Protocol for generating NGN2 iPSC lines and large-scale human neuron production

Large-scale production of induced pluripotent stem cell (iPSC)-derived neurons is valuable in disease modeling and drug discovery. Here, we describe a workflow to engineer a doxycycline-inducible NGN2 (neurogenin 2) cassette into the AAVS1 (adeno-associated virus integration site 1) locus and differentiate positive clones into neurons. iPSCs are electroporated with ribonucleoprotein and a donor plasmid. The positive clone rate is maximized with homology-directed repair enhancement, antibiotic selection, and fluorescence. Validated clones are differentiated into neurons in 5 days at a scale of billions. These neurons can be cryopreserved or maintained for months. For complete details on the use and execution of this protocol, please refer to Shan et al.¹.