Diversification of glial lineages: a novel method to clone brain cells in vitro on nitrocellulose substratum

Abstract

We have developed a novel in vitro method to analyze the diversification of glial cells during development. The primary advantage of the approach is that glial lineages are formed in discrete clones on a nitrocellulose substratum where the relationship of the progeny is strictly defined. This method facilitates the comparison of a large complement of astrocyte and oligodendrocyte lineages under controlled conditions. Clones were formed by plating a brain dissociate on nitrocellulose at very low density (5,000-40,000 cells/154 mm2). However, growth depended on diffusible factors produced by brain cells growing under the nitrocellulose support at high density (feeder layer). The cloning efficiency of cells from mouse forebrain (P0) was 1-3%. This means we can detect 100,000 to 300,000 clonal progenitors in the dissociate (10(7) cells per forebrain) using the clonal culture technique. Cell phenotypes were determined by immunocytochemical staining with anti-glial fibrillary acidic protein (GFAP) to label astrocytes and anti-galactocerebroside (GC) and anti-myelin basic protein (MBP) to label oligodendrocytes. There was a remarkable diversity of glia represented in different lineages. The number of astrocyte clones was greater than the number of oligodendrocyte clones but combined their total was 90%. Clone sizes were distributed over a wide range, which indicated that growth rates varied. Clones appeared compact or dispersed but astrocyte clones exhibited three different morphologies-fibroblast-like, stellate, and elongated. Oligodendrocytes had different morphologies distinct from astrocytes. Although there were different glial lineages the cells in most clones were homogeneous, indicating the progeny had the same fate. However, a small number of the clones, approximately 2%, were heterogeneous and contained both astrocytes and oligodendrocytes. The application of this technique to glial lineages demonstrates that intrinsic factors have a role in determining cell fate since different clones formed under the same external conditions. Finally, these results are consistent with the existence of multiple glial progenitors or the continued presence of multipotential progenitors at the time of birth.