May 22, 2025
iPSC-derived vmDAn
- Valentina Baruffi1,2,
- Irene Fernández-Carasa1,2,
- Antonella Consiglio1,2,
- Angel Raya1,2,
- Miquel Vila3
- 1Department of Pathology and Experimental Therapeutics, Bellvitge University Hospital- IDIBELL, 08908 Hospitalet de Llobregat, Spain;
- 2Institute of Biomedicine of the University of Barcelona (IBUB), Carrer Baldiri Reixac 15-21, Barcelona 08028, Spain.;
- 3VHIR-CIBERNED-ASAP
- Vilalab Public
Protocol Citation: Valentina Baruffi, Irene Fernández-Carasa, Antonella Consiglio, Angel Raya, Miquel Vila 2025. iPSC-derived vmDAn. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpqk68lzp/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: May 22, 2025
Last Modified: May 22, 2025
Protocol Integer ID: 218737
Keywords: derived vmdan ipsc, vmdan ipsc, derived vmdan, ipsc
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
iPSC-derived vmDAn
Troubleshooting
iPSC medium is changed to Serum Replacement Medium (SRM), which is based on KO-DMEM with
15% of KO Serum Replacement (KO-SR; Gibco‱), 1 % of P/S, 1% of Glut, 1% of Non-Essential Amino Acids (NEAA; Lonza), and 10 μM of β-Mercaptoethanol (BME; Gibco‱).
The first day, SRM are supplemented with 100 nM of LDN193189 hydrochloride (LDN; Sigma-Aldrich) and 10 μM of SB 431542 (SB; Tocris Bioscience). From there and during all the differentiation, cells are maintained under hypoxic conditions (37 ºC Temperature, 5% CO2 and 5% O2).
From day 1 to day 5, SRM medium is supplemented with same concentrations of LDN and SB and adding 1 μM of Smoothened Agonist (SAG; Merck Millipore), 2 μM of Purmorphamine (Tocris Bioscience), and 100 ng/mL of FGF-8. From day 3 onwards, 3 μM of StemMACS‱ CHIR99021 (CHIR; Miltenyi Biotec) are also added.
From day 5, medium is progressively switched from SRM to N2, which consists of Neurobasal‱ Medium (Gibco‱), 1x N2 supplement (Gibco‱), 1% of P/S and 1% of Glut. On day 11, medium was changed with N2 supplemented with LDN and CHIR and cells could be either expanded as FP progenitors or matured to vmDAn.
At day 11, Cells were maintained in N2 supplemented with LDN and CHIR, until reaching 90% of confluency within the plate. To differentiate FP progenitors into vmDAn, medium is changed to B27-vitA, which contained Neurobasal‱, 2x B27‱- vitamin A (Gibco‱), 1% of P/S and 1% of Glut. Every two days, medium is fully changed with B27 supplemented with 20 ng/mL of BDNF, 20 ng/mL of Glial cell-line Derived Neurotrophic Factor (GDNF), 1 ng/mL of TGF-β3, 10 μM of Deoxyadenosine Triphosphate (dAPT; Calbiochem), 0.5 mM of Dibutyryl cyclic-Adenosine Monophosphate sodium salt (cAMP; Sigma-Aldrich) and 0.2 mM of L-Ascorbic Acid (AA; Sigma-Aldrich).
On day 20, cells are detached with Accutase (Merck Millipore), counted and centrifuged at 300xg for 5 minutes. Cells are plated on 6-well or glass coverslips in 24-well plastic plates with a triple-coating of 15 μg/mL Poly- L-Ornithine solution (Sigma-Aldrich), 3 μg/mL of Laminin (Sigma-Aldrich) and 2 μg/mL of Fibronectin (Sigma-Aldrich) to a density of 100,000 cells/cm2.
From day 20 onwards, half of B27 with factors is changed every 3-4 days until vmDAn are used for experiments.