BMP, MEK and WNT modulation with NGN2 expression for rapid neuron differentiation from hiPSC amenable to regional patterning [BMWi]

hiPSCs aid in studying neurological diseases and developing therapies. Interventions, such as dual SMAD inhibition or NGN2 overexpression (“iNGN2”), can direct hiPSCs towards neurons. Starting directly from hiPSCs, iNGN2 shortens the time to a neuronal stage but leads to neurons resembling peripheral or posterior fates. We applied an accelerated induction paradigm that is only dependent on inhibition of BMP, MEK and WNT pathways (“BMWi”), to commit iPSCs before to rostral neuroectoderm. The resulting neurons showed strong expression of telencephalic, cortical markers, with decreased levels of peripheral and posterior marker genes compared to iNGN2 alone. The resulting cortical neurons are suitable for a tau aggregation assay. Furthermore, we could demonstrate that during BMWi treatment, the cells are amenable to regional patterning clues. This allowed the generation of neurons from different regions of the CNS and PNS, which will significantly improve existing in vitro models for neurodegenerative disorders. We performed a comparison of iPSCs, iNGN2 and BMWi differentiated neurons at different days of differentiation. Neurons were plated in either NMM+ or NMM- (Neurobasal and B27 with or without retinoids). BMWi cells were replated in NMM- and the medium was changed after 7 days.