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. Overall design: 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.