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

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 genedelivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

“…For the quantification of transduction efficiency, we introduced GFP to MEF as above and more than 70% of cells expressed GFP. To quantify efficiency of iPS generation, total number of Nanog-positive colonies were counted and calculated as percentage of infected cell numbers, which was calculated as GFP+ cells [28].…”

Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells

“…More importantly, the generation of patient-specific and disease-specific (4) iPS cells has the potential to greatly impact the future of regenerative medicine, drug development, as well as our basic understanding of specific disease mechanisms. With the fast progress in this field, different reprogramming strategies have been developed, including using nonintegrating adenoviruses (5), two factors with small molecules (6), reprogramming with a polycistronic cassette containing all four factors (7,8), excisable transposons (9,10), and more recently, virus-free plasmid (11,12). However, the majority of these studies use skin fibroblasts as the parental cells, which usually requires at least 4 weeks to expand from a single skin biopsy to get enough starting cells for reprogramming.…”

New Techniques in the Generation of Induced Pluripotent Stem Cells

“…Indeed, Esrrb has been used as a factor to reprogram somatic cells back to a pluripotent state. In the presence of Oct4 and Sox2, Feng et al (2009) demonstrated that Esrrb could replace Klf4 as an enhancer to reprogram mouse fibroblasts into iPSC, albeit with lower reprogramming efficiency (Feng et al, 2009a). Furthermore, another family member Esrrg also possesses a similar reprogramming ability when used in conjunction with Oct4 and Sox2 (Feng et al, 2009a).…”