Simplified 3D protocol capable of generating early cortical neuroepithelium.
Here, we report a 3D cerebellar differentiation protocol with 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 granule cells). Follow-up tests indicated that addition of FGFs, 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 cost-efficient method for 3D culture, whether to research development of the early cerebellar neuroepithelium, a base to generate mature cortical structures, or to optimize minimal-factor protocols for other brain regions.
Department of Pediatrics/Child Neurology, Amsterdam Neuroscience, VU University Medical Center, Amsterdam 1081 HV, The Netherlands.
Dwayne B
DB
Holmes
Department of Pediatrics/Child Neurology, Amsterdam Neuroscience, VU University Medical Center, Amsterdam 1081 HV, The Netherlands vm.heine@vumc.nl.|Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, Amsterdam 1081 HV, The Netherlands.
Vivi M
http://orcid.org/0000-0003-4416-3875
VM
Heine
3D; Cerebellum; Cortex; Organoid; Pluripotent stem cell