Figure 1. Neural Differentiation Timeline.

(A) Timeline of neural differentiation includes culturing iPS cell colonies (B) to confluency for 7 days, followed by culture in suspension for 4 days to allow for the formation of embryoid bodies, followed by incubation in neural induction media and growth on a laminin substrate to trigger the formation of neuroepithelial cells displaying neural tube-like rosette structures, which are expanded in suspension (C) culture for several days prior to plating dissociated cells on coverslips. Dissociated cells are cultured in a neural differentiation media containing the growth factors BDNF, GDNF, and IGF to promote the differentiation of neural cells (D, arrows indicate neural cell bodies, red arrow indicates recording electrode for performing electrophysiology). Dissociated neural cells were then cultured for 12–18 weeks in neural differentiation media. At 10-weeks post-plating neural cultures displayed neuronal lineage marker beta III-tubulin and glial cell lineage s100β positive cells (E), indicating that iPS cells differentiated into cultures containing a mixed culture of neurons and glia. Focal synapsin-1 immunostaining along beta III tubulin (F) positive processes were observed indicating the formation of synaptic specializations within the culture.