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
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.
"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). "
"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. "
[Show abstract][Hide abstract] 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.
Brain and Behavior 03/2014; 4(2):187-200. DOI:10.1002/brb3.172 · 2.24 Impact Factor
"Before experimental procedure cells were trypsinized using 0.5% trypsin-EDTA (GIBCO). Under these conditions, neurons, oligodendrocytes and microglia rapidly die or do not adhere (Imura et al., 2006). The type 2 astrocyte mouse cell line C8-S, the mouse embryo-derived teratocarcinoma cell line P19, the mouse neuroblastoma cell line N1E-115, and the mouse fibroblast cell line NIH/3T3 were purchased from the American Tissue Culture Collection and cultured in Dulbecco's Modified eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS), streptomycin, penicillin and glutamine, in a 5% CO 2 humidified atmosphere at 37 C. P19 cells were, if indicated, incubated with retinoic acid (1 lM, R2625, Sigma) for 24 h. "
[Show abstract][Hide abstract] ABSTRACT: Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance. GLIA 2013.
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