Reproducible Differentiation of Human Pluripotent Stem Cells into Two‐Dimensional Cortical Neuron Cultures with Checkpoints for Success

Abstract

AbstractThe patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR‐modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two‐dimensional cultures of hPSC‐derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC.Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neuronsSupport Protocol 1: Harvesting and fixing cells for flow cytometry analysesSupport Protocol 2: Performing flow cytometry analysesSupport Protocol 3: Thawing NPCs from a cryopreserved stockAlternate Protocol 1: Continuing Expansion of NPCsAlternate Protocol 2: Treatment of neurons with Ara‐C to ablate radial gliaSupport Protocol 4: Experimental methods for validation of excitatory cortical neurons