Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition

By José C. R. Silva, Ornella Barrandon, Jennifer Nichols, Jitsutaro Kawaguchi, T. Theunissen, Austin G Smith. PLoS Biology, 2008

873 citations recorded by the source corpus.

Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency—including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras—were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1–2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/fd8200395aecb29095f738cf813b2eda5c24579c

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Citations in the corpus

Wnt/ b -Catenin Signaling, Disease, and Emerging Therapeutic Modalities (2017, background): “Thus, the ES phenotype can be maintained in culture by just two small molecules, one being the Wnt activating GSK3 inhibitor CHIR (Silva et al., 2008).”
Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. (2010, background): “suggested that it may promote the transition from pre-iPSCs to iPSCs as described previously ( Silva et al., 2008 ).”
Nanog Is the Gateway to the Pluripotent Ground State (2009, positive): “Incompletely reprogrammed cells may be promoted to pluripotency by exposure to 2i/LIF (Silva et al., 2008).”
Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination (2016, background): “Although the role of PI3K/AKT/mTOR in this process has not been fully uncovered, several reports have demonstrated that PI3K/ AKT signalling promotes iPSC generation through inhibition of GSK3β and FOXO1, while concurrently enhancing the glycolytic metabolism (Silva et al., 2008; Yu et al., 2014; Zh”
A Small Molecule Inhibitor of Tgf-β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog (2009, background): “It has been shown that certain non-pluripotent, partially reprogrammed cell lines derived from MEFs transduced with Oct4, Klf4, cMyc, and Sox2 can be fully reprogrammed with AZA or a combination of chemical inhibitors of Glycogen Synthase Kinase 3β (GSK-3β) and the Mek signaling pathway (2i conditio”
Epigenetic reprogramming and induced pluripotency (2009, background): “…reprogrammed iPS cells with inhibitors of the ERK kinase and GSK3 signalling cascades, both of which are crucial for the derivation of ES cells (Ying et al., 2008), results in the efficient conversion of partially reprogrammed cell lines into fully reprogrammed iPS cells (Silva et al., 2008).”
Epigenetic memory in induced pluripotent stem cells (2010, methodology): “These data highlight our limited understanding of the epigenetic heterogeneity of pluripotent stem cells, and the need for improved methods to ensure reprogramming of somatic cells to a fully naïve, “ground state” of pluripotency 48 .”
Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation (2009, result): “To test if residual factor expression biases the ability of individual cells to differentiate, we simultaneously monitored exogenous Sox2 and endoderm marker CXCR4 expression upon directed differentiation of hiPSC lines 50 and 59 (Fig.”
Capture of authentic embryonic stem cells from rat blastocysts. (2008, positive): “Additionally, 2i facilitates isolation of induced pluripotent stem (iPS) cells during transfectioninduced reprogramming (Silva et al., 2008).”
Naive and primed pluripotent states. (2009, methodology): “This competence appears to be mediated through a combination of both selection for and stabilization of the ground state (Silva et al., 2008; Silva and Smith, 2008).”
Senescence impairs successful reprogramming to pluripotent stem cells. (2009, background): “Inhibition of DNA methylation, knockdown of lineage-specific genes, or treatment with MEK and GSK3 inhibitors (Mikkelsen et al., 2008; Silva et al., 2008) can either convert some of these pre-iPS to iPS or increase the proportion of fully reprogrammed iPS versus pre-iPS (Figure 4.10).”
Role of the murine reprogramming factors in the induction of pluripotency (2009, positive): “, 2007), and upon addition of a specific MEK inhibitor used in recent reprogramming experiments (Silva et al., 2008) an efficient transition to GFP-positive colonies is observed (Figure S6).”
Klf4 reverts developmentally programmed restriction of ground state pluripotency (2009, background): “We then examined the effect of Klf4 transfection in 2i/Lif conditions, which have been shown to promote the final stages of iPS cell generation from neural stem cells and fibroblasts (Silva et al., 2008).”
A fresh look at iPS cells. (2009, background): “…mouse iPS cells have been generated from bone marrow cells (Takahashi and Yamanaka, 2006), hepatocytes and gastric epithelial cells (Aoi et al., 2008), pancreatic cells (Stadtfeld et al., 2008a), neural stem cells (Kim et al., 2008; Silva et al., 2008), and B lymphocytes (Hanna et al., 2008).”