Capture of Neuroepithelial-Like Stem Cells from Pluripotent Stem Cells Provides a Versatile System for In Vitro Production of Human Neurons

Department of Biochemistry, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, United Kingdom.
PLoS ONE (Impact Factor: 3.23). 01/2012; 7(1):e29597. DOI: 10.1371/journal.pone.0029597
Source: PubMed


Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide new prospects for studying human neurodevelopment and modeling neurological disease. In particular, iPSC-derived neural cells permit a direct comparison of disease-relevant molecular pathways in neurons and glia derived from patients and healthy individuals. A prerequisite for such comparative studies are robust protocols that efficiently yield standardized populations of neural cell types. Here we show that long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from 3 hESC and 6 iPSC lines in two independent laboratories exhibit consistent characteristics including i) continuous expandability in the presence of FGF2 and EGF; ii) stable neuronal and glial differentiation competence; iii) characteristic transcription factor profile; iv) hindbrain specification amenable to regional patterning; v) capacity to generate functionally mature human neurons. We further show that lt-NES cells are developmentally distinct from fetal tissue-derived radial glia-like stem cells. We propose that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds.

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    • "However, continued competency to respond to morphogens has permitted their targeted differentiation into distinct neuronal subtypes of the central and peripheral nervous system (Chambers et al., 2012; Elkabetz et al., 2008; Falk et al., 2012; Koch et al., 2009; Lee et al., 2010; Li et al., 2011; Reinhardt et al., 2013). Recently, proliferative gliogenic intermediates giving rise to large numbers of astrocytes have also been described (Krencik et al., 2011; Roybon et al., 2013; Serio et al., 2013; Shaltouki et al., 2013). "
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    ABSTRACT: Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor-mediated expansion with pre-exposure to the differentiation-inducing agent retinoic acid and subsequent immunoisolation of CD133-positive cells. This protocol yields an adherent and self-renewing population of hindbrain/spinal cord radial glia (RG)-like neural precursor cells (RGL-NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL-NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate-determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL-NPCs efficiently convert into NG2-positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL-NPCs expedite the generation of PSC-derived oligodendrocytes with O4-, 4860-, and myelin basic protein (MBP)-positive cells that already appear within 7 weeks following growth factor withdrawal-induced differentiation. Thus, RGL-NPCs may serve as robust tool for time-efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. GLIA 2015. © 2015 Wiley Periodicals, Inc.
    Glia 06/2015; 63(12). DOI:10.1002/glia.22882 · 6.03 Impact Factor
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    • "Combinatorial cell culture is a powerful empirical ‘search engine’ that can be used to discover completely new methods for stem cell differentiation, as well as to optimize or further develop existing protocols, for example by varying the input cell type (e.g. iPS [52], [53] cells, adult stem cells, stable mesenchymal [54], [55] or neuroepithelial [56], [57] intermediates, or differentiated cultures derived by previously determined protocols comprising growth factors or small molecules [19], [58]), the cell culture media components or their concentration, the timing of media changes and many other factors important in cell culture. It can be used to devise protocols that are faster, more productive or cost-effective; protocols that generate higher quality cells, eliminate undefined components such as serum or substitute growth factors with small molecules. "
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    ABSTRACT: We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.
    PLoS ONE 09/2014; 9(9):e104301. DOI:10.1371/journal.pone.0104301 · 3.23 Impact Factor
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    • "It is now possible to investigate developmental processes of the human central nervous system (CNS) and the pathogenesis of neural disorders using human PSCs [3] [4] [5] [6]. iPSC-derived NPCs (iPSC-NPCs) can produce both neuronal and glial cells; however, they tend to remain neurogenic for a prolonged period [7] [8]. Extensive research has led to methods for promoting iPSC differentiation toward specific lineages. "
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    ABSTRACT: Here we established a unique human glial cell line, GDC90, derived from a human glioma and demonstrated its utility as a glial scaffold for the polarization and differentiation of human induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs). When co-cultured with GDC90 cells, iPSC-NPCs underwent rapid polarization and neurite extension along the radially spreading processes of the GDC90 cells, and showed migratory behavior. This method is potentially useful for detailed examination of neurites or for controlling neurites behavior for regenerative medicine.
    Biochemical and Biophysical Research Communications 04/2014; 447(4). DOI:10.1016/j.bbrc.2014.04.070 · 2.30 Impact Factor
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