Apr 08, 2026
NPC-iGLUT: NGN2-neuronal induction from NPCs
- PJ Michael Deans1,
- Kristen Brennand1
- 1Yale University School of Medicine
- Brennand Laboratory
Protocol Citation: PJ Michael Deans, Kristen Brennand 2026. NPC-iGLUT: NGN2-neuronal induction from NPCs. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3nw9ev25/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 21, 2026
Last Modified: April 08, 2026
Protocol Integer ID: 243768
Keywords: hiPSCs, NPCs, neuron, MPRA, CRISPR, FACS, morphology, derived neural progenitor cell, neurons from hipsc, neural progenitor cell, neuronal induction from npc, induced pluripotent stem cell, lentiviral transduction of neurogenin, pluripotent stem cell, generating neuron, derived neuron, neuronal phenotype, heterogeneity of the neuronal phenotype, neuronal induction, maturation of functional excitatory neuron, neuronal induction via the forced expression, populations of functional neuron, neurogenin, population of hipsc npc, functional neuron, neuron, exogenous transcription factor, functional excitatory neuron, lentiviral transduction, hipsc npc, discovery of somatic reprogramming, somatic reprogramming
Funders Acknowledgements:
NIMH
Grant ID: R01MH101454
Abstract
Since the discovery of somatic reprogramming, human induced pluripotent stem cells (hiPSCs) have been exploited to model a variety of neurological and psychiatric disorders. Because hiPSCs represent an almost limitless source of patient-derived neurons that retain the genetic variations thought to contribute to disease etiology, they have been heralded as a patient-specific platform for high throughput drug screening. However, the utility of current protocols for generating neurons from hiPSCs remains limited by protracted differentiation timelines and heterogeneity of the neuronal phenotypes produced. Neuronal induction via the forced expression of exogenous transcription factors rapidly induces defined populations of functional neurons from fibroblasts and hiPSCs. Here, we describe an adapted protocol that accelerates maturation of functional excitatory neurons from hiPSC-derived neural progenitor cells (NPCs) via lentiviral transduction of Neurogenin 2 (using both mNgn2 and hNGN2). This methodology, relying upon a robust and scalable starting population of hiPSC NPCs, should be readily amenable to scaling for hiPSC-based high-throughput drug screening.
Materials
**NPC Media:**
- DMEM/F-12;
- 2% B-27-RA supplement 50x;
- 1% N-2 supplement 100x;
- 1% antibiotic-antimycotic 100x.
- 20 ug/mL FGF2
- 1 ug/mL Doxycycline (1:1000).
**Brainphys neuronal media:**
- Brainphys media;
- 2% B-27-RA supplement 50x;
- 1% N-2 supplement 100x;
- 1 ug/mL Natural Mouse Laminin;
- 20 ng/mL BDNF;
- 20 ng/mL GDNF;
- 250 ug/mL dbcAMP;
- 200 uM L-ascorbic acid;
- 1% antibiotic-antimycotic 100x.
**Additional Reagents:**
- Puromycin 1:3500 0.3 ug/ml
- G418/geneticin (for neomycin vectors) 1:100 1mg/ml
- HygroB 1:100 1mg/ml
Day -3 Prepare Geltrex plates coated with 1x Geltrex
Thaw Geltrex aliquot and dissolve it in ice-cold DMEM rapidly (waiting too long causes geltrex to congeal). 100ul 15 mg/ml stock in 25ml DMEM = ~60ug/ml final conc
Distribute Matrigel-DMEM at 2ml/well of a 6 well plate.
Incubate plates minimum 1hr in incubator to coat, don’t leave more than 2-3 days before use.
Day -2 Seed NPCs
Aspirate medium and add RT Accutase (1ml per well of 6 well plate)
Incubate in the incubator for 3-5 mins.
Collect cells in accutase (use P1000 to collect and gently pipette 2-3 times to break up clumps) and transfer to 15ml tube containing DMEM at a ratio of 3 parts DMEM: 1 part accutase
Spin down at 1000g, 5 mins.
