Mar 19, 2026
Differentiation of iPSCs into NGN2 Neurons Protocol
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Differentiation of iPSCs into NGN2 Neurons Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk12ewg5r/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: 243125
Keywords: iPSCs, neurons, doxycycline, neurogenin 2, differentiation, ngn2 neurons protocol, ngn2 neurons protocol this protocol, inducible expression system for neurogenin, induced pluripotent stem cell, pluripotent stem cell, neuronal development, study of neuronal development, neurogenin, ngn2, neuron, differentiation of ipsc, ipsc
Abstract
This protocol aims to differentiate induced pluripotent stem cells (iPSCs) into neurons using a doxycycline-inducible expression system for neurogenin 2 (NGN2), facilitating the study of neuronal development and function.
Materials
Geltrex™ (Growth factor-reduced, 1 mL per well)
StemFlex™ media (Complete medium for iPSC culture)
Neurobasal media (Basal medium for neuronal culture)
Poly-L-ornithine (20 µg/mL solution in DPBS)
Laminin (1 µg/mL solution in DPBS)
Doxycycline (5 µg/mL solution)
SB431542 (10 µM solution)
LDN193189 (100 nM solution)
Puromycin (1 µg/mL solution)
Ara-C (0.5 µM solution)
Accutase (10 mL for cell harvesting) - 10 mL
Reagents:
Small Molecule SB431542 - TGF-β inhibitor used for neural induction
Small Molecule LDN193189 - BMP inhibitor for neural induction
Antibiotic Puromycin - Selective antibiotic for transduced cells
Small Molecule Ara-C - Antimetabolite used to inhibit cell proliferation
Troubleshooting
Problem
Low transfection efficiency
Solution
Optimize DNA and Lipofectamine ratios, ensure cells are healthy and at optimal confluency.
Problem
Poor neuronal differentiation
Solution
Verify reagent concentrations and ensure timely media changes.
Safety warnings
Follow biosafety guidelines when handling iPSCs and reagents. Use appropriate personal protective equipment (PPE) including gloves, lab coat, and safety goggles.
iPSC Transfection
Transfect iPSCs with PiggyBac plasmids using Lipofectamine Stem Transfection Reagent.
Preparation of iPSCs
Dissociate iPSCs and seed at 104,000 cells/cm² onto Geltrex™-coated plates in StemFlex™ media supplemented with 10 µM Y27632 and 5 µg/mL doxycycline.
Neural Induction
Transition media to Neurobasal N2B27 with specific supplements.
Media Transition
On day 1, replace media with Neurobasal N2B27 containing 10 µM SB431542, 100 nM LDN193189, 5 µg/mL doxycycline, and 5 µg/mL puromycin.
Daily Media Changes
From days 3 to 6, change media daily with Neurobasal N2B27 supplemented with 1 µg/mL puromycin and 5 µg/mL doxycycline.
Cell Harvesting
On day 6, treat cells with Ara-C and harvest for co-culture.
Ara-C Treatment
Add 0.5 µM Ara-C to the culture for 24 hours.
Cell Harvesting
Use Accutase to dissociate cells and collect for co-culture.
Protocol references
doi: 10.1016/j.neuron.2013.05.029.