Nov 14, 2025
Differentiation of KOLF2.1J hIPSC to midbrain dopaminergic neurons
- Guochang Lyu1,
- Ernest Arenas1
- 1Karolinska Institute
- Ernest Arenas: deceased 15.09.2024
- SOX6 mDA differentiation
Protocol Citation: Guochang Lyu, Ernest Arenas 2025. Differentiation of KOLF2.1J hIPSC to midbrain dopaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld65x7g5b/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: 226835
Keywords: representative markers for ventral midbrain dopaminergic neuron, ventral midbrain dopaminergic neuron, mature midbrain dopaminergic neuron, dopaminergic neuron, dopaminergic neurons this protocol, mature midbrain, resultant cell, neuronal acitivity, sox6
Abstract
This protocol was used to differentiate KOLF2.1J cells into mature midbrain dopaminergic neurons. The resultant cells do not show representative markers for ventral midbrain dopaminergic neurons such as EN1 or SOX6, although they showed neuronal acitivity and other canonical markers.
Protocol materials
Stemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
Stemolecule™ SB431542ReprocellCatalog #04-0010-10
PurmorphamineTocrisCatalog #4551
CHIR99021R&D SystemsCatalog #4423
Y-27632 dihydrochlorideTocrisCatalog #1254
Laminin-511BiolaminaCatalog #LN511
Poly-L-Ornithine Merck MilliporeSigma (Sigma-Aldrich)Catalog #P4957
Ascorbic Acid 500 mg
STEMCELL Technologies Inc.Catalog #72132
Dibutyryl-cAMPSTEMCELL Technologies Inc.Catalog #73884
Neurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049
B27 supplement minus vitamin AGibco - Thermo Fisher ScientificCatalog #12587010
L-Glutamine Gibco - Thermo Fisher ScientificCatalog #25030024
Human Recombinant BDNFSTEMCELL Technologies Inc.Catalog #78005
Human Recombinant GDNFSTEMCELL Technologies Inc.Catalog #78058
Tgf beta 3 (human) Recombinant ProteinInvitrogen - Thermo FisherCatalog #RP8600
PD0325901 1 mg
STEMCELL Technologies Inc.Catalog #72182
SU5402 1 mg
STEMCELL Technologies Inc.Catalog #73912
Geltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1569601
Essential 8 Flex complete medium (E8)Gibco - Thermo Fisher ScientificCatalog #A2858501
N2 supplementGibco - Thermo Fisher ScientificCatalog #17502048
Troubleshooting
Pre-Differentiation (iPSC Expansion & Seeding)
Day -2 (Two Days Before Differentiation Start):
Coat culture plates with:
- Geltrex (Life Technologies)Geltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1569601
- Laminin-511 (Biolamina)Laminin-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) mediumEssential 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).
Neurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049 N2 supplementGibco - Thermo Fisher ScientificCatalog #17502048 B27 supplement minus vitamin AGibco - Thermo Fisher ScientificCatalog #12587010 L-Glutamine Gibco - Thermo Fisher ScientificCatalog #25030024
Small Molecule Additives (Day 0–11):
- LDN193189 – 250 nM (Day 0–7) Stemolecule LDN-193189Stemgent - Bio-connectCatalog #04-0074
- SB431542 – 10 µM (Day 0–7) Stemolecule™ SB431542ReprocellCatalog #04-0010-10
- Purmorphamine – 12 µM (Day 0–7) PurmorphamineTocrisCatalog #4551
- CHIR99021 – 0.7 µM (Day 0–3), increase to 7.5 µM from Day 4–11 CHIR99021R&D SystemsCatalog #4423
- Y27632 – 10 µM (Day 0–1 only) Y-27632 dihydrochlorideTocrisCatalog #1254
Replating Plates
Day 11: First Replating. Dissociate into single cells.
Replate at 800,000 cells/cm² on LN511-coated plates. Add 10 µM Y27632 for 48 hours post-replating. Laminin-511BiolaminaCatalog #LN511 Y-27632 dihydrochlorideTocrisCatalog #1254
Day 16: Second 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. Laminin-511BiolaminaCatalog #LN511 Poly-L-Ornithine Merck MilliporeSigma (Sigma-Aldrich)Catalog #P4957 Y-27632 dihydrochlorideTocrisCatalog #1254
Differentiation Media Transition
Day 10–28: Switch to Differentiation Media.
- Neurobasal / B27 / L-glutamine base, plus: Neurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049 B27 supplement minus vitamin AGibco - Thermo Fisher ScientificCatalog #12587010 L-Glutamine Gibco - Thermo Fisher ScientificCatalog #25030024
- BDNF – 20 ng/mL Human Recombinant BDNFSTEMCELL Technologies Inc.Catalog #78005
- GDNF – 20 ng/mL Human Recombinant GDNFSTEMCELL Technologies Inc.Catalog #78058
- TGF-β3 – 1 ng/mL Tgf beta 3 (human) Recombinant ProteinInvitrogen - Thermo FisherCatalog #RP8600
- Ascorbic acid – 0.2 mM Ascorbic Acid 500 mg STEMCELL Technologies Inc.Catalog #72132
- Dibutyryl cAMP – 0.2 mM Dibutyryl-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 16–21: Administer Dual Inhibitor Cocktail.
- PD0325901 – 1 µM PD0325901 1 mg STEMCELL Technologies Inc.Catalog #72182
- SU5402 – 5 µM SU5402 1 mg STEMCELL Technologies Inc.Catalog #73912
Day 26–28: Neurogenesis Promotion.
Add DAPT at 10 µM to promote neuronal maturation.