Apr 09, 2026
hiPSC-iGABA: ASCL1 and DLX2-neuron induction
- Sam Powell1
- 1Icahn School of Medicine at Mount Sinai
- Brennand Laboratory
Protocol Citation: Sam Powell 2026. hiPSC-iGABA: ASCL1 and DLX2-neuron induction. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2zpoxl1y/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: February 21, 2026
Last Modified: April 09, 2026
Protocol Integer ID: 243767
Keywords: hiPSCs, NPCs, neuron, MPRA, CRISPR, FACS, morphology, neuron induction igaba, µm rock inhibitor, cell solution, accutase, inducible lentivirus vector, inhibitory neuron, idan generation, neuron media on div14, ml hygromycin, stemflex, specific identity for igaba
Funders Acknowledgements:
Kristen Brennand
Grant ID: R01MH106056
Kristen Brennand
Grant ID: U01DA047880
Kristen Brennand
Grant ID: R01DA048279
Abstract
iGABAs were generated via transduction with two separate doxycycline-inducible lentivirus vectors encoding tetO-ASCL1-PuroR and tetO-DLX2-HygroR according to Yang et al. In brief, hiPSCs were harvested in Accutase, dissociated into a single-cell solution, quenched in DMEM, pelleted via centrifugation for 5 min at 800 × g, and resuspended in StemFlex with 10 µM ROCK Inhibitor. Volumetric equivalents of tetO-ASCL1-PuroR, tetO-DLX2-HygroR, and pUBIQ-rtTA were added to the suspension, mixed gently by inversion, dispensed onto Matrigel-coated plates, and incubated overnight at 37 °C. The next day, media was changed to Induction Media (identical recipe to that used for iDAN generation) with 1.0 µg/mL doxycycline (DIV1). 1.0 µg/mL puromycin and 250 µg/mL hygromycin (Thermo, #10687010) were added the next day (DIV2) and continued for four days. We included 4.0 µM Ara-C in the media from ~DIV4-8. Cells were harvested, dissociated, and replated on 0.1% PEI and 80 µg/mL Matrigel-coated plates around DIV5-7. Media was switched to Neuron Media on DIV14, and doxycycline was withdrawn at that time. Half media changes were performed every other day from DIV14 until the time of harvest at DIV42 for the samples used for RNA-sequencing library-generation. Importantly, these inhibitory neurons are reported to be predominantly SST+, with some double staining for CB+ and/or CR+, and little PV+ staining, thus indicating somewhat of a GABAergic neuron subtype-specific identity for iGABA.
Materials
- BrainPhys: StemCell Technologies, #05790
- DMEM F12+ with Glutamax and Sodium Pyruvate: Thermo Fisher, #10565018
- Matrigel, growth factor reduced: Corning #354230
- Polyethylenimine: Sigma/Millipore #P3143
- Pierce Borate buffer 20X (dilute 1:10, then make 0.1% PEI soln in it): Thermo Fisher #28314
- DMEM: Thermo Fisher #11966025
- BDNF: Peprotech, #45002 (media concentration of 20 ng/mL)
- GDNF: Peprotech, #45010 (media concentration of 20 ng/mL)
- cAMP: Sigma #D0627 (media concentration of 500 ug/mL)
- Ascorbic Acid: Sigma #A0278 (media concentration of 200nM)
- N2: Thermo Fisher, #17502-048
- B27, Thermo Fisher
- Natural mouse laminin, Thermo Fisher #23017-015
- Rock inhibitor (THX) Y-26732
- StemFlex
- Accutase: Innovative Cell Technologies #AT104
- Antibiotic-antimycotic: Thermo Fisher #15240062
- Glutamax: Thermo Fisher #35050061
- Sodium pyruvate: Thermo Fisher #11360070
Induction Media
DMEM/F12+ (already contains sodium pyruvate and glutamax)
Anti-anti
N2
B27
Neuron Media
BrainPhys
BDNF
GDNF
cAMP
Ascorbic Acid
N2
B27
Mouse laminin
Sodium Pyruvate
Glutamax
Anti-anti
Harvest iPSCs by incubating in 1mL per well Accutase at 37 degrees C for at least 5-10 minutes. Max of 20 minutes
Resuspend the iPSCs in the accutase in each well and then mix in a 50mL falcon tube with at least 1:3 volumetric ratio of room temp. DMEM
Spin for 5 minutes (Sam likes 6 b/c he’s paranoid and it makes him feel better) at RT at 800g
Aspirate supernatant and resuspend pellet in 1 mL of Stemflex with 1:1000 rock inhibitor THX. Count. Dilute in Stemflex with THX to a cell suspension concentration of 1e6 cells/mL
Add the appropriate amount of Ascl1-PuroR, Dlx2-HygroR, and rTTA viruses
A safe amount to add of each is 50 uL per 1e6 cells (and therefore 50uL of each per 10mL) of suspension for Sam’s current aliquots of virus
Mix well by inversion and gentle, repeated pipetting with a large strip pipet
Dispense 1.0-1.5 mL of suspension per well (6 well size) coated with 1x matrigel
Incubate in incubator overnight (min 12 hours; max 36 hours)
DIV1: aspirate the media in the virus hood and replace with Induction media with Dox 1ug/mL
DIV2: replace media with Induction media with dox (1ug/mL), puromycin (2ug/mL), and hygromycin (250ug/mL which means 1:250 dilution in the media with our aliquots)
DIV3: only if there is a significant amount of cell death, repeat step from DIV2
DIV4: Start AraC 4uM treatment on this day. Continue puromycin 2ug/mL and hygromycin 250ug/mL until DIV6
DIV6: harvest by incubating in 1mL Accutase at 37degrees for 5 minutes. Gently resuspend in the well with the Accutase so it break up clumps. Quench with at least 1:3 DMEM. Spin for 5mins at RT at 800g. Resuspend pellet in 1mL of Neuron media with Dox 1ug/mL, puromycin 2ug/mL, hygromycin 250ug/mL, AraC 4uM, and THX 1:1000. Count. Dilute to a concentration of 1e6 cells/mL. Mix well but gently by inversion and pipetting with large pipet. Dispense 2-4 million cells per well (and therefore 2-4mL of the suspension) on either 0.1% PEI + 2x matrigel OR 4x matrigel-coated plates
DIV7: replace media with neuron+Dox 1ug/mL +AraC 4uM
DIV8: at this time you may stop AraC, so replace media accordingly
Note: if there is still a concern about flat cells, the AraC can be continued up until DIV14 at the latest. In those cases, I often will decrease it to 2uM AraC at DIV8 and keep until DIV14
DIV10: repeat step from DIV8. Note, you may continue using AraC at this time
DIV12: repeat step from DIV10
DIV14: replace media with just neuron media WITHOUT dox or AraC
Perform ½ media changes 3 times a week or full media changes twice a week until desired time point for harvest
Note: Sam typically harvests iGABA at DIV35