17-P022 Virus-free induction of pluripotency and subsequentexcision of reprogramming factors

Reprogramming of somatic cells to pluripotent cells promises to transform regenerative medicine. Recently many groups have achieved direct reprogramming of somatic cells by forced expression of defined factors using multiple viral vectors. However, such induced pluripotent stem (iPS) cells contain a number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Moreover, viral vectors silenced in iPS cells can be re-activated when the cells differentiate. Here we show non-viral transfection with a single multiprotein expression vector can generate iPS cells from non-genetically modified mouse embryonic fibroblasts. This one vector system can achieve reprogramming from one integration site, enabling subsequent elimination of the reprogramming cassette by Cre-mediated excision. These non-viral iPS cells show robust expression of pluripotent markers and genuine pluripotency was confirmed by invitro differentiation assays and formation of adult chimeric mice. When the single vector reprogramming system was combined with a piggyBac transposon we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This non-viral single vector system minimizes genome modification and eliminates the unpredictable reactivation of reprogramming factors, providing iPS cells more applicable to regenerative medicine, reliable drug screening and establishment of trustworthy disease models.