Resuspend cells in 1ml of NPC medium (use P1000 to gently resuspend pellet)
Count cells using haemocytometer/countess II if needed
Further dilute in NPC medium to desired density. (2ml NPC medium/well of a 6 well plate)
Seed desirable number of cells in a well. Cell density in a single well of 6-well plate is around 1.5-2M cells/well depending on cell growth of individual NPC lines.
Note: Usually I just perform a 1:2 split of confluent NPCs the day before transduction and don’t bother with counting cells. Generally NPCs can be passaged anything from 1:2-1:6 while expanding, though they prefer to be crowded! If plated too sparse they are at risk of spontaneous differentiation and can no longer divide/be expanded.
Day -1 Transduction
Check the condition of NPCs seeded at Day -2.
Prepare virus-containing NPC medium. Note: I typically use NGN2-neo-GFP and rtta lentiviruses; when prepared from 15 cm HEK dish transfection and resuspended in 600ul DMEM, usually 150ul of each virus is sufficient to transduce 2 x 6 well plates of NPCs. Add 150ul of each virus to 24ml of RT NPC media for the media change.
Aspirate medium from a plate and add fresh medium with virus (2ml/well of a 6 well plate).
Incubate the plate in an incubator 3-4 hours before replacing with fresh NPC media.
Day 0 Dox treatment
Prepare induction media: NPC media + 1ug/ml doxycycline
Perform whole media change
Day 1 Antibiotic selection
Prepare selection media: fresh NPC medium with Dox at 1ug/ml and antibiotics at following concentrations (adjust depending on antibiotic resistance of NGN2 vector)
Perform whole media change
Note: GFP signal should be visible at this stage if using a GFP construct; helpful for assessing early neuron health/differentiation.
Day 3 Switch to neuron medium
Prepare Brainphys neuronal medium (see above) with Dox at 1ug/ml.
Aspirate NPC medium in a plate and add neuron medium.
D5-D7 Replate neurons in 12 well plates
Prepare 12 well plates for seeding NGN2 neurons by coating with 4x geltrex at least 4 hours prior to plating (see day -3; preferably the day before replating). 400ul 15 mg/ml stock in 25ml DMEM = ~240ug/ml final conc
Aspirate medium from NGN2 neurons and add RT Accutase (1ml per well of 6 well plate)
Incubate in the incubator for 5 mins until neurites retract.
Collect cells in accutase (use P1000 to collect and gently pipette 2-3 times to break up clumps) and transfer to 15ml tube containing DMEM at a ratio of 3 parts DMEM: 1 part accutase. Note: cells should come off easily! If they do not detach or break up into a single cell suspension you need to leave accutase on for longer. “Stickiness” of cells is also an indicator of poor culture health (dead cells release chromosomal DNA which causes sticking)
Spin down at 1000g, 5 mins.
Resuspend cells in 1ml of neuron medium (use P1000 to gently resuspend pellet)
Count cells using haemocytometer/countess II and calculate needed density of suspension.
Further dilute in NPC medium to desired density. (2ml NPC medium/well of a 6 well plate)
Seed desirable number of cells in a well. Cell density in a single well of 12-well plate is around 1.2M neurons/well. If using 6/24/96 well plates scale as necessary.
Immature neurons can be plated any point between D5 and D7 – I usually adjust according to the weekday/weekend configuration relative to induction start. More mature cells may be too damaged by the replating process due to longer neurites.
D7 - D11.
Remove half of media and replace with fresh neuron media (do not include any dox at this point (leaving dox in for too long keeps neurons in a more immature state); GFP signal will gradually fade)
D11 - D13 Ara-C treatment to reduce flat/proliferating cells
Add Ara-C (200nM at working con.) by half volume change, e.g. use 400nM Ara-C in neuron medium so that the Ara-C con. in a well becomes 200nM Ara-C.
D13-21+
Continuing feeding every 2-3 days until harvest. Neurons show activity by D21 and robust synaptic activity at D28,