Nov 21, 2025
Differentiation of iPSCs with the NGN2 construct into cortical neurons protocol
- Christian Tarr1,
- Claire Clelland1,2
- 1University of California, San Francisco, Weill Institute for Neurosciences, San Francisco, CA, United States;
- 2Contact: claire.clelland@ucsf.edu
Protocol Citation: Christian Tarr, Claire Clelland 2025. Differentiation of iPSCs with the NGN2 construct into cortical neurons protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx4d5ol8j/v1
Manuscript citation:
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
Created: November 19, 2025
Last Modified: November 21, 2025
Protocol Integer ID: 232980
Keywords: ngn2 construct into cortical neurons protocol, induced pluripotent stem cell, pluripotent stem cell, inducible neurogenin, ngn2 construct, cortical neuron, cortical neurons protocol, ngn2, differentiation of ipsc, ipsc
Abstract
This protocol describes the differentiation of induced pluripotent stem cells (iPSCs) into cortical neurons using a doxycycline-inducible Neurogenin 2 (NGN2) construct.
This protocol is adapted from Wang and Ward et. al., 2017.
Attachments
Materials
Table 8. Reagents
| A | B | C | |
| Item | Manufacturer | Catalog Number | |
| Knockout DMEM/F-12 | ThermoFisher Scientific | 12660012 | |
| Growth Factor Reduced Matrigel | Corning | 356231 | |
| mTeSR Plus and supplement | Stemcell Technologies | 1000276 | |
| Gibco DPBS, no Ca, no Mg | ThermoFisher Scientific | 14-190-235 | |
| Accutase | Stemcell Technologies | 07920 | |
| DMEM/F-12, HEPES | ThermoFisher Scientific | 11330032 | |
| N-2 Supplemen | ThermoFisher Scientific | 17502048 | |
| MEM Non-Essential Amino Acids Solution (NEAA) | ThermoFisher Scientific | 11140050 | |
| GlutaMAX Supplement | ThermoFisher Scientific | 35050061 | |
| B-27 Supplement, serum free | ThermoFisher Scientific | 17504044 | |
| Poly-D-Lysine (PDL) | ThermoFisher Scientific | A3890401 | |
| Laminin Mouse Protein, Natural | ThermoFisher Scientific | 23017015 | |
| Doxycycline Hyclate (reconstituted in water) | Millipore Sigma | D3447 | |
| ROCK1 Inhibitor (Y-27632 2HCl) | Selleckchem | S1049 | |
| 5-Bromo-2ʹ-deoxyuridine (BrdU) | Millipore Sigma | B9285 | |
| Neurobasal-A | ThermoFisher Scientific | 10888022 | |
| Recombinant Human BDNF | Peprotech | 450-10 | |
| Recombinant Human NT3 | Peprotech | 450-03 |
Table 9. Plates
| A | B | C | D | E | |
| Well Format | Coating | Item | Manufacturer | Catalog Number | |
| 96-well | Standard | Falcon 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate with Lid | Corning | 353219 | |
| Pre-coated | Corning BioCoat 96-Well, Poly-D Lysine-Treated, Flat-Bottom Microplate | Corning | 354640 | ||
| 24-well | Standard | Clear, Cellstar, TC, Lid with Condensation Rings, Sterile, Single Packed | Greiner Bio-One | 662160 | |
| Pre-coated | Corning BioCoat Poly-D Lysine 24-well Clear Flat Bottom TC treated Multiwell Plate, with Lid | Corning | 354414 | ||
| 12-well | Standard | Clear, Cellstar, TC, Lid with Condensation Rings, Sterile, Single Packed | Greiner Bio-One | 665180 | |
| Pre-coated | Corning BioCoat Poly-D Lysine 12-well Clear Flat Bottom TC treated Multiwell Plate, with Lid | Corning | 354470 | ||
| 6-well | Standard | Clear, Cellstar, TC, Lid with Condensation Rings, Sterile, Single Packed | Greiner Bio-One | 657160 | |
| Pre-coated | Corning BioCoat Poly-D Lysine 6-well Clear Flat Bottom TC-treated Multiwell Plate, with Lid | Corning | 354413 |
Table 10. Equipment
| A | B | C | |
| Item | Manufacturer | Catalog Number | |
