In order to study neuronal function and connectivity in vitro, we adapted a previously established cortical differentiation protocol (Fig. 1a)25 for differentiating hiPSCs into cerebral cortex neurons. Starting from commercially available hiPSCs (Sigma or Cellectis) (Fig. 1b) neural differentiation was induced. Neural progenitor phenotype was confirmed by PAX6, Nestin and OTX2 staining (Fig. 1c) at 25 days after starting neural induction. After proliferation and purification, neural precursor cells (NPCs) were cryopreserved until further use. Final plating of NPCs in neural maintenance medium on a laminin-coated surface further differentiated cells into cortical neurons expressing neuronal marker class III β-tubulin and cortical markers TBR1, CTIP2, and SATB2 (Fig. 1d) at 80 days after neural induction (7 weeks after final plating). However, also non-differentiated progenitor cells were still present in the cultures. Furthermore, the cells clustered and detached easily (Fig. 1d), thereby complicating downstream analyses.
