iAstrocytes model cytokine influences on complement expression and neuronal network synchronization

Astrocytes play essential roles in neuronal development, function, and disease, yet existing methods to derive astrocytes from human pluripotent stem cells (hPSCs) are complex and can involve months of in vitro maturation. We developed a genomic safe-harbor knock-in system for inducible expression of the astrogenic transcription factors NFIA, NFIB, and SOX9, enabling rapid and robust generation of functional induced astrocytes (iAstrocytes). Across five hPSC lines, NFIB-SOX9 and NFIA-NFIB-SOX9 combinations efficiently generated highly pure populations expressing astrocyte-specific and synaptogenic genes. iAstrocytes displayed cytokine-induced expression of complement factors C3 and C4 and were amenable to CRISPR interference (CRISPRi) gene expression knockdown. Optimization of culture conditions enabled survival of NFIB-SOX9 iAstrocytes in co-culture with human induced neurons (iNeurons). Through pharmacological and genetic perturbations, we uncovered a previously undescribed phenomenon in which co-culture with iAstrocytes promoted the development of synchronized iNeuron network calcium activity mediated by specific gap junction proteins. This rapid and genetically tractable iAstrocyte platform provides a robust model to dissect human genetic and environmental effects on astrocyte-neuron interactions.