Sep 29, 2025
Generation of human iPSC-derived cortical neurons, astrocytes, and microglia
- Maria Jose Perez J.1
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Generation of human iPSC-derived cortical neurons, astrocytes, and microglia. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4o97lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 25, 2025
Last Modified: September 29, 2025
Protocol Integer ID: 228162
Keywords: microglia generation of human ipsc, microglia generation, derived cortical neuron, generation of human ipsc, cortical neuron, human ipsc, ipsc
Funders Acknowledgements:
ASAP
Abstract
Generation of human iPSC-derived cortical neurons, astrocytes, and microglia
Troubleshooting
Generation of human iPSC-derived cortical neurons, astrocytes, and microglia
The control neuronal precursor cell (NPC) lines were previously generated and characterized (Ref main text n 8, 86)
For developing NPCs, select first iPSC colonies and culture them for 4 days in embryoid body (EB) medium containing 20% KO serum replacement, 80% DMEM/F-12, 1% nonessential amino acids (NEAAs), 1% penicillin–streptomycin (PS), 10 μM SB431542 (SB), and 2.5 μM dorsomorphin.
Beginning on day 5, culture EBs for 4 additional days in N2B27 medium containing 100% DMEM/F-12, 1% N2, 1% B27 without vitamin A, 1% NEAAs, 1% PS, 10 μM SB, 2.5 μM dorsomorphin, and 20 ng/mL FGF2.
Isolate neural rosettes with Accutase, replate on Corning‱ Matrigel‱ Growth Factor Reduced (GFR) Basement Membrane Matrix, and maintain in N2B27 medium.
Manually dissect secondary or tertiary rosettes to purify NPCs.
Maintain NPCs in basal NPC medium consisting of 1:1 DMEM-Ham’s F-12 and Neurobasal medium, 0.5% N2, 1% B27 without vitamin A, 1% PS, and 1% GlutaMAX, supplemented with 3 μM CHIR99021, 0.5 μM purmorphamine, and 150 μM ascorbic acid.
Split NPCs into Matrigel-coated wells every 5–6 days at a 1:10 ratio.
Cortical neuron differentiation
Seed NPCs at 80% confluence in neuronal differentiation medium consisting of basal neuronal medium (1:1 DMEM-Ham’s F-12 and Neurobasal medium), 0.5% N2, 1% B27 without vitamin A, 1% PS, and 1% GlutaMAX, supplemented with 200 μM ascorbic acid, 1 μM purmorphamine, and 100 ng/mL FGF8.
Culture for 7 days, then split and seed in neuronal maturation medium consisting of basal neuronal medium supplemented with 20 ng/mL BDNF and 1 mM dibutyryl-cyclic AMP (dcAMP).
Replace maturation medium every other day.
Perform a final split after 14 days in vitro (DIV).
Conduct experiments after 21 DIV.
Astrocyte differentiation
Seed NPCs at 15,000 cells/cm² in commercial astrocyte differentiation medium (ScienCell Research Laboratories).
Passage cells every 5–6 days at 15,000 cells/cm² using Accutase and plate on Matrigel-coated wells in differentiation medium.
After 30 days, replace medium with astrocyte maturation medium. (Ref 87 main text)
Split cells at 90% confluence, maintain in astrocyte maturation medium, and use for experiments between passages 3 and 10 after the medium change.
Microglia differentiation
Seed 3 × 10^6 iPSCs into a well of an AggreWell 800 plate to form EBs.
Culture EBs for 4 days in mTeSR‱ Plus medium supplemented daily with 50 ng/mL BMP4, 50 ng/mL VEGF, and 20 ng/mL SCF.
Transfer EBs to 6-well plates (15 EBs/well) and culture in X-VIVO15 medium supplemented with 1% GlutaMAX, 1% PS, 100 ng/mL M-CSF, 25 ng/mL IL-3, and 0.055 mM β-mercaptoethanol.
Add fresh medium weekly.
After 3–4 weeks, harvest macrophage precursors appearing in the supernatant.
Plate harvested macrophage precursors at 100,000 cells/cm² and culture for 10 days in advanced DMEM/F12 medium supplemented with 1% N2, 1% GlutaMAX, 1% PS, 100 ng/mL M-CSF, 100 ng/mL IL-34, 10 ng/mL GM-CSF, and 0.055 mM β-mercaptoethanol.