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
ABSTRACT 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.
- SourceAvailable from: Giacomo Masserdotti
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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). "
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.Cell stem cell 06/2015; DOI:10.1016/j.stem.2015.05.014 · 22.15 Impact Factor
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- "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. "
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.Glia 08/2013; DOI:10.1002/glia.22569 · 6.03 Impact Factor
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- "serum and adding EGF and FGF to the media. Moreover, it is also well known that there are populations of GFAPexpressing radial cells present in the postnatal and adult brain in vivo that are neural stem cells and characterized by expression of LeX/CD15 (Garcia et al., 2004; Imura et al., 2006). It would be interesting to analyze Ezh2 expression in these GFAP expressing progenitors. "
ABSTRACT: Recently, we have demonstrated the expression of the polycomb group protein Ezh2 in embryonic and adult neural stem cells. Although Ezh2 remained highly expressed when neural stem cells differentiate into oligodendrocyte precursor cells, it is downregulated during the differentiation into neurons or astrocytes. This is in accordance with the differentiation repressive role Ezh2 is thought to play in the maintenance and self-renewal of stem cells. To establish the importance of downregulation of Ezh2 for becoming astrocytes, we have studied the effect of forced Ezh2 expression in postnatal mouse astrocytes. Upon forced expression of this polycomb group protein, cultured astrocytes retracted their cell extensions and became proliferating round/bipolar cells that occasionally formed small neurosphere-like clusters. Analysis of the expression profile of these Ezh2-expressing astrocytes reveal downregulation of typical astrocytic genes, like GFAP and S100, and upregulation of genes that are generally expressed in neural stem cells, like nestin, Sox2, musashi, and CD133. However, these neural stem cell-like cells lack a differentiation potential, indicating that overexpression of Ezh2 alone is insufficient for a complete dedifferentiation.10/2010; 13(1):1-6. DOI:10.1089/cell.2010.0052