| Eppendorf Centrifuge 5810/5810R | Millipore Sigma | EP022628168 | |
| Invitrogen Countess II automated cell counter | ThermoFisher Scientific | AMQAX1000 | |
| Countess Cell Counting Chamber Slides and Holder, disposable | ThermoFisher Scientific | C10312 |
Troubleshooting
Maintenance of iPSC
Table 1. Recommended reagent volume and seeding densities for standard cell culture vessels
| A | B | C | D | E | F | G | |
| Well Format | Matrigel Coating Volume | DPBS/PDL | Accutase Volume | Media Volume | Suggested Day 0 Seeding Density/well | Suggeste Day 3 Seeding Density/well | |
| 96-well | 50uL | 100uL | 30uL | 100uL | N/A | ICC: 10-20k Drug: 20-40k | |
| 24-well | 250uL | 250uL | 125uL | 500uL | N/A | 100-250k | |
| 12-well | 500uL | 500uL | 250uL | 1mL | N/A | 500-750k | |
| 6-well | 1mL | 1mL | 500uL | 2mL | 750k | 750k-1 million | |
| 10-cm dish | 4mL | 6mL | 3mL | 6mL | 4 million | 7 million | |
| 15-cm dish | 12mL | 18mL | 9mL | 18mL | N/A | 20 million |
Culture iPSCs changing the media every day, once cells are over 20% confluent.
Passage cells once they reach 60-75% confluency. Do not exceed 90% confluency.
Note
Cells should not be differentiated from thaw. Passage 1-3 times, or until cells reach growth phase and appear healthy (Fig. 1A)
Before starting the differentiation, ensure you have the proper number of wells to differentiate, while keeping an additional well to maintain the cell line.
Note
Ensure wells have enough cells to differentiate, around 75% confluency
NGN2 Protocol Overview
Figure 1. Timeline and overview of NGN2 differentiation. (A) iPSCs should appear
healthy in large multicell colonies. (B) Early NPCs exhibit signs of neuritic extensions
with “spikey” morphology and do not form homogeneous colonies. (C) Confluence
nearly doubles with a similar morphology to day 1. (D) Plate is nearly 100%
confluent with single cell NPCs that have defined, small neuritic processes. (E)
Neuron morphology is apparent with clear somas and extended axon processes. (F)
Neurons maintain defined somas with a robust axon network. (G) Timeline of NGN2
differentiation. (H) Neuronal media corresponding to each day within NGN2 timeline.
Scale bars, 50um
NGN2 Day 0 Differentiation - Replating iPSCs
Figure 2. After 3 days in NIM + Dox iPSCs differentiate to neural progenitor cells (NPCs).
Table 2. Neural Induction Media (NIM)
| A | B | C | D | |
| Reagent | Media Dilution Factor | Stock Concentration | 100mL Recipe | |
| KO DMEM/F12 | 1 | 1X | 100mL | |
| N2 Supp. | 1:100 | 100X | 1mL | |
| NEAA | 1:100 | 100X | 1mL |
Table 3. Day 0 NGN2 media
| A | B | C | D | |
| Reagent | Media Dilution Factor | Stock Concentration | 50mL Recipe | |
| NIM | 1 | 1 | 50mL | |
| Doxycycline* | 1:1000 | 2mg/mL | 50uL | |
| Rock Inhibitor | 1:1000 | 10mM | 50uL | |
| Laminin | 1:1000 | 1mg/mL | 50uL | |
| BDNF | 1:2000 | 20ug/mL | 25uL | |
| NT3 | 1:2000 | 20ug/mL | 25uL |
*Doxycycline is light sensitive and should be added immediately before using media
Matrigel coat plates (6-well or 10cm recommended) and incubate at 37 °C for at least 00:30:00 or Overnight
Prepare NIM then Day 0 NGN2 media
Note
Make enough NIM media for day 0 replating, day 1 feeding and cell resuspension. Store at 4 °C for the next day media change
Aspirate media from iPSCs and wash with 1X DPBS
Add accutase and incubate at 37 °C for 00:05:00 to 00:07:00
Dissociate iPSCs by quenching with 1X DPBS at a volume 5 times that of accutase. Collect cell suspension in a 15mL or 50mL conical tube.
