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 ...
lineages. Because Nanog is required for the development of primordial germ cells (PGCs) in mice and axolotls, we propose that the deletion of Nanog in species often employed as experimental models, such as frogs and teleosts (Zebrafish; Danio rerio), has been compensated by the evolution of predetermined germ cells in these organisms. Our data highlights Nanog as a major component of the gene regulatory network that governs early development in the major trunk of chordate evolution. Maintenance of multipotency is a central question in stem cell biology. In the mouse embryo, mesodermal cells of the dorsal somite, the dermomyotome, give rise to different lineages such as vascular smooth and skeletal muscle. These multipotent stem cells express the Pax3 and Pax7 transcription factors, essential for the myogenic lineage. Here we identify a novel negative feedback loop between Pax3/7 and Foxc2. Using both genetic approaches and manipulation of external signals in somite explants, we demonstrate that the Pax3/7:Foxc2 ratio modulates myogenic versus vascular cell fates of dermomyotomal cells. This exemplifies the importance of such a mechanism of reciprocal repression in the maintenance and lineage commitment of multipotent stem cells. Fgf10 has been implicated in maintenance of stem cells in various embryonic and non-neural organs such as teeth and stomach. We have previously used Fgf10-LacZ reporter mice to show that the localization and fate of Fgf10-expressing cells in the adult mouse brain points to an involvement of Fgf10 in the control of neurogenesis in quiescent neurogenic niches (Hajihosseini etal., 2008. MCN 37: 857-868). In the adult hypothalamus, expression of Fgf10 is restricted to tanycytes which reside in the median eminence, at the floor of the third ventricle. Here, we have used a set of immunolabelling and neurosphere culture studies to study whether these Fgf10-expressing cells in the adult hypothalamus are neural stem cells or lineage-restricted neural progenitors.We find that in addition to Fgf10, tanycytes express BLBP, Nestin and Musashi. Fgf10 is downregulated as descendants of tanycytes migrate into the surrounding hypothalamic nuclei and express DARPP32 or the mature neuronal marker, NeuN. In invitro assays, we found that cells derived from the median eminence can generate neurospheres as efficiently as those derived from the lateral subventricular zones. Moreover, in differentiation assays, these neurospheres give rise to all three neural lineages: neurons (TuJ1+), astrocytes (GFAP+) and oligodendrocytes (olig2+). These data suggest that the adult hypothalamus contains a quiescent population of neural stem cells and, as in non-neural tissues, Fgf10 may play a critical role in the maintenance and/or differentiation of subpopulations of neural cells. Further studies are underway to verify and dissect the role of Fgf10 in this system. Pluripotency defines the ability of cells to produce all somatic cells and germ line cells. In mammals, primordial germ cells (PGCs) are induced from pluripotent c...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.