Imura T, Nakano I, Kornblum HI, Sofroniew MV.. Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes. Glia 53: 277-293

Department of Neurobiology, University of California, Los Angeles, California 90095-1763, USA.
Glia (Impact Factor: 6.03). 02/2006; 53(3):277-93. DOI: 10.1002/glia.20281
Source: PubMed


Recent findings show that the predominant multipotent neural stem cells (NSCs) isolated from postnatal and adult mouse brain express glial fibrillary acid protein (GFAP), a protein commonly associated with astrocytes, and that primary astrocyte cultures can contain GFAP-expressing cells that act as multipotent NSCs when transferred to neurogenic conditions. The relationship of GFAP-expressing NSCs to GFAP-expressing astrocytes is unclear, but has important implications. We compared the phenotype and neurogenic potential of GFAP-expressing cells derived from different CNS regions and maintained in vitro under different conditions. Multiple labeling immunohistochemistry revealed that both primary astrocyte cultures and adherent neurogenic cultures derived from postnatal or adult periventricular tissue contained subpopulations of GFAP-expressing cells that co-expressed nestin and LeX/CD15, two molecules associated with NSCs. In contrast, GFAP-expressing cells in similar cultures prepared from adult cerebral cortex did not express detectable levels of LeX/CD15, and exhibited no neurogenic potential. Fluorescence-activated cell sorting (FACS) of both primary astrocyte cultures and adherent neurogenic cultures for LeX/CD15 showed that GFAP-expressing cells competent to act as multipotent NSCs were concentrated in the LeX-positive fraction. Using neurosphere assays and a transgenic ablation strategy, we confirmed that the predominant NSCs in primary astrocyte and adherent neurogenic cultures were GFAP-expressing cells. These findings demonstrate that GFAP-expressing cells derived from postnatal and adult forebrain are heterogeneous in both molecular phenotype and neurogenic potential in vitro, and that this heterogeneity exists before exposure to neurogenic conditions. The findings provide evidence that GFAP-expressing NSCs are phenotypically and functionally distinct from non-neurogenic astrocytes.

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    • "It seems likely that the effects of different factors on GLT-1 expression may generalize to other markers of astrocyte differentiation. There is evidence for morphologically distinct subtypes of astrocytes (Oberheim et al. 2009) and several groups have begun to define molecular subtypes in vitro and in vivo (Bachoo et al. 2004;Imura et al. 2006;Yeh et al. 2009;Stahlberg et al. 2011;Lau et al. 2012;Benesova et al. 2012;Rusnakova et al. 2013;Kasymov et al. 2013; for reviews, see Chaboub and Deneen 2012). As the molecular subtypes of astrocytes are identified, it will be interesting to learn if Pax6 contributes to broader differences in subtypes of astrocytes. "
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    ABSTRACT: The Na(+) -dependent glutamate transporter, GLT-1 (EAAT2), shows selective expression in astrocytes, and neurons induce expression of GLT-1 in astrocytes. In unpublished analyses of GLT-1 promoter reporter mice, we identified an evolutionarily conserved domain of 467 nucleotides ~8 kb upstream of the GLT-1 translation start site that is required for astrocytic expression. Using in silico approaches, we identified Pax6 as a transcription factor that could contribute to the control of GLT-1 expression by binding within this region. We demonstrated expression of Pax6 protein in astrocytes in vivo. Lentiviral transduction of astrocytes with exogenous Pax6 increased expression of enhanced green fluorescent protein (eGFP) in astrocytes prepared from transgenic mice that use a bacterial artificial chromosome (BAC) containing a large genomic region surrounding the GLT-1 gene to control expression of eGFP. It also increased GLT-1 protein, and GLT-1-mediated activity, while there was no effect on the levels of astroglial glutamate transporter, GLAST. Transduction of astrocytes with an shRNA directed against Pax6 reduced neuron-dependent induction of GLT-1 or eGFP. Finally, we confirmed Pax6 interaction with the predicted DNA binding site in electrophoretic mobility assays (EMSA) and chromatin immunoprecipitation (ChIP). Together, these studies show that Pax6 contributes to regulation of GLT-1 through an interaction with these distal elements and identify a novel role of Pax6 in astrocyte biology. This article is protected by copyright. All rights reserved.
    Full-text · Article · Oct 2015 · Journal of Neurochemistry
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    • "In order to investigate the early events of direct reprogramming, the cDNA of Neurog2 and Ascl1 was fused to the modified estrogen receptor ligand binding domain ERT2 (Raposo et al., 2015) and sub-cloned into a retroviral construct, together with the red fluorescent protein (DsRed-Expressed2, hereafter indicated as DsRed) (Berninger et al., 2007; Heinrich et al., 2010; Heins et al., 2002). Proliferating astrocytes were obtained from postnatal day (P)6–7 mouse cerebral cortex Gray Matter (GM), avoiding the White Matter (WM) and ventricular regions comprising endogenous neural stem cells (Imura et al., 2006). The purity of these cultures was previously assessed with various astrocytic markers and genetic fate mapping (Berninger et al., 2007; Heinrich et al., 2010; Heins et al., 2002) (see also Figures S1I and S1J). "
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    ABSTRACT: Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jun 2015 · Cell stem cell
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    • "Fgf2 treatment shifts the glial population from cells with astroglial morphology toward cells with radial and bipolar morphology. Similarly, Fgf signaling changes glia morphology in the zebrafish spinal cord (Goldshmit et al. 2012) or in mammalian astrocytes in vitro (Imura et al. 2006; Goldshmit et al. 2012; Lichtenstein et al. 2012). The radial and bipolar glia cells promote the formation of bridges that support axonal regeneration through the lesion. "
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    ABSTRACT: A major impediment for recovery after mammalian spinal cord injury (SCI) is the glial scar formed by proliferating reactive astrocytes. Finding factors that may reduce glial scarring, increase neuronal survival, and promote neurite outgrowth are of major importance for improving the outcome after SCI. Exogenous fibroblast growth factor (Fgf) has been shown to decrease injury volume and improve functional outcome; however, the mechanisms by which this is mediated are still largely unknown. In this study, Fgf2 was administered for 2 weeks in mice subcutaneously, starting 30 min after spinal cord hemisection. Fgf2 treatment decreased the expression of TNF-a at the lesion site, decreased monocyte/macrophage infiltration, and decreased gliosis. Fgf2 induced astrocytes to adopt a polarized morphology and increased expression of radial markers such as Pax6 and nestin. In addition, the levels of chondroitin sulfate proteoglycans (CSPGs), expressed by glia, were markedly decreased. Furthermore, Fgf2 treatment promotes the formation of parallel glial processes, "bridges," at the lesion site that enable regenerating axons through the injury site. Additionally, Fgf2 treatment increased Sox2-expressing cells in the gray matter and neurogenesis around and at the lesion site. Importantly, these effects were correlated with enhanced functional recovery of the left paretic hind limb. Thus, early pharmacological intervention with Fgf2 following SCI is neuroprotective and creates a proregenerative environment by the modulation of the glia response.
    Full-text · Article · Mar 2014 · Brain and Behavior
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