Controlled differentiation of mES cells into neural precursors

During normal development, neural precursors undergo a stepwise transition from a multipotent to a more lineage-restricted neuronal or glial progenitor stage. Although neural differentiation has been considered a default pathway of ES cell differentiation (15, 16), the generation of high numbers of neurons and glia requires protocols that promote the survival and proliferation of neural precursors. The experimentally induced differentiation pathway presented here (Protocol 1) recapitulates the gradual transition of neurogenic into gliogenic precursors observed during CNS maturation. Based on a differentiation paradigm described by Okabe et al. (6,20), EB-derived neural precursors are sequentially propagated with discrete growth factors, and develop from a multipotent into a predominantly glial precursor fate (7,20). Figure 1 shows a schematic of the temporal sequence of events.

An important prerequisite for the successful application of this protocol is the use of mES cell cultures that are well maintained, that is with little or no signs of spontaneous differentiation. Undifferentiated mES cells are refractile and small, and the cell clusters are rounded and well defined (Figure 2A). On withdrawal of LIF from the medium and reseeding ofmES cells onto a non-adhesive substratum, EB formation is initiated (Figure 2B). After 4 to 5 days, the newly formed EBs are plated onto an adhesive substratum and propagated in a serum-free medium (ITSFn; Figure 2C), which favours the survival of neuroepithelial cells and inhibits the growth of other cell types (6). Following this selection step, the resulting

Plating in ITSFn medium

Embryoid body formation Mouse ES cell proliferation

Lineage selection

4 FGF2+EGF

Neurons

\ FGF2+PDGF

Lineage selection -►

Lineage selection -►

Oligodendrocytes

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