LAUSR

and oligodendrocyte precursors as adherent cells. Based on high expression level of Pax6, Sox1, Sox2 and low expression of Pax3 NESCs likely have primitive neural plate identity. Besides spontaneous differentiation in adherent culture, hNESCs are capable of spontaneously forming cerebral organoids or “minibrains” in suspension. These organoids contain functional neurons and glial cells. We also obeserve self-organized structures resembling developing brain cortex with occasional folding. We demonstrate the importance of WNT and FGF2 signaling during organoids expansion phase for the formation of these cortex-like structures. Cultivation of cerebral organoids as floating aggregates on orbital shaker for more than one month leads to downregulation of Sox2 and DCX and to neuronal maturation. After 2 months after aggregation, we were able to detect various types of neurons including GABA, dopaminergic and serotoninergic, as well as astrocytes. Two-photon microscopy revealed an intricate network of MAP2-positive neuronal dendrites. Calcium imaging demonstrated spontaneous calcium waves characteristic of both neuronal and astroglial activity. Regionalization of organoids by FGF8 and SHH-signaling activation lead to significant increase in tyrosine-hydroxylase-positive cell number and to the detection of dopamine in conditioned culture media. Drug testing of marketed drugs demonstrated the adverse effects of one compound on cerebral organoids growth and development. The presented approach allows efficient derivation of hNESC that can differentiate into neuronal cells with high efficiency both in 2D and in 3D culture and can serve as a platform for human brain development studies and for studies of drugs teratogenicity.