Apr 23, 2026

Differentiation of Human iPSCs into Forebrain Neurons Using Dual-SMAD Inhibition

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm84d5g3p/v1
  • 1Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA;
  • 2Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA
Pilar Alvarez Jerez
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm84d5g3p/v1
Protocol Citation: Xylena Reed, Alexandra Beilina, Mark Cookson 2026. Differentiation of Human iPSCs into Forebrain Neurons Using Dual-SMAD Inhibition. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm84d5g3p/v1
Manuscript citation:
Forebrain Neuron Differentiation from Human iPSCs (Dual-SMAD Inhibition; Burkhardt et al. 2013)
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: April 23, 2026
Last Modified: April 23, 2026
Protocol  Integer ID: 315636
Keywords: pluripotency culture into neuronal differentiation medium, neuronal cultures suitable for downstream application, forebrain neuronal lineage, induced pluripotent stem cell, pluripotent stem cell, neuronal differentiation medium, neuronal culture, forebrain neuron, neuronal patterning with retinoic acid, differentiation of human ipsc, smad inhibition, smad inhibition strategy, smad inhibition this protocol, maturation in neurotrophic factor, sb431542 for neural induction, pluripotency culture, neuronal patterning, gene expression profiling, neurotrophic factor, including gene expression profiling, retinoic acid, human ipsc, neural induction
Disclaimer
This protocol is for research purposes only. Follow all relevant guidelines and regulations.
Abstract
This protocol describes the differentiation of human induced pluripotent stem cells (iPSCs) into forebrain neuronal lineage using a dual-SMAD inhibition strategy originally described by Burkhardt et al. 2013. iPSCs are transitioned from pluripotency culture into neuronal differentiation medium (N3) supplemented with dorsomorphin and SB431542 for neural induction, followed by neuronal patterning with retinoic acid and maturation in neurotrophic factor-supplemented medium (N4). This protocol yields neuronal cultures suitable for downstream applications including gene expression profiling, immunocytochemistry, and functional assays.
Guidelines
- All incubations are performed at 37°C, 5% CO₂, unless otherwise stated.
- Use high-quality iPSC cultures with minimal spontaneous differentiation.
- Begin differentiation only when iPSCs are 95–100% confluent.
- During differentiation, feed daily unless otherwise specified.
- Use gentle pipetting during media changes to avoid lifting neural monolayers.
- Retinoic acid is light-sensitive; protect from light at all times.
- During differentiation, use 5 mL media per well of a 6-well plate.
Materials
Materials
Reagents
ABC
ReagentCompanyCatalog #
Essential 8 (E8) MediumThermo Fisher ScientificA1517001
MatrigelCorning354277
DMEM/F12Thermo Fisher Scientific11320-033
Neurobasal (TM) MediumThermo Fisher Scientific21103-049
Penicillin-StreptomycinThermo Fisher Scientific15140-122
B-27 (TM) Supplement minus vitamin AThermo Fisher Scientific12587010
N2 SupplementThermo Fisher Scientific17502-048
GlutaMAX (TM)Thermo Fisher Scientific35050-061
NEAA (Non-essential amino acids)Thermo Fisher Scientific11140-050
β-mercaptoethanol (2-ME)Thermo Fisher Scientific21985023
InsulinSigma 12643
DorsomorphinTocris Bioscience3093
SB431542Stemgent04-0010
Retinoic acid (RA)SigmaR2625
BDNFThermo Fisher ScientificPHC7074
GDNFThermo Fisher Scientific450-10
PBSThermo Fisher Scientific20012-027
Cell dissociation reagent (Accutase/TrypLE)Thermo Fisher ScientificA11105-01/12605010 
Poly-L-ornithine (PLO)SigmaA-004-C
FibronectinThermo Fisher Scientific33016015
LamininThermo Fisher Scientific23017015
Safety warnings
- Add growth factors and RA fresh when possible.
- Protect RA-containing media from light.
- Ensure full well coverage.
- Do not allow coated plates to dry.
- Avoid harsh trituration; neuronal progenitors are fragile.

