Isolation, cloning and characterization of a putative type-1 astrocyte cell line

We have established a permanent cell line (1H91) of putative type-1 astrocyte precursor cells that were clonally derived from a single cell isolated from E16 mouse cerebellum. Epidermal growth factor (EGF) and transforming growth factor (TGF alpha) are strong mitogens for 1H91 cells (ED50 of 9.02 + 1.74 ng/ml and 15.98 +/- 2.34 ng/ml, respectively), while basic fibroblast growth factor (bFGF) is only weakly mitogenic and platelet derived growth factor (PDGF) has no mitogenic activity. In the proliferative state, the 1H91 cells are immunohistochemically positive for nestin and vimentin, and negative for A2B5, CNPase, neurofilament (NF), and neuron specific enolase (NSE). The majority of EGF-treated 1H91 cells are not immunoreactive for glial acid fibrillary protein (GFAP). In the presence of 5 ng/ml bFGF, 1H91 cells become non-mitotic and develop a morphology consistent with a fibrous astrocyte. In contrast to the proliferating cultures, the bFGF treated cultures were strongly immunoreactive for GFAP, only mildly immunoreactive for nestin and vimentin, and negative for A2B5, CNPase, NF, and NSE. Type-1 astrocytes are known to proliferate in response to EGF, and are immunohistochemically GFAP positive, A2B5 negative, and CNPase negative [38]. However, type-1 astrocytes only develop a fibrous morphology during the process of reactive gliosis [31]. Since EGF is a strong mitogen for 1H91 cells, and these cells may be differentiated into GFAP positive, A2B5 negative, CNPase negative astrocytes, we conclude that 1H91 cells conform to a type-1 astrocyte precursor phenotype. In addition, the fibrous morphology of the bFGF treated 1H91 cells suggests that these cells follow the process of reactive gliosis. Therefore, the 1H91 clonal cell line may provide an in vitro model for studying the underlying cellular mechanisms of the type-1 astrocyte in reactive gliosis.