Simplified 3D protocol capable of generating early cortical neuroepithelium

Abstract

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.

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

“…Based on results reported by Muguruma et al, and our own recent study, it appears that it is possible to achieve differentiation towards cerebellar fates without concerted efforts to reproduce in vivo conditions, as earlier studies have done 1 2 3 4 8 10 . The intriguing part is that the two studies used different sets of growth factors, suggesting that neither set were necessary, though both used FGF2.…”

“…For FGF2, the down arrow refers to lower concentrations (4 ng/mL), and the up arrow refers to higher concentrations (20 ng/mL). This figure has been modified from Holmes and Heine 10 . Please click here to view a larger version of this figure.…”

“…Scale bar = 100 µm. This figure has been modified from Holmes and Heine 10 . Please click here to view a larger version of this figure.…”

“…NOTE: hESCs were obtained from a non-commercial organization (line H01, see Table of Materials ). Three hiPSC control lines (hvs51, 60, and 88) were generated by reprogramming fibroblasts from three healthy human patients (fibroblasts were derived from anonymous, non-identifiable donors and therefore exempt from IRB approval) 10 17 .…”

“…We recently reported a 3D cerebellar differentiation protocol using a reduced number of patterning factors (namely, FGF2, 4, and 8), as well as a simplified setup by keeping cells in 6-well plates (6WPs) throughout to minimize medium requirements 10 . To assist production of granule cells, smoothened agonist (SAG) was used during the final maturation step.…”