17-P024 Nanog is a master regulator of axolotl development

Abstract

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. The transcription factor Sox2 (SRY-related HMG-box gene 2), in synergy with Oct4, plays a pivotal role in maintaining embryonic stem (ES) cells self-renewing and pluripotent. After knock-down of Sox2 in mouse ES cells with three different shRNA constructs, alterations in transcript and protein expression of genes associated with pluripotency, commitment to trophectoderm, mesendoderm, neuroectoderm and cell-cycle regulation were detected. Absence of Sox2 resulted in loss of self-renewal (with down-regulation of Oct4, Nanog, Rex1 and Utf1), severe compromise of ES cell growth rate and cell-cycle arrest (as indicated by increased levels of p21 and p27, as well as decreased levels of Nucleostemin and Cyclin D2). However, apoptosis in Sox2 shRNA ES cells was not significantly different to mock-transfected control ES cells. In a culture system designed to promote neurogenesis, cells did not form neural precursors after Sox2 shRNA but acquired a more glial (astrocytic) phenotype, as demonstrated by increased levels of Musashi1 and Gfap by day 14, with parallel decreased levels of Sox1, Beta III tubulin and Pax6, compared to mock-transfected control ES cells. The most striking impact of knocking down Sox2 in mouse ES cells was observed within the first 3 days of the neural differentiation protocol with significant up-regulation of the trophectoderm markers Cdx2, Eomes and Fgfr2, as well as of the mesendoderm markers Mixl1, Goosecoid and Brachyury. Concomitant up-regulation of b-catenin and down-regulation of Lef1 in the absence of Sox2, suggests that ...