Fig. 4.

Generation of neurons and glia from colonies formed at clonal density. (A and B) Colonies were generated from E14.5 forebrain at clonal density or high density in the presence or absence of exogenous Wnt protein. BrdU was added to the cultures overnight to label dividing cells. Colonies were then plated into differentiation conditions on laminin-coated chamber slides. After 1 to 2 weeks, the differentiated cells were fixed and stained to detect neurons, oligodendrocytes, and astrocytes. Clonal-density colonies yielded cells capable of generating astrocytes (GFAP, green in A), neurons (doublecortin, white in A) and oligodendrocytes (NG2, green in B). BrdU incorporation (red) is detected in the nuclei (blue) of the differentiated neurons and glia (arrows for each inset), indicating that the differentiated cells were derived from proliferative stem cells that were dividing in vitro, and not simply contaminating postmitotic cells from the fetal brain.