Highlights d We establish a single-cell model to study synapses in iPSCderived neurons d This platform allows quantitative analysis of synaptic transmission and plasticity d The platform is validated for GABA-or glutamatergic iPSCderived human neurons d The platform is scalable and suitable for compound screening and disease modeling

Highlights d Quality of human autapses depends on astrocyte type and fetal bovine serum additives d Standardized assays of key morphological, biophysical, and synaptic properties d Standardized assays of short-term plasticity, synaptic depression, and synapse recovery d Some NGN2-induced neurons show multiple/long axons and glutamate-GABA corelease

Summary This protocol describes a highly standardized pipeline for transcription factor-mediated forward programming of human pluripotent stem cells into highly enriched glutamatergic or GABAergic neurons followed by a cryopreservation step that enables the generation of large quality-controlled batches. This approach is particularly useful for reducing interexperimental variability in the context of collaborative studies across different locations and time points. For complete details on the use and execution of this protocol, please refer to Meijer et al. (2019) and Rhee et al. (2019) .