Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation
Xiwei Shan, Ai Zhang, Mitchell G. Rezzonico, Ming‐Chi Tsai, Carlos Sánchez-Priego, Yingjie Zhang, Michelle B. Chen, Meena Choi, José Miguel Andrade López, Lilian Phu, Amber Cramer, Qiao Zhang, Jillian M. Pattison, Christopher M. Rose, Casper C. Hoogenraad, Claire G. Jeong
Abstract
NGN2-driven induced pluripotent stem cell (iPSC)-to-neuron conversion is a popular method for human neurological disease modeling. In this study, we present a standardized approach for generating neurons utilizing clonal, targeted-engineered iPSC lines with defined reagents. We demonstrate consistent production of excitatory neurons at scale and long-term maintenance for at least 150 days. Temporal omics, electrophysiological, and morphological profiling indicate continued maturation to postnatal-like neurons. Quantitative characterizations through transcriptomic, imaging, and functional assays reveal coordinated actions of multiple pathways that drive neuronal maturation. We also show the expression of disease-related genes in these neurons to demonstrate the relevance of our protocol for modeling neurological disorders. Finally, we demonstrate efficient generation of NGN2-integrated iPSC lines. These workflows, profiling data, and functional characterizations enable the development of reproducible human in vitro models of neurological disorders.