Note
Less DPBS can be used to avoid overfilling the conical tube if pooling from multiple wells
Spin down cells at 300 x g for 00:03:00
Aspirate the supernatant and resuspend cells in Day 0 NGN2 media
Count cells and calculate your desired cell amount (see Table 1 for suggestions)
Note
Cell count roughly doubles from day 0 to 3. Plate enough cells to account for all downstream experiments when replating on day 3
Add media to the well plate(s) being used and plate the calculated cell volume
NGN2 Day 1 Differentiation - Feeding
Table 4. Day 1 NGN2 media
| A | B | C | D | |
| Reagent | Media Dilution Factor | Stock Concentration | 50mL Recipe | |
| NIM | 1 | 1 | 50mL | |
| Doxycycline | 1:1000 | 2mg/mL | 50uL | |
| Laminin | 1:1000 | 1mg/mL | 50uL | |
| BDNF | 1:2000 | 20ug/mL | 25uL | |
| NT3 | 1:2000 | 20ug/mL | 25uL |
Prepare Day 1 NGN2 media
Note
Media should be prepared on the day of use only
Perform a full media change, being careful not to let the cell surface dry out
NGN2 Day 2 Differentiation - Coat Plates
No media change is required on day 2
Use 1X PDL to coat as many plates as needed and incubate at 37 °C for at least 02:00:00 but ideally Overnight
NGN2 Day 3 Differentiation - Replate NPCs
Figure 3. After 4 days in NMM + Dox NPCs fully differentiate into induced neurons (iNeurons).
Table 5. Neural Maturation Media (NMM)
| A | B | C | D | |
| Reagent | Media Dilution Factor | Stock Concentration | 100mL Recipe | |
| DMEM/F-12 | 1:2 | 1X | 50mL | |
| Neurobasal-A | 1:2 | 1X | 50mL | |
| NEAA | 1:100 | 100X | 1mL | |
| GlutaMAX | 1:100 | 100X | 1mL | |
| N2 Supplement | 1:100 | 100X | 1mL | |
| B27 Supplement | 1:50 | 50X | 2mL |
Table 6. Day 3 NGN2 media*
| A | B | C | D | |
| Reagent | Media Dilution Factor | Stock Concentration | 50mL Recipe | |
| NMM | 1 | 1 | 50mL | |
| Doxycycline | 1:1000 | 2mg/mL | 50uL | |
| BrdU** | 1:1000 | 40mM | 50uL | |
| Laminin | 1:1000 | 1mg/mL | 50uL | |
| BDNF | 1:2000 | 20ug/mL | 25uL | |
| NT3 | 1:2000 | 20ug/mL | 25uL |
*Rock inhibitor at 1:1000 can be added to media on day 3 replating if the cell line or assay is prone to cell death
**BrdU is light sensitive and should be added immediately before using media
Part 1
Aspirate PDL from coated plates and wash 3 times with sterile distilled water
Note
PDL is toxic and residual coating can be harmful to cultures
Let plates dry in the hood with the lid askew until the well surface is completely dry (00:30:00 to 01:00:00 )
Part 2
While plates dry, make NMM and Day 3 NGN2 media
Once plates are dry, aspirate media from the neural progenitor cells (NPCs)
Wash cells with 1X DPBS
Add accutase and incubate at 37 °C for 00:05:00 to 00:07:00
Note
NPCs tend to detach less readily from plates. Longer incubation times up to around 00:10:00 may be needed
Dissociate NPCs by quenching with 1X DPBS at a volume 5 times that of accutase. Collect cell suspension in a 15mL or 50mL conical tube.
Centrifuge the cells at 200 x g for 00:05:00
Aspirate the supernatant and resuspend in Day 3 NGN2 media
Count cells and calculate your desired cell amount:
Note
The number of NPCs plated depends on your specific experiment and desired end result. Experiments that you expect cell death due to treatment or require high extraction yields should use the higher density suggested in Table 1. Experiments where single cell resolution is necessary (e.g. ICC) use lower cell suggestion.
Add media to the well plate(s) being used and plate the calculated cell volume
NGN2 Day 7+ Differentiation
Table 7. Day 7 NGN2 media
| A | B | C | D | |
| Reagent | Media Dilution Factor | Stock Concentration | 50mL Recipe | |
| NMM | 1 | 1 | 50mL | |
| Laminin | 1:1000 | 1mg/mL | 50uL | |
| BDNF | 1:2000 | 20ug/mL | 25uL | |
| NT3 | 1:2000 | 20ug/mL | 25uL |
Prepare Day 7 NGN2 media
Perform a full media change, being extra careful not to lift or dry out the cell surface
For the remainder of your experiment, use a weekly half-media change (e.g. day 14, day 21, etc.)
