Generating iAstrocytes from hiPSCs by combining low-density passaging of NPCs and NFIA transdifferentiation

Astrocytes are key regulators of CNS homeostasis and their dysfunction is implicated in neurological and neurodegenerative disorders. Here, we describe a two-step protocol to generate astrocytes from iPSCs using a bankable neural progenitor cell (NPC) intermediate, followed by low-density passaging and overexpression of the gliogenic transcription factor NFIA. A bankable NPC intermediates allows for facile differentiation into both purified neuronal and astrocyte cell types in parallel from the same genetic background, depending on the experimental needs. This article presents a protocol to generate NPCs from iPSCs (Basic Protocol 1), which are then differentiated into iPSC-derived astrocytes, termed iAstrocytes (Basic Protocol 2). The resulting iAstrocytes express key markers of astrocyte identity at transcript and protein levels by bulk RNA-seq and immunocytochemistry respectively. Additionally, they respond to the inflammatory stimuli poly(I:C) and generate waves of calcium activity in response to either physical activity or addition of ATP. Our approach offers a simple and robust method to generate and characterize human astrocytes which can be used to model human disease affecting this cell type. Describing new differentiation protocol for Astrocytes from IPSC. FA10 NPC No NFIA (1-3) is previous described differentiation protocol. FA10 NPC NFIA (1-3) is new protocol (Basic Protocol 1) and IMR90-iPSC-NFIA-SOX9 (1-3) is another protocol we generated for generating iAstrocytes from IPS cell lines.