📌 ROCK inhibitor improves survival after replating.
Before start
1. Grow human iPSCs on Matrigel-coated plates in E8 medium.
2. Plate iPSCs so that each iPSC line occupies one well of a 6-well plate.
3. Allow cultures to reach 95–100% confluency before initiating differentiation.
4. Plan to feed differentiation cultures using 5 mL media per well per day.
Before Start
Grow human iPSCs on Matrigel-coated plates in E8 medium.
Plate iPSCs so that each iPSC line occupies one well of a 6-well plate.
Allow cultures to reach 95–100% confluency before initiating differentiation.
Plan to feed differentiation cultures using 5 mL media per well per day.
Media Recipes
N3 Medium (Neuronal Differentiation Medium)
  • 50% DMEM/F12
  • 50% Neurobasal™**
  • Penicillin-Streptomycin: 1×
  • B-27™ minus vitamin A: 0.5×
  • N2 supplement: 0.5×
  • GlutaMAX™: 1×
  • NEAA: 1×
  • β-mercaptoethanol: 0.055 mM
  • Insulin: 1 µg/mL
Store at 4°C up to 1 week.
N4 medium (Neuronal Maturation Medium)
N4 = N3 plus:
  • BDNF: 2 ng/mL (25 µg/mL stock, 40 µL added to 500 mL)
  • GDNF: 2 ng/mL (10 µg/mL stock, 100 µL added to 500 mL)
  • Retinoic acid (RA): 0.05 µM (10 mM stock, 2.5 µL added to 500 mL)
‱ Add growth factors and RA fresh when possible.
Part 1: Neural Induction (Dual-SMAD Inhibition)
20m
Step 1: Start differentiation (Day 1):
1. Aspirate E8 medium from iPSCs at 95–100% confluency.
2. Add 5 mL per well of N3 medium supplemented with:
  • Dorsomorphin: 1.5 µM
  • SB431542: 10 µM
00:10:00 Incubate for 10 minutes.

10m
Step 2: Feed daily with N3 + dual-SMAD inhibitors (Days 1–11):
1. Replace media daily with fresh N3 + dorsomorphin + SB431542.
2. Use 5 mL per well.
00:10:00 Incubate for 10 minutes per day.
‱ Expected result: cells transition into neuroepithelial-like morphology over time.
10m
Dual-SMAD inhibitor preparation (500 mL N3):
Factor | Final Concentration | Stock | Volume for 500 mL
FactorFinal ConcentrationStockVolume for 500 mL
Dorsomorphin1.5 µM10 mM75 µL
SB43154210 µM10 mM500 µL
(For 300 mL N3: dorsomorphin 45 µL, SB431542 300 µL)
Part 2: Neural Maintenance
10m
Step 3: Feed daily with N3 only (Days 12–15):
1. From Day 12 to Day 15, replace media daily using N3 medium only (no dorsomorphin, no SB431542).
2. Use 5 mL per well.
00:10:00 Incubate for 10 minutes per day.

10m
Part 3: Retinoic Acid Patterning
10m
Step 4: Feed daily with N3 + RA (Days 16–19):
1. From Day 16 through Day 19, replace media daily using N3 medium supplemented with RA:
  • Retinoic acid: 0.05 µM

00:10:00 Incubate for 10 minutes per day.
‱ Protect RA-containing media from light.
‱RA addition guideline: stock RA = 10 mM
  • Example: 1 µL RA stock in 200 mL N3
10m
Part 4: Plate Coating for Neuronal Replating
4h
Step 5: Coat plates with Poly-L-ornithine (Day 17):
1. On Day 17, coat two 6-well plates per original 6-well plate (splitting 1:2).
2. Add 1 mL PLO per well.
3. Incubate overnight at 37°C.
Overnight
‱ Ensure full well coverage.
Part 4: Plate Coating for Neuronal Replating
4h
Step 6: Coat plates with Fibronectin + Laminin (Day 18):
1. Remove PLO.
2. Wash wells 3× with DPBS.
3. Prepare Fibronectin/Laminin coating solution in DPBS:

