Nov 14, 2025

Public workspaceImproved differentiation of KOLF2.1J hIPSC to midbrain dopaminergic neurons informed by multiomics

 Forked from Differentiation of KOLF2.1J hIPSC to midbrain dopaminergic neurons
DOI
https://dx.doi.org/10.17504/protocols.io.3byl46z48go5/v1
  • Guochang Lyu1,
  • Ernest Arenas1
  • 1Karolinska Institute
  • Ernest Arenas: deceased 15.09.2024
  • SOX6 mDA differentiation
Roberto Garcia Swinburn
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl46z48go5/v1
Protocol Citation: Guochang Lyu, Ernest Arenas 2025. Improved differentiation of KOLF2.1J hIPSC to midbrain dopaminergic neurons informed by multiomics. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46z48go5/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: September 09, 2025
Last Modified: November 14, 2025
Protocol Integer ID: 226841
Keywords: representative markers for ventral midbrain dopaminergic neuron, ventral midbrain dopaminergic neuron, mature midbrain dopaminergic neuron, dopaminergic neuron, mature midbrain, ventral identity, neuronal activity, sox6, resultant cell, multiomic
Abstract
This protocol was used to differentiate KOLF2.1J cells into mature midbrain dopaminergic neurons with a more ventral identity. The resultant cells show representative markers for ventral midbrain dopaminergic neurons such as EN1 or SOX6, as well as neuronal activity and other canonical markers.
Protocol materials
ReagentGeltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1569601
ReagentLaminin-511BiolaminaCatalog #LN511
ReagentEssential 8 Flex complete medium (E8)Gibco - Thermo Fisher ScientificCatalog #A2858501
ReagentL-Glutamine Gibco - Thermo Fisher ScientificCatalog #25030024
ReagentNeurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049
ReagentN2 supplementGibco - Thermo Fisher ScientificCatalog #17502048
ReagentB27 supplement minus vitamin AGibco - Thermo Fisher ScientificCatalog #12587010
ReagentHuman Recombinant BDNFSTEMCELL Technologies Inc.Catalog #78005
ReagentHuman Recombinant GDNFSTEMCELL Technologies Inc.Catalog #78058
ReagentTgf beta 3 (human) Recombinant ProteinInvitrogen - Thermo FisherCatalog #RP8600
ReagentAscorbic Acid 500 mg STEMCELL Technologies Inc.Catalog #72132
ReagentDibutyryl-cAMPSTEMCELL Technologies Inc.Catalog #73884
ReagentY-27632 dihydrochlorideTocrisCatalog #1254
ReagentPoly-L-Ornithine Merck MilliporeSigma (Sigma-Aldrich)Catalog #P4957
ReagentStemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
ReagentStemolecule™ SB431542ReprocellCatalog #04-0010-10
ReagentPurmorphamineTocrisCatalog #4551
ReagentCHIR99021R&D SystemsCatalog #4423
Troubleshooting
Pre-Differentiation (iPSC Expansion & Seeding)
Day -2 (Two Days Before Differentiation Start):
Coat culture plates with:
  • Geltrex (Life Technologies)ReagentGeltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1569601
  • Laminin-511 (Biolamina)ReagentLaminin-511BiolaminaCatalog #LN511
Seed iPSCs:
  • Plate adapted iPSCs at a density of 200,000 cells/cm² onto the coated plates. Maintain cells in Essential 8 Flex (Thermo Fisher Scientific) mediumReagentEssential 8 Flex complete medium (E8)Gibco - Thermo Fisher ScientificCatalog #A2858501 . Continue until cells reach ~80% confluency.
Initiation of Differentiation
Day 0: Start of Differentiation:
Culture Medium: Switch to Neurobasal/N2/B27 medium supplemented with 2 mM L-glutamine (Invitrogen).
ReagentNeurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049 ReagentN2 supplementGibco - Thermo Fisher ScientificCatalog #17502048 ReagentB27 supplement minus vitamin AGibco - Thermo Fisher ScientificCatalog #12587010 ReagentL-Glutamine Gibco - Thermo Fisher ScientificCatalog #25030024
Small Molecule Additives (Day 0–11):
  • LDN193189 – 250 nM (Day 0–7) ReagentStemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
  • SB431542 – 10 µM (Day 0–7) ReagentStemolecule™ SB431542ReprocellCatalog #04-0010-10
  • Purmorphamine – 2 µM (Day 0–7) ReagentPurmorphamineTocrisCatalog #4551
  • CHIR99021 – 0.7 µM (Day 0–3), increase to 7.5 µM from Day 4–14 ReagentCHIR99021R&D SystemsCatalog #4423
  • Y27632 – 10 µM (Day 0–1 only) ReagentY-27632 dihydrochlorideTocrisCatalog #1254
Replating Plates
Day 14: Replating. Dissociate into single cells.
Replate at 800,000 cells/cm² on Poly-L-ornithine+LN511-coated plates. Add 10 µM Y27632 for 48 hours post-replating. ReagentLaminin-511BiolaminaCatalog #LN511 ReagentPoly-L-Ornithine Merck MilliporeSigma (Sigma-Aldrich)Catalog #P4957 ReagentY-27632 dihydrochlorideTocrisCatalog #1254

Differentiation Media Transition
Day 10–28: Switch to Differentiation Media.
  • Neurobasal / B27 / L-glutamine base, plus: ReagentNeurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049 ReagentB27 supplement minus vitamin AGibco - Thermo Fisher ScientificCatalog #12587010 ReagentL-Glutamine Gibco - Thermo Fisher ScientificCatalog #25030024
  • BDNF – 20 ng/mL ReagentHuman Recombinant BDNFSTEMCELL Technologies Inc.Catalog #78005
  • GDNF – 20 ng/mL ReagentHuman Recombinant GDNFSTEMCELL Technologies Inc.Catalog #78058
  • TGF-β3 – 1 ng/mL ReagentTgf beta 3 (human) Recombinant ProteinInvitrogen - Thermo FisherCatalog #RP8600
  • Ascorbic acid – 0.2 mM ReagentAscorbic Acid 500 mg STEMCELL Technologies Inc.Catalog #72132
  • Dibutyryl cAMP – 0.2 mM ReagentDibutyryl-cAMPSTEMCELL Technologies Inc.Catalog #73884
Note on Supplements Withdrawal:
  • LDN193189, SB431542, and Purmorphamine are withdrawn after Day 7.
  • N2 supplement is withdrawn after Day 10.
Additional Small Molecule Treatments
Day 12–15: Add GW3965 at 10 µM to promote mDA identity.
Day 26–28: Neurogenesis Promotion.
Add DAPT at 10 µM to promote neuronal maturation.