Jan 23, 2026
Excitatory neuron differentiation from inducible Ngn2 iPSCs (WTC11)
- Boxun Li1,
- Charles Gersbach1
- 1Duke University
- Gersbach Lab
Protocol Citation: Boxun Li, Charles Gersbach 2026. Excitatory neuron differentiation from inducible Ngn2 iPSCs (WTC11). protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1kqxpgr2/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: January 16, 2026
Last Modified: January 23, 2026
Protocol Integer ID: 238821
Keywords: iPSC differentiation, excitatory neuron differentiation, WTC11, Ngn2, excitatory neuron differentiation from inducible ngn2 ipsc, inducible ngn2 ipsc, generating excitatory neuron, ngn2, inducible mouse
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from work in the Yin Shen lab and the Li Gan lab and used by Boxun Li in the Gersbach lab at Duke University.
Abstract
This protocol describes methods for generating excitatory neurons from a wild type male iPSC line (WTC11) containing doxycycline-inducible mouse neurogenin-2 (Ngn2) integrated at the AAVS1 safe-harbor locus.
Guidelines
All work must be performed in a sterile environment.
Materials
| A | B | C | |
| Reagent | Catalog # | Vendor | |
| ROCK Inhibitor Y-27632 | 72304 | STEMCELL | |
| Corning‱ Matrigel‱ hESC-Qualified Matrix, LDEV-free | 354277 | Corning | |
| AccutaseTM Cell detachment solution | 07922 | STEMCELL | |
| MEM Non-Essential Amino Acids Solution (100X) (NEAA) | 11140-050 | Life Technologies | |
| GlutaMAX‱ Supplement (100X) | 35050-061 | Life Technologies | |
| Recombinant Human/Murine/Rat BDNF | 450-02 | PeproTech | |
| Recombinant Human NT-3 | 450-03 | PeproTech | |
| Laminin Mouse Protein, Natural | 23017-015 | Life Technologies | |
| Doxycycline hyclate | D9891 | Sigma-Aldrich | |
| B-27‱ Supplement (50X) | 17504-044 | Life Technologies | |
| N-2 Supplement (100X) | 17502-048 | Life Technologies | |
| DMEM/F-12, HEPES | 11330-032 | Life Technologies | |
| KnockOut‱ DMEM/F-12 | 12660-012 | Life Technologies | |
| Neurobasal‱-A Medium | 12349-015 | Life Technologies | |
| DPBS, no calcium, no magnesium | 14190-144 | Life Technologies | |
| Poly-D-Lysine | P6407 | Sigma-Aldrich |
Troubleshooting
Differentiation protocol
Differentiation media: Prepare the pre-differentiation medium and maturation medium according to the table below. BDNF, NT-3, laminin, and doxycycline should be added immediately before use.
| A | B | C | D | |
| Medium | Reagents | FinalConcentration | Example(100 mL) | |
| Pre-differentiation Medium | KnockOut‱ DMEM/F-12 | 100% | 98 mL | |
| Pre-differentiation Medium | N-2 Supplement (100X) | 1X | 1 mL | |
| Pre-differentiation Medium | NEAA (100X) | 1X | 1 mL | |
| Pre-differentiation Medium | BDNF | 10 ng/mL | Add before use | |
| Pre-differentiation Medium | NT-3 | 10 ng/mL | Add before use | |
| Pre-differentiation Medium | Laminin | 1 µg/mL | Add before use | |
| Pre-differentiation Medium | Doxycycline | 2 µg/mL | Add before use | |
| Maturation Medium | Neurobasal‱-A Medium | 50% | 48.5 mL | |
| Maturation Medium | DMEM/F-12, HEPES | 50% | 48.5 mL | |
| Maturation Medium | NEAA (100X) | 1X | 1 mL | |
| Maturation Medium | B-27‱ Supplement (50X) | 0.5X | 1 mL | |
| Maturation Medium | N-2 Supplement (100X) | 0.5X | 0.5 mL | |
| Maturation Medium | GlutaMAX‱ Supplement (100X) | 0.5X | 0.5 mL | |
| Maturation Medium | BDNF | 10 ng/mL | Add before use | |
| Maturation Medium | NT-3 | 10 ng/mL | Add before use | |
| Maturation Medium | Laminin | 1 µg/mL | Add before use |
Pre-differentiation (Day -3 to Day 0)
Day -4:
(1) Use Accutase to dissociate the WTC11 inducible Ngn2 iPSCs following the Gladstone Stem Cell Core iPSC Maintenance protocol.
