Simplified 3D protocol capable of generating early cortical neuroepithelium

Abstract

Here,we report a 3Dcerebellar differentiation protocolwith quick startup method, defined medium and no special materials or handling requirements. Three fibroblast growth factors (FGF2, 4 and 8) were used for cerebellar patterning and smoothened agonist (SAG) for granule cell development. After 35 days, differentiation products exhibited similar structures and neuronal markers reported in prior 'organoid' and 'spheroid' protocols. This included cells positive for KIRREL2 (a marker of early cerebellar neuroepithelium) and ZIC1 (a marker for granulecells).Follow-uptestsindicatedthatadditionofFGFs, if helpful, was not required to generate observed structures and cell types. This suggests that intrinsic production of patterning factors by aggregates themselves may be adequate for region-specific 3D modeling. This protocol may be used as a quick, easy and costefficientmethod for 3D culture, whether to research development of the early cerebellar neuroepithelium, a base to generate mature cortical structures,or tooptimizeminimal-factorprotocols forother brainregions.