Lentiviral vectors for reprogramming of somatic cells into induced pluripotent stem (iPS) cells

Recently, Yamanaka and colleagues showed that the retroviral transduction of murine fibroblasts with 4 transcription factors (Oct3/4, Sox2, Klf4, c-Myc), which are highly expressed in ES cells, resulted in the generation of induced pluripotent stem cells (iPS; Takahashi and Yamanaka, Cell 2006, 126(4)). The ultimate goal is the generation of patient- or disease-specific iPS cells to create new avenues for disease models, drug discovery and regenerative medicine. Here, we describe a new modular lentiviral vector system for these 4 reprogramming factors. First, for constitutive expression we used the strong retroviral promoter SFFV, which mediates high transgene expression in a variety of cells and is frequently silenced in ES cells. Optionally, we linked each transcription factor to a fluorescent marker (choice of green, red and blue fluorescence genes) to follow their gene marking and expression kinetics. Second, to express the transcription factors in a regulated manner, we designed vectors under control of the tet-on system. Third, our modular system allows the transient, non-integrating delivery of the transcription factors using integrase-deficient lentiviral vectors, avoiding the risks of permanent transcription factor expression and insertional mutagenesis. Finally, we tested various co-expression strategies. In total, we derived 30 vectors encoding reprogramming factors. Proof of concept for lentiviral reprogramming was obtained using murine embryonic fibroblasts (MEFs). We used OG-2 MEFs, which express GFP under the control of the Oct3/4 promoter, to demonstrate reprogramming using the 4 (with myc) or the 3 (without myc, albeit with less efficiency) transcription factors expressed from integrating lentiviral vectors. We were capable to reprogram MEFs into GFP expressing iPS-like cells, similar to ES cells in terms of morphology and proliferation. Further pluripotency testing is currently ongoing. The described vector system may ease the generation of murine and human iPS-like cells and also contribute to the identification of the underlying mechanisms.