Jan 26, 2026

Public workspaceNeural Progenitor Cell (NPC) differentiation

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvje1nwgk5/v1
  • Lexi Bounds1,
  • Alejandro Barrera1,
  • Maria ter Weele1,
  • Siyan Liu1,
  • Euphy Wu2,
  • Shengyu Li1,
  • Revathy Venukuttan1,
  • Ruhi Rai1,
  • Wancen Mu2,
  • Nahid Iglesias1,
  • Paola Giusti-Rodriguez3,
  • Timothy E. Reddy1,
  • Yun Li2,
  • Raluca Gordan4,
  • Andrew Allen1,
  • Michael Love2,
  • Patrick Sullivan2,
  • Greg Crawford1,
  • Charles Gersbach1
  • 1Duke University;
  • 2University of North Carolina;
  • 3University of Florida;
  • 4University of Massachusetts
  • Gersbach Lab
Andrea R Daniel
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvje1nwgk5/v1
Protocol Citation: Lexi Bounds, Alejandro Barrera, Maria ter Weele, Siyan Liu, Euphy Wu, Shengyu Li, Revathy Venukuttan, Ruhi Rai, Wancen Mu, Nahid Iglesias, Paola Giusti-Rodriguez, Timothy E. Reddy, Yun Li, Raluca Gordan, Andrew Allen, Michael Love, Patrick Sullivan, Greg Crawford, Charles Gersbach 2026. Neural Progenitor Cell (NPC) differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvje1nwgk5/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: October 30, 2025
Last Modified: January 26, 2026
Protocol Integer ID: 231163
Keywords: iPSC, NPC, Neural progenitor cells, differentiations, differentiation into neural progenitor cell, neural progenitor cell, npc, cell, differentiation this protocol, ipsc culture, differentiation
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Lexi Bounds in the Gersbach lab at Duke University.
Abstract
This protocol describes methods for WTC11 iPSC culture and differentiation into neural progenitor cells.
Materials
WTC11 iPSCs from the Coriell Institute for Biomedical Research (GM25256)
mTeSR1 (StemCell Technologies #85850)
Matrigel (Corning #354230)
Versene (ThermoFisher #15040066)
KnockOut‱ Serum Replacement (ThermoFisher #10828028)
Y-27632 (Dihydrochloride) (StemCell Technologies (#72302)
6-well culture plates (Corning #3471)
SB431542 (StemCell Technologies #72234): Resuspend 10 mg in 2.06 mL DMSO.
LDN193189 (StemCell Technologies #72147): Resuspend 10 mg in 2.09 mL of DMSO.
STEMdiff‱ Neural Rosette Selection Reagent (StemCell Technologies #05832)
Penicillin-Streptomycin (10,000 units/mL) (ThermoFisher #15140122)
B27 Supplement w/o Vitamin A (50X) (ThermoFisher #12587010)
N-2 Supplement (100X) (ThermoFisher #12587010)
Animal-Free Recombinant Human EGF (Peprotech #AF-100-15): Resuspend 100 ug in 1 mL ultra-pure H20. 
Animal-Free Recombinant Human FGF-basic (Peprotech #AF-100-18B): o   Resuspend 50 ug in 500 uL ultra-pure H20.

