Mar 15, 2024
Forebrain neural progenitor cells (fbNPCs) differentiation from hiPSCs (dual SMAD inhibition)
- 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
Protocol Citation: anita.adami 2024. Forebrain neural progenitor cells (fbNPCs) differentiation from hiPSCs (dual SMAD inhibition). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9ozzg8j/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: January 31, 2023
Last Modified: March 15, 2024
Protocol Integer ID: 76132
Funders Acknowledgement:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Swedish Research Council
Grant ID: 2018-02694
Swedish Brain Foundation
Grant ID: FO2019-0098
Cancerfonden
Grant ID: 190326
Barncancerfonden
Grant ID: PR2017-0053
NIHR Cambridge Biomedical Research Centre
Grant ID: NIHR203312
Swedish Society for Medical Research
Grant ID: S19-0100
National Institutes of Health
Grant ID: HG002385
Swedish Research Council
Grant ID: 2021-03494
Swedish Research Council
Grant ID: 2020-01660
Abstract
This protocol described how to differentiate hiPSCs to forebrain neural progenitor cells using dual SMAD inhibition
hiPSCs dissociation
hiPSCs dissociation
15m
Human induced pluripotent stem cells (hiPSCs) are rinsed once with DPBS (GIBCO) and dissociated when 70-90% confluent with 0.5 mM EDTA (75 ml/cm2; GIBCO) at 37 °C for 00:07:00
7m
Dissociated cells are then carefully washed away from the wells and collected in wash medium (9.5 mL DMEM/F-12 (31330-038; GIBCO) and 0.5 mL knockout serum replacement (GIBCO)).
The cells are then centrifuged at 400 x g for 00:05:00 and resuspended in iPS brew medium (StemMACS iPS-Brew XF and 0.5% penicillin/streptomycin (GIBCO)).
5m
hiPSCs differentiation into fbNPCs via dual SMAD inhibition
hiPSCs differentiation into fbNPCs via dual SMAD inhibition
2w
Day 0 of differentiation. The resuspended cells are then plated on LN111-coated (1.14µg/cm2; Biolamina) coated Nunc multidishes at a density of 10000 cells/cm2 and grown in N2 medium (1:1 DMEM/F-12 (21331020; GIBCO) and Neurobasal (21103049; GIBCO) supplemented with 1% N2 (GIBCO), 2 mM L-glutamine (GIBCO), and 0.2% penicillin/streptomycin). 10 μM of Y27632 (Rock inhibitor; Miltenyi) are also added to the cells when plating. Finally, 10 μM SB431542 (Axon) and 100 ng/ml noggin (Miltenyi) for dual SMAD inhibition are supplemented to the media.
The media, supplemented with dual SMAD inhibitors, is changed every 2-3 days until day 9 of differentiation. The amount of media is slightly increased at every feeding as the cells' confluency increases.
Day 9 of differentiation. The cells are fed with N2 media without dual SMAD inhibitors.
Day 11 of differentiation. The cells are dissociated, centrifuged, and resuspended as described above (hiPSCs dissociation SECTION). They are then replated on LN111-coated Nunc multidishes at a density of 800000 cells/cm2 in B27 medium (Neurobasal supplemented with 1% B27 without vitamin A (GIBCO), 2 mM L-glutamine and 0.2% penicillin/streptomycin Y27632 (10 μM), BDNF (20 ng/ml; R&D), and L-ascorbic acid (0.2 mM; Sigma)). 10 μM of Y27632 are also added. The cells are kept in the same medium until day 14 of differentiation, when they are harvested for downstream analyses (e.g. bulk RNA sequencing).