Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame.

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.