  • Neural basal medium (NBM): 500 mL DMEM/F-12 + 5 mL N2 + 10 mL B27 + 5 mL P/S
  • Neural induction medium (NIM): 50 mL NBM + 0.5 uL LDN + 50 uL SB431542
  • Neural expansion medium (NEM): 50 mL NBM + 10 uL bFGF + 10 uL EGF
Troubleshooting
iPSC culture
All cell lines were cultured at 37C and 5% CO2.
WTC11 iPSCs were obtained from the Coriell Institute for Biomedical Research (GM25256)
Cells are propagated in mTeSR1 (StemCell Technologies #85850) on Matrigel (Corning #354230)-coated tissue culture plates and passaged as colonies using Versene (ThermoFisher #15040066).
Cells are frozen in freezing solution comprised of 60% mTeSR1 media, 10% DMSO, and 30% KnockOut‱ Serum Replacement (ThermoFisher #10828028), with 10uM ROCK Inhibitor (RI) Y-27632 (Dihydrochloride) (StemCell Technologies (#72302).
NPC Differentiation
WTC11 iPSCs are differentiated into NPCs using embryoid-body (EB) formation and small molecules as previously described with minor modifications (Refs 1, 2) 
WTC11 iPSCs are seeded in low-adhesion 6-well culture plates (Corning #3471) in NBM containing 10 uM SB431542 (StemCell Technologies #72234) and 0.1 uM LDN193189 (StemCell Technologies #72147) (NIM) with 10 uM Y-27632.
24 hours later (Day 1), 2 mL of media is removed and replaced with 2 mL NIM. 
On Days 2-4, 3 mL of media is removed and replaced with 3 mL NIM. 
On Day 5, EBs are transferred onto Matrigel-coated 6-well plates containing 1 mL NIM per well.
24 hours later (Day 6), all media is removed and replaced with 3 mL NIM.
On Days 7-12, 3 mL NIM was changed daily. 
On Day 13, neural rosettes are harvested using STEMdiff‱ Neural Rosette Selection Reagent according to the manufacturer’s protocol (StemCell Technologies #05832) and plated onto Matrigel-coated plates in NEM (‘Passage 0’):
  1.      Aspirate media.
  2.      Wash w/ 1 mL DMEM/F-12.
  3.      Aspirate.
  4.      Add 1 mL neural rosette selection reagent.
  5.      Incubate 1 hour at 37C. (may need longer incubation, check at 30min, 45min)
  6.      Aspirate selection reagent.
  7.      Using P1000, wash each well 2x w/ 1 mL DMEM/F-12 and collect cells.
a.     Try not to break up the rosettes.
8.     Centrifuge 200-300g for 5min.
9.      Aspirate supernatant.
10.   Resuspend cells in 2 mL NEM.
11. Plate to one well of 6-well.
Repeat for all wells.
NPCs are propagated for five passages in NEM before characterization as described below. All experiments are performed with NPCs of passage number 7-15. 
NPC culture
WTC11 NPCs are cultured in NEM. Change media daily with 2 mL NEM.
Passage cells w/ Accutase when 80-90% confluent (include RI in media when passaging, like iPSCs)
Seed cells at 200-500K cells per well (cell line/differentiate dependent).
Collect cells for RNA at P2, P5. Freeze cells when possible in NBM + KSR + DMSO + RI.
Protocol references
1. Generation, Expansion, and Differentiation of Human Pluripotent Stem Cell(hPSC) Derived Neural Progenitor Cells (NPCs). Brafman DA. Methods Mol Biol. 2015;1212:87-102. doi: 10.1007/7651_2014_90.PMID: 25063499

2. Inhibition of STEP(61) ameliorates deficits in mouse and hiPSC-based schizophrenia models. Xu J, Hartley BJ, Kurup P, Phillips A, Topol A, Xu M, Ononenyi C, Foscue E, Ho SM, Baguley TD, Carty N, Barros CS, Müller U, Gupta S, Gochman P, Rapoport J, Ellman JA, Pittenger C, Aronow B, Nairn AC, Nestor MW, Lombroso PJ, Brennand KJ.Mol Psychiatry. 2018 Feb;23(2):271-281. doi: 10.1038/mp.2016.163. Epub 2016 Oct 18.PMID: 27752082 

3.  Transcriptional signatures of schizophrenia in hiPSC-derived NPCs and neurons are concordant with post-mortem adult brains. Hoffman GE, Hartley BJ, Flaherty E, Ladran I, Gochman P, Ruderfer DM, Stahl EA, Rapoport J, Sklar P, Brennand KJ. Nat Commun. 2017 Dec 20;8(1):2225. doi: 10.1038/s41467-017-02330-5. PMCID: PMC5738408