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.

Transforming growth factor α promotes sequential conversion of mature astrocytes into neural progenitors and stem cells

“…The size of the population affected by TGFa and the progressiveness of the morphological alterations allowed a clear identification of GFAP-immunoreactive polygonal astrocytes as the target cells. However, previous reports, using growth factors cocktails including EGF, the functional analog of TGFa, have detected the presence of neural stem cells at a low frequency (0.1-10%) in astrocyte cultures (Laywell et al, 2000;Imura et al, 2006). We thus verified whether residual radial glia and/or neural stem cells could account for part of our observations.…”

“…The demonstration that neurospheres, considered as signing for the presence of neural stem cells (Reynolds et al, 1992), can be derived from astrocytes sub-cultured for a week in EGF-or EGF-and FGF-containing medium (Laywell et al, 2000;Imura et al, 2003), has additionally pointed to the possibility that mature astrocytes themselves might behave as stem cells at least in vitro. But the brevity of the time course required to obtain neurospheres from astrocyte cultures (7 DIV) and its similarity with that required to obtain neurospheres from brain germinal zones (Reynolds et al, 1992) suggest that they correspond to GFAPexpressing neural stem cells preserved in the cortical astrocyte cultures used (Imura et al, 2006). In contrast, in neural stem cell and progenitor-deprived cultures, we observed a slow and sequential conversion of a population of astrocytes into functional neural progenitors.…”

Integrin-Kindlin3 requirements for microglial motility in vivo are distinct from those for macrophages

“…Moreover, all Kindlin3-positive cells also expressed CD11b, the α subunit of integrin α m β 2 , which serves as a microglia marker ( Figure 1C). Brain astrocytes identified by GFAP staining (26) did not express Kindlin3, as evidenced by costaining of brain tissue sections or mixed cultures of microglia and astrocytes ( Figure 1D). Thus, Kindlin3 was almost exclusively expressed by microglia, both in retinas ( Figure 1E) and the brain.…”

TGFβ1 and TNFα potentiate nitric oxide production in astrocyte cultures by recruiting distinct subpopulations of cells to express NOS-2

“…(Bowman et al 2003;Conti et al 1994;Hosli and Hosli 1993;Imura et al 2006;Matthias et al 2003;Miller and Szigeti 1991;Nikcevich et al 1997;Porter and McCarthy 1995;Shao et al 1994;St-Pierre et al 2000;Swanson et al 1997;Venance et al 1998;Whitaker-Azmitia et al 1993;Zhou and Kimelberg 2001). For example, both in vitro and in vivo studies show that the expression of glutamate transporter isoforms GLT-1 and GLAST segregate to different hippocampal GFAP-positive astrocytes (Conti et al 1998;Perego et al 2000;van Landeghem et al 2006).…”