript. KN, MO, MN: contributed key reagents and interpreted the data. BDS, DR-M, AM-L, MCC, AB, LA, JC, AH, OQ-B, JCS, MFF, AF, IG: performed experiments. TG: conceived the study. PM: conceived the study, designed experiments, analyzed data and wrote the manuscript.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website
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Application of induced pluripotent stem cell technology for the investigation of hematological disorders
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