High-Throughput Identification of Genes Promoting Neuron Formation and Lineage Choice in Mouse Embryonic Stem Cells

Karolinska Institute, Cell and Developmental Biology, Box 285, Stockholm 17177, Sweden.
Stem Cells (Impact Factor: 6.52). 07/2007; 25(6):1539-45. DOI: 10.1634/stemcells.2006-0485
Source: PubMed


The potential of embryonic stem cells to differentiate to all cell types makes them an attractive model for development and a potential source of cells for transplantation therapies. Candidate approaches have identified individual genes and proteins that promote the differentiation of embryonic stem cells to desired fates. Here, we describe a rapid large-scale screening strategy for the identification of genes that influence the pluripotency and differentiation of embryonic stem cells to specific fates, and we use this approach to identify genes that induce neuron formation. The power of the strategy is validated by the fact that, of the 15 genes that resulted in the largest increase in neuron number, 8 have previously been implicated in neuronal differentiation or survival, whereas 7 represent novel genes or known genes not previously implicated in neuronal development. This is a simple, fast, and generally applicable strategy for the identification of genes promoting the formation of any specific cell type from embryonic stem cells. Disclosure of potential conflicts of interest is found at the end of this article.

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Available from: Anna Falk, Nov 03, 2014
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    • "During the course of this study a report was published by Falk et al. (2007) describing a high throughput transfection screen for neuronal differentiation factors. They identified 15 genes whose expression weakly induced differentiation, none of which overlapped with ours. "
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    ABSTRACT: Neuronal differentiation is a complex process that involves a plethora of regulatory steps. To identify transcription factors that influence neuronal differentiation we developed a high throughput screen using embryonic stem (ES) cells. Seven-hundred human transcription factor clones were stably introduced into mouse ES (mES) cells and screened for their ability to induce neuronal differentiation of mES cells. Twenty-four factors that are capable of inducing neuronal differentiation were identified, including four known effectors of neuronal differentiation, 11 factors with limited evidence of involvement in regulating neuronal differentiation, and nine novel factors. One transcription factor, Oct-2, was studied in detail and found to be a bifunctional regulator: It can either repress or induce neuronal differentiation, depending on the particular isoform. Ectopic expression experiments demonstrate that isoform Oct-2.4 represses neuronal differentiation, whereas Oct-2.2 activates neuron formation. Consistent with a role in neuronal differentiation, Oct-2.2 expression is induced during differentiation, and cells depleted of Oct-2 and its homolog Oct-1 have a reduced capacity to differentiate into neurons. Our results reveal a number of transcription factors potentially important for mammalian neuronal differentiation, and indicate that Oct-2 may serve as a binary switch to repress differentiation in precursor cells and induce neuronal differentiation later during neuronal development.
    Preview · Article · Apr 2009 · Genes & development
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    • "Therefore, the membrane protrusions formed in HEK293 cells may bear resemblance to polarized neurites (Shaw et al., 2002; Nalvarte et al., 2004; Govek et al., 2005). In this context it is noteworthy that a close homolog of ORP3, ORP6, was identified as one of the fifteen proteins that promote the differentiation of neurons from murine embryonic stem cells (Falk et al., 2007). Protrusion formation similar to that induced by ORP3 has been reported to occur upon overexpression of several other proteins, all of which represent regulators of the actin cytoskeleton: Cdc42 effector protein 1 (CEP1/MSE55) (Burbelo et al., 1999), ATPbinding cassette transporter-1 (ABCA1) (Wang, N. et al., 2000; Tsukamoto et al., 2001), calpain 2 (CAPN2) (Franco et al., 2004; Perrin et al., 2006), downstream of kinase 2 (Dok-2, Dok-R or FRIP) (Master et al., 2003) and c-Abl tyrosine kinase (Master et al., 2003). "
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    ABSTRACT: Oxysterol-binding protein (OSBP)-related protein 3 (ORP3) is highly expressed in epithelial, neuronal and hematopoietic cells, as well as in certain forms of cancer. We assessed the function of ORP3 in HEK293 cells and in human macrophages. We show that ORP3 interacts with R-Ras, a small GTPase regulating cell adhesion, spreading and migration. Gene silencing of ORP3 in HEK293 cells results in altered organization of the actin cytoskeleton, impaired cell-cell adhesion, enhanced cell spreading and an increase of beta1 integrin activity--effects similar to those of constitutively active R-Ras(38V). Overexpression of ORP3 leads to formation of polarized cell-surface protrusions, impaired cell spreading and decreased beta1 integrin activity. In primary macrophages, overexpression of ORP3 leads to the disappearance of podosomal structures and decreased phagocytotic uptake of latex beads, consistent with a role in actin regulation. ORP3 is phosphorylated when cells lose adhesive contacts, suggesting that it is subject to regulation by outside-in signals mediated by adhesion receptors. The present findings demonstrate a new function of ORP3 as part of the machinery that controls the actin cytoskeleton, cell polarity and cell adhesion.
    Full-text · Article · Apr 2008 · Journal of Cell Science
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    ABSTRACT: Recently, intense interest in the potential use of neural stem cells (NSC) in the clinical therapy of brain disease and injury has resulted in rapid progress in research on the properties of NSC, their innate and directed differentiation potential and the induced reprogramming of differentiated somatic cells to revert to a pluripotent NSC-like state. The aim of this review is to give an overview of our current operational definitions of the NSC lineage, the growing understanding of extrinsic and intrinsic mechanisms, including heritable but reversible epigenetic chromatin modifications that regulate the maintenance and differentiation of NSC in vivo, and to emphasize ground-breaking efforts of cellular reprogramming with the view to generating patient-specific stem cells for cell replacement therapy. This is set against a summary of current practical procedures for the isolation, research and application of NSC, and of the state of the art in NSC-based regenerative medicine of the nervous system. Both provide the backdrop for the translation of recent findings into innovative clinical applications, with the hope of increasing the safety, efficiency and ethical acceptability of NSC-based therapies in the near future.
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