Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation.

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.

Shan X ，Zhang A ，Rezzonico MG ，Tsai MC ，Sanchez-Priego C ，Zhang Y ，Chen MB ，Choi M ，Andrade López JM ，Phu L ，Cramer AL ，Zhang Q ，Pattison JM ，Rose CM ，Hoogenraad CC ，Jeong CG ... -  《-》

NGN2 induces diverse neuron types from human pluripotency.

Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.

Lin HC ，He Z ，Ebert S ，Schörnig M ，Santel M ，Nikolova MT ，Weigert A ，Hevers W ，Kasri NN ，Taverna E ，Camp JG ，Treutlein B ... -  《Stem Cell Reports》

Making neurons, made easy: The use of Neurogenin-2 in neuronal differentiation.

Directed neuronal differentiation of human pluripotent stem cells (hPSCs), neural progenitors, or fibroblasts using transcription factors has allowed for the rapid and highly reproducible differentiation of mature and functional neurons. Exogenous expression of the transcription factor Neurogenin-2 (NGN2) has been widely used to generate different populations of neurons, which have been used in neurodevelopment studies, disease modeling, drug screening, and neuronal replacement therapies. Could NGN2 be a "one-glove-fits-all" approach for neuronal differentiations? This review summarizes the cellular roles of NGN2 and describes the applications and limitations of using NGN2 for the rapid and directed differentiation of neurons.

Hulme AJ ，Maksour S ，St-Clair Glover M ，Miellet S ，Dottori M ... -  《Stem Cell Reports》

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.

Frega M ，van Gestel SH ，Linda K ，van der Raadt J ，Keller J ，Van Rhijn JR ，Schubert D ，Albers CA ，Nadif Kasri N ... -  《-》

A Single-Cell Model for Synaptic Transmission and Plasticity in Human iPSC-Derived Neurons.

Synaptic dysfunction is associated with many brain disorders, but robust human cell models to study synaptic transmission and plasticity are lacking. Instead, current in vitro studies on human neurons typically rely on spontaneous synaptic events as a proxy for synapse function. Here, we describe a standardized in vitro approach using human neurons cultured individually on glia microdot arrays that allow single-cell analysis of synapse formation and function. We show that single glutamatergic or GABAergic forebrain neurons differentiated from human induced pluripotent stem cells form mature synapses that exhibit robust evoked synaptic transmission. These neurons show plasticity features such as synaptic facilitation, depression, and recovery. Finally, we show that spontaneous events are a poor predictor of synaptic maturity and do not correlate with the robustness of evoked responses. This methodology can be deployed directly to evaluate disease models for synaptic dysfunction and can be leveraged for drug development and precision medicine.

Meijer M ，Rehbach K ，Brunner JW ，Classen JA ，Lammertse HCA ，van Linge LA ，Schut D ，Krutenko T ，Hebisch M ，Cornelisse LN ，Sullivan PF ，Peitz M ，Toonen RF ，Brüstle O ，Verhage M ... -  《-》