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Despite the well\u2010known role of astrocytes in the pathophysiology of neurologic disorders, the utilization of induced pluripotent stem cell (iPSC)\u2010derived astrocytes in neuronal networks remains limited. Here, we present a streamlined one\u2010step protocol for the differentiation of iPSCs directly into functional astrocytes without the need for ectopic gene expression or neural progenitor cell generation. We found that culturing iPSCs directly in commercial astrocyte medium, was sufficient to differentiate iPSCs into functional astrocytes within 5\u2009weeks. More than 60 iPSC lines were successfully differentiated into astrocytes by independent researchers across 10 independent laboratories. Validation of the iPSC\u2010astrocyte cultures demonstrated consistent astrocyte differentiation with minimal batch\u2010to\u2010batch variability. In dept. characterization of a subset of iPSC lines confirmed astrocyte identity and functionality of the iPSC\u2010astrocyte monocultures by immunofluorescence, flow cytometry, RNA sequencing, glutamate uptake assays and calcium signaling recordings. Optimization of the protocol enabled co\u2010culture of iPSC\u2010astrocytes with Ngn2 iPSC\u2010derived neurons (iNeurons), promoting neuronal differentiation and synapse formation. Lastly, we used single\u2010cell electrophysiology and multi\u2010electrode arrays, by four independent researchers, to confirm robust neuronal network development in 5\u2010week\u2010old iPSC\u2010astrocyte and iNeuron co\u2010cultures. This protocol offers a rapid and efficient method to establish all\u2010human astrocyte\u2010neuron co\u2010cultures, facilitating the investigation of cell\u2010type\u2010specific contributions to disease pathogenesis. Its validation across numerous iPSC lines in 10 independent laboratories highlights the reproducibility of the protocol and positions it as a platform for advancing disease modeling in human neural networks.<\/jats:p>","DOI":"10.1002\/glia.70202","type":"journal-article","created":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T07:09:56Z","timestamp":1784099396000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Navigating Human Astrocyte Differentiation: Direct and Rapid One\u2010Step Differentiation of Induced Pluripotent Stem Cells to Functional Astrocytes Supporting Neuronal Network Development"],"prefix":"10.1002","volume":"74","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6294-9471","authenticated-orcid":false,"given":"Imke M. 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