Protocol for purifying primitive neural progenitors by fractional passaging to generate astroglia-enriched cerebral organoid.

Human pluripotent stem cell (hPSC)-derived primitive neural progenitor cells (pNPCs) and cerebral organoids provide a powerful platform for studying human brain development. Here, we present a fractional passaging protocol that yields nearly pure PAX6+/SOX2+ pNPCs. We describe steps for neuroepithelial differentiation, purification, and production of astroglia-enriched organoids. We then detail procedures for downstream analysis. The organoids contain over 15% astrocytes alongside maturing neurons, providing a scalable and reproducible system to model human glial-neuronal interactions in development and disease. For complete details on the use and execution of this protocol, please refer to Dang et al.1.