Abstract
Generation of induced pluripotent stem (iPS) cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors (TFs). However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame (ORF) via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.
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Acknowledgements
We thank Dr Liangyou Rui for providing the lentivirus expression vector pLentG and thank our colleagues at Maine Medical Center Research Institute for their critical review. We also thank the MMCRI Cell Separation & Analysis Core, the Histology Core, and the Mouse Transgenic Core for their services. This work was supported in part by an NIH grant from the National Center for Research Resources (P20 RR018789), USA. W.S.W was supported by a K01 award from the National Institute of Diabetes and Digestive and Kidney Disease (K01DK078180-01), USA.
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Supplementary video s1 (download AVI )
The beating of the cardiac muscle cells differentiated from the induced iPS cell colony (clone #1). iPS cells were cultured in the ES cell medium in a ultra-low 6-well plate for 9 days, and the formed embryoid bodies (EBs) were transferred to gelatin-coated plate and cultured in the same medium for another 6 days. The beating of the cardiac muscle cells was microscopically observed and recorded by a video recorder. (AVI 1008 kb)
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Shao, L., Feng, W., Sun, Y. et al. Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame. Cell Res 19, 296–306 (2009). https://doi.org/10.1038/cr.2009.20
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DOI: https://doi.org/10.1038/cr.2009.20
