Differentiation of induced pluripotent stem cells (iPSCs) into astrocytes

Table of Contents

• Introduction
• Materials
• Protocol
• References

Introduction

Hello there! Differentiating induced pluripotent stem cells (iPSCs) into astrocytes is a fantastic way to generate highly relevant glial cell models for drug discovery, neurotoxicity testing, and studying neurological diseases in vitro. By carefully directing the developmental pathways, you can efficiently derive functional astrocytes. This protocol provides a straightforward, step-by-step guide to help you achieve robust astrocyte differentiation in your lab!

Materials

Component	Concentration
DMEM/F12 and Neurobasal Medium	1:1 ratio
N2 Supplement	1x
B27 Supplement	1x
L-Ascorbic acid	200 µM
FGF2	10 ng/ml
CNTF	10 ng/ml

Protocol

1. Coat culture plates with Matrigel or Vitronectin and incubate at 37°C for at least 1 hour.
2. Detach iPSCs using Accutase and plate them onto the coated dishes in Essential 8 medium containing 10 µM ROCK inhibitor Y-27632 (HB2297).
3. Once cells reach 80-90% confluence, transition the cultures to Neural Induction Medium (DMEM/F12 and Neurobasal medium supplemented with N2, B27, and FGF2). Note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs.
4. Perform daily media changes to maintain healthy neural progenitor cells (NPCs).
5. After 7-10 days, passage the NPCs and switch to Astrocyte Maturation Medium containing L-Ascorbic acid (HB1238) and CNTF (HB8968).
6. Culture the cells for an additional 20-30 days, refreshing the maturation medium every 2-3 days, until characteristic astrocyte morphology is observed.
7. Confirm astrocyte generation via immunocytochemistry using Anti-GFAP antibody ValidAbTM (HB8267) and counterstain nuclei with DAPI (HB0747).

References