Apr 08, 2026
hiPSCs-SNaP: NGN2-induced NPCs
- Sophie Cohen1,
- Meilin Fernandez Garcia1,
- Kristen Brennand1
- 1Yale University School of Medicine
- Brennand Lab
Protocol Citation: Sophie Cohen, Meilin Fernandez Garcia, Kristen Brennand 2026. hiPSCs-SNaP: NGN2-induced NPCs. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl84o26g2w/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
We use this protocol and it's working
Created: April 07, 2026
Last Modified: May 18, 2026
Protocol Integer ID: 314676
Keywords: CRISPR, stem cells, neurogenomics, npcs rapid generation of npc, induced npcs rapid generation, ngn2, npc, hipscs via transient overexpression, hipsc
Funders Acknowledgements:
NIMH
Grant ID: RM1MH132648
Abstract
Rapid generation of NPCs from hiPSCs via transient overexpression of NGN2.
Materials
**Cell Lines:**
- 3182-3-C5^^ngn2-eGFP-neo, rtTA hiPSCs(P36)-SNaPs P14, stable Cas9v2-Hygro
- 2607-2-C9 ngn2-eGFP-neo, rtTA hiPSCs(P38)-SNaPs P14, stable Cas9v2-Hygro
**Media**
- DMEM (ThermoFisher Scientific, #10566-016)
- DMEM/F12,GLUTAMAX (Thermofisher, #10565018)
- Brainphys (STEMCELL, # 05790)
**Supplements**
- 100X N-2 (Thermofisher, #17502048)
- 50X B27-RA (Thermofisher, #12587010)
- 1 mg/mL Natural Mouse Laminin (Thermofisher, # 23017015)
- 20 ug/mL BDNF stock (R 26D, #248,248-BD-025)
- 20 ug/mL GDNF stock (R 26D, #212)
- 250 mg/mL Dibutyryl cyclic-AMP (cAMP) stock (Sigma, # D0627)
- 200 mM L-ascorbic acid stock (Sigma, # A4403)
- 100X Pen/Strep
- 100X MEM NEAA (Thermofisher #11140050)
- 100 ug/mL EGF stock(final 10 ng/mL 1:10,000)
- 20 ug/mL bFGF stock (final 10 ng/mL 1:2,000)
**Selection/antibiotics**
- 1 mg/mL Doxycycline stock (1:500-1000 working dilution)
- 50 mg/mL Geneticin stock (Thermofisher, #10131035) (1:100, 0.5mg/mL final working dilution)
- 50 mg/mL Hygromycin (Invitrogen, #10687010)
- 1mg/mL Puromycin (Final 1ug/mL 1:1000 dil)(Sigma, no. P7255)
**Lentivirus**
- Cas9v2-hygro (addgene #98291)
- lox-ASD-Puro CRISPRko library (Boston Children’s Viral Core)
**Other reagents**
- Chroman1 (Tocris #7163; working dilution 1:10,000)
- Accutase Cell Detachment Solution (Innovative Cell Technologies, # AT-104)
**Media Recipes:**
- SNaP Media**
- NES base media
- DMEM/F12 + Glutamax
- Pen/Strep (1:100)
- MEM NEAA (1:100)
- B27 w/o Vitamin A (1:50)
- N2 supplement (1:100)
- EGF (1:10,000)
- bFGF (1:2,000)
- Neuron Media**
- Brainphys neuron medium
- 1% N2 (1:100)
- 2% B27-RA (1:50)
- Natural Mouse Laminin (1:1000)
- BDNF (1:1000)
- GDNF (1:1000)
- cAMP (1:1000)
- L-ascorbic acid (1:1000)
- 1 mM Sodium Pyruvate
- 1% Glutamax
- 1% Anti-Anti
Experimental Design
Seed 1M/w12wp
Induce Ngn2-eGFP-Neo(+Dox) Cas9v2 maintenance (+low Hygro) in SNaP Medium
Induce Ngn2-eGFP-Neo(+Dox), Cas9v2 maintainance(+low hygro) in SNaP Medium
Selection (+Geneticin) in SNaP medium
Selection (+Geneticin) 26 Switch to Neuron Medium
Split into 2X GTx, seeded 1M/w12wp, Infect with ASD gRNAs-Puro
gRNA selection (Puro)
gRNA selection (Puro)
Accutase Harvest
Protocol
Replate SNaPs into 12wp at 1 million cells per well
Coat 12wp with 1x geltrex. Incubate for ~2 hrs
Split cells using accutase – 5-7 minutes at room temp
Transfer to 3x volume of DMEM
Spin 5 minutes at .8 rcf
Aspirate and resuspend in media + chroman
Count and seed 1M cells/w12wp into SNaP media with chroman 1
*1 million was slightly too dense in my opinion. If I were to run this again, I would say ~750K is ideal.
Change media to SNaP media with:
+dox (1:500)
+hygro (1:3500)
Change media to SNaP media with:
+dox (1:500)
+hygro (1:3500)
Change media to SNaP media with:
+dox (1:500)
+hygro (1:3500)
+geneticin (1:100)
Neuron media
+dox (1:500)
+hygro (1:3500)
+geneticin (1:100)
Split to new plates
Coat 12wp with 2x geltrex. Incubate for ~2 hrs
Split cells using accutase – for 15-20 minutes