Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Abstract

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed "glia-like" cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that "glia-like" cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5-10 days approximately 5-10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3-6 weeks in culture. At confluence the cultures contained 2-5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95-98% GFAP-negative "glia-like" cells. This population of flat "glia-like" cells was positively stained for vimentin, fibronectin, and 20-30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat "glia-like" cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that "glia-like" cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.

GFAP-positive astrocytes are rare or absent in primary adult human brain tissue cultures

“…Table 2 shows discrepant results obtained by immunocytochemical staining of adult human brain cultures. Although minor differences in the percentage of GFAP-positive cells may be attributed to random cell counting errors, it remains unclear why GFAP-positive cells were rare or even absent in adult human brain cultures reported by Osborn et al (1981), Rutka et al (1986) and Perzelova et al (2007). In contrast, Estes et al (1990) and Van der Laan et al (1997) showed a high percentage (more than 80%) of GFAP-positive cells in explanted or dissociated adult human brain cultures.…”

“…The morphological features of primary cultures were described previously (Perzelova et al 2007). Briefly, in primary cultures we found three main morphologically distinct cell types: ameboid cells with grainy cytoplasm, process-bearing cells (PBC) and flat or spindle shaped cells (Fig.…”

“…Previously, we have shown that initially GFAP-negative flat or spindle shaped "glia-like" cells became GFAP-positive during the period of spontaneously decelerated growth (Perzelova & Mares 1993). We supposed that "glia-like" cells which predominate in adult human brain cultures are glial precursor cells (Perzelova et al 2007). On the other hand, significant discrepancies have been observed in the expression of GFAP, fibronectin, vimentin and neuronal markers in adult human brain culture (Rutka et al 1986;Estes et al 1990; Van der Laan et al 1997).…”

Unexpected presence of cytokeratins in human “glia-like” cells

“…Most of immunocytochemical studies use GFAP as the best marker for astrocytes under normal and pathological conditions (10,6,11). However, human brain tissue cultures contain only a small percentage of GFAP-positive astrocytes in early passages (12,13,14). On the other hand, some authors have shown a high percentage of fl at GFAP-positive stained cells in adult human brain cultures (15,16).…”

“…To characterize the glial cell types we used the same marker antibodies as described previously (14,18), particularly, GFAP for astrocytes, O4 for oligodendrocytes, and CD11c for microglia/ macrophages. Indirect immunofl uorescence was used for identifi cation of glial cell types, IF proteins and fi bronectin in primary and secondary cultures up to passage number 10.…”