Abstract
Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation.
Highlights
Human fibroblasts were the first cell type successfully reprogrammed by ectopic expression of OCT4, SOX2, KLF4 and cMYC (OKSM) [1,2,3,4,5,6]
Human induced pluripotent stem cells have been derived from different tissues, and further studies have shown that the age, origin and cell type used as target for reprogramming affects the reprogramming efficiency, eventually requiring the expression of fewer factor [7,8,9]
Haase et al [11] derived Human induced pluripotent stem cells (hiPSC) from Cord blood (CB)-derived endothelial cells while Giorgetti et al [7] succeeded in the derivation of hiPSC from a CB-derived CD133+ cell subset which contains hematopoietic progenitor cells committed to myeloid but not lymphoid lineages [12,13,14,15]
Summary
Human fibroblasts were the first cell type successfully reprogrammed by ectopic expression of OCT4, SOX2, KLF4 and cMYC (OKSM) [1,2,3,4,5,6]. Human induced pluripotent stem cells (iPSC) have been derived from different tissues, and further studies have shown that the age, origin and cell type used as target for reprogramming affects the reprogramming efficiency, eventually requiring the expression of fewer factor [7,8,9]. Takenaka et al [16] very recently derived hiPSC from CB-derived CD34+ cells upon retroviral over-expression of OKSM factors and showed that further p53 depletion robustly enhanced reprogramming efficiency. Ye et al [17] derived hiPSC from blood cells and CB-derived CD34+ cells using four independent retroviral vectors: pMXs-Oct, pMXs-Sox, pMXs-Klf and pMXs-c-myc
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