Long-lasting coexpression of nestin and glial fibrillary acidic protein in primary cultures of astroglial cells with a major participation of nestin(+)/GFAP(-) cells in cell proliferation

Nestin, a currently used marker of neural stem cells, is transiently coexpressed with glial fibrillary acidic protein (GFAP) during development and is induced in reactive astrocytes following brain injury. Nestin expression has also been found in cultures of astroglial cells, but little is known about the fate and the mitotic activity of nestin-expressing cells in this in vitro model. The present study reveals a long-lasting expression of nestin in primary cultures of astroglial cells derived from the rat brain. Over 70% of the cells were nestin(+) at 12 weeks, with a large majority coexpressing the GFAP astrocytic marker. Time-course analyses supported a transition from a nestin(+)/GFAP(-) to a nestin(+)/GFAP(+) phenotype over time, which was further increased by cell cycle arrest. Interestingly, double staining with Ki67 revealed that over 90% of cycling cells were nestin(+) whereas only 28% were GFAP(+) in a population consisting of almost equivalent numbers of nestin(+) and GFAP(+) cells. These observations indicated that nestin(+)/GFAP(-) cells are actively engaged in mitotic activity, even after 2 weeks in vitro. Part of these cells might have retained properties of neural stem cells, insofar as 10% of cells in a primary culture of glial cells were able to generate neurospheres that gave rise to both neurons and astrocytes. Further studies will be necessary to characterize fully the proliferating cells in primary cultures of glial cells, but our present results reveal a major contribution of the nestin(+)/GFAP(-) cells to the increase in the number of astrocytes, even though nestin(+)/GFAP(+) cells proliferate also.