Gene expression in astrocytes is affected by subculture

Abstract

We have investigated the effects of cell passaging and time in culture on astrocyte morphology, transferrin expression and the expression of two main astrocyte markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS; EC 6.3.1.2). When primary astrocytes were subcultured, giving rise to secondary and tertiary cultures, their morphology changed, regardless of the split ratio used to passage the cells. Correlating with this morphological change, a dramatic increase in the accumulation of GFAP and GS mRNAs was observed after cells had been passaged. This effect was in marked contrast to the moderate increase in the levels of GFAP and GS mRNAs observed over several weeks in primary culture. Hydrocortisone induction of GS gene expression was not affected by cell passage. Transferrin mRNA, which is not normally found in astrocytes in vivo, was expressed at a high level in primary cultures of astrocytes. However, transferring mRNA almost completely disappeared after the second passage. Astrocyte-conditioned media, or co-cultures with oligodendrocytes, modified transferrin gene expression. Taken together, these results show that subculturing of primary rat astrocytes leads to a dramatic change in the genetic expression of several proteins and provides a new approach to modify astrocyte differentiation in vitro.