BMP, MEK and WNT modulation with NGN2 expression for rapid generation of hiPSC-derived neurons amenable to regional patterning [snRNA-seq]

hiPSC are a promising tool for studying neurological diseases and developing therapies for neurodegenerative diseases. Differentiation of hiPSC into neurons can be achieved by dual SMAD inhibition (dSMADi) or induced NGN2 overexpression (“iNGN2”). Starting directly from hiPSC, iNGN2 shortens the time to a neuronal stage but leads to neurons partially resembling peripheral or posterior fates while dSMADi more faithfully recapitulates forebrain development. To modify the iNGN2 paradigm, we applied an accelerated induction paradigm that is dependent on inhibition of BMP, MEK and WNT pathways (“BMWi”), to commit hiPSC into a rostral neuroectoderm fate before iNGN2. The resulting neurons showed strong expression of telencephalic and 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 additional regional patterning cues. This allowed the generation of neurons from different regions of the CNS and PNS, which will significantly facilitate in vitro modeling of a range of neurodevelopmental and neurodegenerative disorders. The aim of this study was to observe the progression of BMWi differentiation (hiPSC, day 6 of the protocol and final neurons) using snRNA sequencing and thus to examine the heterogeneity of the culture. For comparison, iNGN2 neurons were also examined.