Generating iAstrocytes From Human Induced Pluripotent Stem Cells by Combining Low-Density Passaging of Neural Progenitor Cells and Transcription Factor NFIA Transdifferentiation

Abstract

Astrocytes are key regulators of central nervous system (CNS) homeostasis, and their dysfunction is implicated in neurological and neurodegenerative disorders. Here, we describe a two-step protocol to generate astrocytes from human induced pluripotent stem cells (hiPSCs) using a bankable neural progenitor cell (NPC) intermediate, followed by low-density passaging and overexpression of the gliogenic transcription factor NFIA. A bankable NPC intermediate 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 hiPSCs, which are then differentiated into hiPSC-derived astrocytes, termed iAstrocytes. 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 the 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. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Differentiation of hiPSCs to NPCs

Basic Protocol 2: Differentiation of NPCs into iAstrocytes

Support Protocol 1: Molecular validation of iAstrocytes

Support Protocol 2: Calcium imaging-based validation of iAstrocyte function

Support Protocol 3: Differentiation of NPCs into neurons