Generation of Various Telencephalic Regions from Human Embryonic Stem Cells in Three-Dimensional Culture.

In the developing embryo, telencephalon arises from the rostral portion of the neural tube. The telencephalon further subdivides into distinct brain regions along the dorsal-ventral (DV) axis by exogenous patterning signals. Here, we describe a protocol for in vitro generation of various telencephalic regions from human embryonic stem cells (ESCs). Dissociated human ESCs are reaggregated in a low-cell-adhesion 96-well plate and cultured as floating aggregates. Telencephalic neural progenitors are efficiently generated when ESC aggregates are cultured in serum-free medium containing TGFβ inhibitor and Wnt inhibitor. In long-term culture, the telencephalic neural progenitors acquire cortical identities and self-organize a stratified cortical structure as seen in human fetal cortex. By treatment with Shh signal, the telencephalic progenitors acquire ventral (subpallial) identities and generate lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE). In contrast, by treatment with Wnt and BMP signals, their regional identities shift to more dorsal side that generates choroid plexus and medial palllium (hippocampal primordium).