(2) Seed the cells in Matrigel-coated plates using mTeSR plus medium with 5 μM ROCK Inhibitor. Use the following table as a guideline:
| A | B | C | |
| Plate Type | Media Volume | Cell Density | |
| 12-well plate | 1 mL / well | 500K / well | |
| 6-well plate | 2 mL / well | 1M / well | |
| 10-cm dish | 10 mL / dish | 5M / dish |
(3) Optional: Transduce cells with lentivirus (by adding the appropriate amount for target MOI to the cells) immediately after plating. Rock plate to mix well. Lentivirus can also be introduced later.
Day -3:
Change media using pre-differentiation medium without ROCK inhibitor.
Day -2:
Change media using pre-differentiation medium without ROCK inhibitor.
Day -1:
• Change media using pre-differentiation medium without ROCK inhibitor.
• Coat new plates for differentiation using 10 μg/mL Poly-D-Lysine in molecular biology-grade water, incubating at 37°C overnight.
Post-differentiation (Day 0+)
Day 0:
(1) Wash the Poly-D-Lysine-coated plates three times using DPBS. Air dry in
a tissue culture hood until all the DPBS is evaporated.
(2) Dissociate the pre-differentiated cells using Accutase. For example, using
6-well plates:
(2.1) Add 0.5 mL Accutase to each well and incubate at 37°C until cells are
dissociated into single cells (~5 minutes).
(2.2) Add DPBS and collect the cells (do not pipette at this stage).
o First, add 1 mL DPBS and collect the cells without pipetting.
o Add 1.5 mL DPBS to rinse the wells and collect all the cells.
(2.3) Centrifuge for 5 minutes at 200 RCF at room temperature.
(2.4) Aspirate the supernatant.
(2.5) Resuspend in 1 mL Maturation Media and use a P1000 pipette to
dissociate the pellet into single cells. Triturate at least 30X.
(2.6) Count the number of live cells using Trypan blue. At least 90% of the
cells should be single cells.
(3) Plate the cells using Maturation Medium with 2 μg/mL doxycycline using the
following table:
| A | B | C | |
| Plate Type | Media Volume | Cell Density | |
| 24-well plate | 0.5 mL / well | 250K / well | |
| 12-well plate | 1 mL / well | 500K / well | |
| 6-well plate | 2 mL / well | 1M / well | |
| 10-cm dish | 10 mL / dish | 5M / dish | |
| 15-cm dish | 25 mL / dish | 12.5M / dish |
(4) Optional: Transduce cells with lentivirus (by adding the appropriate amount of target MOI to the cells) immediately after plating. Rock plate to mix well.
(5) Optional: Activate dCas9-VPH by adding 20uM trimethoprim (TMP) to the medium after plating. Rock plate to mix well. TMP can also be included or excluded during media changes later depending on experimental design.
Day 1:
Optional: Add mouse astrocytes prepared from P1 C57BL/6 mouse cerebral cortices (that have been cultured in astrocyte growth medium in vitro for 7-8 days) at a ratio of 5 neurons to 1 astrocyte. The astrocytes are dissociated with Trypsin-EDTA and resuspended in neuronal maturation medium (listed at the start of this protocol) to 500K/mL. The added volume should be 1/5 of the existing medium.
Day 7:
Remove half the volume of media from each well or dish and add the
same amount of fresh Maturation Medium without doxycycline.
Day 14:
• Remove half the volume of media from each well or dish and add twice
the amount of fresh Maturation Medium without doxycycline.
• For example, using 12-well plates, first remove 0.5 mL then add 1 mL for a
total of 1.5 mL.
Day 21:
• Remove 1/3 of the media and replace twice the amount of fresh Maturation
Medium without doxycycline.
• At this point, the total volume of Maturation Medium should be double the
initial volume. For example, using 12-well plates, there should be 2 mL
total.
After Day 21:
• Change 1/3 of the media using fresh Maturation Medium without
doxycycline every week.