Fibronectin/Laminin coating solution (10 mL total)

ComponentFinal ConcentrationStockVolume
Fibronectin2 µg/mL1 mg/mL20 µL
Laminin0.2 µg/mL0.5 mg/mL4 µL
DPBS——up to 10 mL

4. Add coating solution to wells (1 mL per well).
5. Incubate 4 hours to overnight at 37°C.
6. Wash wells 3× with DPBS.
7. Leave DPBS in the final wash until ready to plate cells (do not allow wells to dry).
04:00:00 4 hours - Overnight
‱ Do not allow coated plates to dry.

4h
Part 5: Splitting and Replating
Step 7: Split cells 1:2 (Day 19):
1. Aspirate media and rinse cells with 2 mL DPBS per well.
2. Add 1 mL Accutase per well.
3. Incubate 9 min at room temperature (check under microscope; extend if needed).
4. Add 1 mL DPBS, gently pipette to detach cells, and transfer to a 15 mL tube.
5. Rinse well with an additional 1 mL DPBS and combine in tube.
6. Centrifuge at 150 × g for 4 min.
7. Aspirate supernatant.
8. Resuspend pellet in 2 mL N3 + RA + ROCK inhibitor.
9. Plate cells 1:2 by adding 1 mL cell suspension into each prepared well containing 4 mL N3 + RA + ROCK inhibitor.
‱ Avoid harsh trituration; neuronal progenitors are fragile.
‱ ROCK inhibitor improves survival after replating.
Part 6: Neuronal Maturation
10m
Step 8: Feed daily with N4 (Days 20–28):
1. Starting Day 20, replace media daily using N4 medium.
2. Continue daily feeding until Day 28 (or longer if desired).
3. Use 5 mL per well.
00:10:00 Incubate for 10 minutes per day.
‱ Expected result: increasing neurite extension and neuronal network formation.
10m
Optional: Freezing / Plating for Experiments
Step 9: Freeze or transfer neurons (Day 24 or later):
1. From Day 24 onward, neurons may be:
  • frozen down
  • replated for assays
  • maintained in culture (feeding every other day)
‱ Freezing guideline:
  • Freeze 1 well of a 6-well plate into 1 vial
  • Thaw 1 vial into 2 wells
Timing
StageDays
Dual-SMAD neural inductionDay 1–11
N3 maintenanceDay 12–15
RA patterningDay 16–19
ReplatingDay 19
N4 maturationDay 20–28
Total protocol duration~28 days
Anticipated Results
  • Neural induction morphology should be visible by Day 7–11.
  • After replating (Day 19–20), cells should recover and begin forming extended processes.
  • By Day 24–28, neuronal networks should be apparent.
  • Expected neuronal marker expression includes:
SOX1 / PAX6 (early neural progenitors)
TUJ1 (βIII-tubulin) and MAP2 (neuronal identity)
Forebrain-associated markers depending on line/conditions (e.g., FOXG1)
Troubleshooting
Problem: Cells detach during early differentiation
Cause: harsh media changes
Solution: add media slowly along the wall of the well, avoid touching the monolayer
Problem: Poor survival after splitting
Cause: over-incubation in Accutase or insufficient ROCK inhibitor
Solution: monitor detachment closely and include ROCK inhibitor during replating
Problem: Weak neurite outgrowth
Cause: growth factors degraded or media not changed consistently
Solution: prepare fresh BDNF/GDNF aliquots, ensure daily N4 feeding
Protocol references
Acknowledgements
We thank the Stem Cell Core Facility for their support and assistance.