Troubleshooting
If iPSCs are unhealthy or spontaneously differentiating, NGN2 differentiation won’t yield high-quality or quantity of induced neurons because alternate iPSC fates have already been chosen. Spontaneously differentiating cells take on a “spikey” or “cobblestone” appearance (Fig. 5B, C). If earlier passage numbers of the cell line are frozen down, it’s usually faster to thaw new vials of the line than to clean a culture dominated by unhealthy cells. However, if the line is precious, these methods can be used to restore the iPSC line.
Figure 4. Comparison of healthy and spontaneously differentiating cells. (A) Healthy iPSCs with isolated multi-cell colonies. (B) Unhealthy iPSCs take on a “cobblestone” appearance. (C) Spontaneously differentiating iPSCs migrate away from colonies and appear flat with translucent cytoplasm and “spikey” cell membrane. Red arrow denotes spontaneously differentiated cells. Scale bars, 100 μm.
Light Passaging
iPSCs should be around 50% confluent, less than the normal confluence for standard line maintenance passaging
Matrigel coat 3 wells of a 6-well plate and incubate at 37 °C for 00:30:00
Aspirate media from the iPSCs
Wash once with 1X DPBS
Add EDTA and incubate the plate at 37 °C for 00:02:00 to 00:03:00
Note
Non-spontaneously differentiated or healthy cells tend to detach from the plate more readily, leaving behind unhealthy cells. Using a shorter incubation time can help select for healthier cells
Aspirate matrigel from 6-well plate and replace with media
Carefully aspirate the EDTA
Tilt the plate towards you and slowly add media to the well wall closest to you
Lay the plate flat and slowly tilt it back towards yourself to pool the media with your detached cells in the corner of the well
Collect the media that contains cells in suspension and plate in a gradient across the 3 wells of the 6-well plate
Return the plate to 37 °C
Colony Picking
If 1-3 rounds of light passaging do not eliminate spontaneous differentiation, a colony picking method can be used
iPSCs should be around 50% confluent or less in order to identify individual colonies
Use a phase contrast microscope, find healthy, isolated colonies in the well (see Fig. 5A)
Using a marker, lift up the well plate and dot the bottom of the well where the colony is
Use the microscope to ensure dot placement properly marks the colony
Find around 3-5 colonies, if possible
Matrigel coat a 48-well, 24-well, or 12-well, depending on the size and number of healthy colonies identified
Incubate matrigel coated plates for at least 00:30:00 at 37 °C
Aspirate media from the iPSCs
Wash once with 1X DPBS
Add EDTA and incubate the plate at 37 °C for 00:04:00 to 00:05:00
Aspirate the matrigel from coated plates and add media to the receiving plate
Carefully aspirate EDTA from the iPSC plate
Using a P2 pipette, load 1-2ul of media into the tip and carefully dispense and collect the media on the marked isolated colony
Deposit the media containing the colony into a single well of your plate
Repeat steps 10.14 and 10.15 until all marked colonies have been picked
Return the plate to 37 °C
As the cells grow, expand the line from the culture vessel used back into a 6-well format
Protocol references
[1] Wang C, Ward ME, Chen R, Liu K, Tracy TE, Chen X, Xie M, Sohn PD, Ludwig C, Meyer-Franke A, Karch CM, Ding S, Gan L. Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening. Stem Cell Reports. 2017.
[2] Fernandopulle MS, Prestil R, Grunseich C, Wang C, Gan L, Ward ME. Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr Protoc Cell Biol. 2018 Jun;79(1):e51. doi: 10.1002/cpcb.51. Epub 2018 May 18. PMID: 29924488; PMCID: PMC6993937.
[3] Merissa Chen*, Nina Draeger*, Martin Kampmann*, Kun Leng*, Emmy Li*, Connor Ludwig*, Greg Mohl*, Avi Samelson*, Syd Sattler*, Ruilin Tian*. 2020. Kampmann Lab iNeuron pre-differentiation 26 differentiation protocol. Protocols.io.