Mar 19, 2026
Differentiation of Human Induced Pluripotent Stem Cells into Midbrain Neurons
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Differentiation of Human Induced Pluripotent Stem Cells into Midbrain Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9bzmml3e/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: February 12, 2026
Last Modified: March 19, 2026
Protocol Integer ID: 243122
Keywords: hiPSCs, midbrain neurons, differentiation, neurobiology, cell culture, differentiation of human induced pluripotent stem cell, human induced pluripotent stem cell, induced pluripotent stem cell, pluripotent stem cell, midbrain neuron, midbrain neurons this protocol, neuron, neurons this protocol
Abstract
This protocol aims to differentiate human induced pluripotent stem cells (hiPSCs) into midbrain neurons using a defined media composition and specific growth factors, ensuring high purity and reproducibility for potential applications in transplantation and disease modeling.
Materials
Geltrex (Growth factor-reduced, 2D matrix for cell culture)
Laminin (1 mg/mL stock solution, for coating plates)
6-well plates (Tissue culture treated)
50 mL conical tubes (Sterile, for media preparation)
100 mm plates (Tissue culture treated)
Reagents:
Media Neurobasal Medium - Basal medium for neuronal culture
Supplement Glutamax - Glutamine supplement for cell culture
Supplement B27 Supplement - Serum-free supplement for neuronal culture
Supplement N2 Supplement - Supplement for neuronal differentiation
Inhibitor Y-27632 - ROCK inhibitor to enhance cell survival
Inhibitor LDN193189 - BMP signaling inhibitor
Inhibitor SB431542 - TGF-β signaling inhibitor
Agonist SAG - Sonic Hedgehog pathway activator
Inhibitor CHIR99021 - GSK-3 inhibitor for WNT signaling
Growth Factor GDNF - Neurotrophic factor for neuronal survival
Growth Factor BDNF - Neurotrophic factor for neuronal differentiation
Chemical Ascorbic Acid - Antioxidant for neuronal cultures
Chemical Dibutyryl cAMP - cAMP analog for neuronal differentiation
Growth Factor TGF-β3 - Growth factor for neuronal differentiation
Troubleshooting
Problem
Low cell viability
Solution
Ensure proper handling during dissociation and avoid over-confluency.
Problem
Poor differentiation
Solution
Verify the concentrations of growth factors and inhibitors; ensure media is fresh.
Safety warnings
Follow standard laboratory safety protocols when handling all reagents and biological materials. Use personal protective equipment (PPE) including gloves, lab coat, and safety goggles.
Preparation of hiPSCs
Dissociate hiPSCs using Accutase and plate them at 400,000 cells / cm2 on Geltrex-coated plates.
Cell Dissociation
1. Thaw a vial of hiPSCs and resuspend in 10 mL of culture medium. 2. Centrifuge at 160 g for 3 minutes. 3. Resuspend the pellet in 1 mL of Accutase and incubate at 37°C for 5 minutes. 4. Neutralize with 10 mL of culture medium and centrifuge again at 160 g for 3 minutes. 5. Resuspend in 5 mL of culture medium and plate on Geltrex-coated plates.
Initial Culture
1. Add 10 µM Y-27632 to the culture medium and incubate for 24 hours.
Differentiation into Midbrain Neurons
Induce differentiation by changing the media composition and adding specific growth factors.
Media Preparation
1. Prepare base media: Combine 500 mL Neurobasal medium, 5 mL Glutamax, 5 mL B27, and 5 mL N2 supplement. 2. Supplement with 250 nM LDN193189, 10 µM SB431542, 1 µM SAG, and CHIR at specified concentrations:0.7µM day 0-3, 7.5µM day 4-9, 3µM day 10-11.
Media Change and Supplementation
Continue to change the media every 2 days, adjusting CHIR concentrations as specified.
Maturation Phase
After day 10, switch to post-patterning maturation media.
Maturation Media Preparation
1. Prepare maturation media: Combine base media with 20 ng/mL GDNF, 20 ng/mL BDNF, 0.2 mM ascorbic acid, 0.1 mM dibutyryl cAMP, and 1 ng/mL TGF-β3.
Media Change
1. Change to maturation media starting on day 10 and continue for 4 weeks.
Protocol references
doi: 10.1016/j.stem.2021.01.005