Established monolayer differentiation of mouse embryonic stem cells generates heterogeneous neocortical-like neurons stalled at a stage equivalent to midcorticogenesis.

Two existing and widely applied protocols of embryonic stem (ES) cell differentiation have been developed to enable in vitro generation of neurons resembling neocortical projection neurons in monolayer culture and from embryoid bodies. The monolayer approach offers advantages for detailed in vitro characterizations and potential mechanistic and therapeutic screening. We investigated whether mouse ES cells undergoing largely undirected neocortical differentiation in monolayer culture recapitulate progressive developmental programs of in vivo progenitor and postmitotic differentiation and whether they develop into specific neocortical subtypes. We find that ES-derived mitotic cells that have been dorsalized by the sonic hedgehog antagonist cyclopamine, and that express, as a total population, cardinal markers of telencephalic progenitors, are, in fact, molecularly heterogeneous. We next show that these progenitors subsequently generate small numbers of heterogeneous neocortical-like neurons that are "stalled" at an immature stage of differentiation, based on multiple developmental criteria. Although some aspects of neocortical development are recapitulated by existing protocols of ES cell differentiation, these data indicate that mouse ES-derived neocortical progenitors both are more heterogeneous than their in vivo counterparts and seemingly include many incorrectly specified progenitors. Furthermore, these ES-derived progenitors spontaneously differentiate into sparse, and incompletely and largely imprecisely differentiated, neocortical-like neurons that fail to adopt specific neuronal identities in vitro. These results provide both foundation and motivation for refining and enhancing directed differentiation of clinically important neocortical projection neuron subtypes.