All iPSCs were characterized as previously described (Brennand et al., 2011 (link)). EBs were formed by mechanical dissociation of iPSC colonies using collagenase and plating onto low-adherence dishes. For EB differentiation, floating EBs were treated in STEMdiff™ Neural Induction Medium + SMADi (StemCell Technologies) for 20 days. To obtain neural progenitor cells, EBs were then plated onto polyornithine/laminin (Sigma)-coated dishes in DMEM/F12 plus 1% N2 and 1% B27. Rosettes were manually collected and dissociated with accutase (Chemicon) after 1 week and plated onto PLO (50 µg/ml) and laminin-coated dishes in neural progenitor cell media (DMEM/F12, 1% N2, 1% B27 (Invitrogen), and 20 ng/ml EGF, and 20 ng/ml FGF2 (Invitrogen)). To obtain hippocampal mature neurons, neural progenitor cells were plated onto dPGA or PLO (50 µg/ml) and laminin (1 µg/ml) coated-dishes and differentiated in BrainPhys neuronal medium (StemCell Technologies) supplemented with 1 × N2, 1 × B27, 20 ng/ml BDNF (Peprotech), 1 mM dibutyrl-cyclicAMP (Sigma), 200 nM ascorbic acid (Sigma), 1 μg/ml laminin and 620 ng/ml Wnt3a (R&D) for 2 weeks. After 2 weeks post differentiation, media was replaced by STEMdiff™ Forebrain Neuron Maturation Kit (StemCell Technologies) in BrainPhys media until use. All cells used in the present study were verified as free from mycoplasma contamination.
Stemdiff forebrain neuron maturation kit
Manufactured by STEMCELL
The STEMdiff™ Forebrain Neuron Maturation Kit is a cell culture system designed to facilitate the differentiation and maturation of human pluripotent stem cell-derived forebrain neurons. The kit provides a defined medium and associated reagents to support the transition of neural progenitor cells towards a mature neuronal phenotype.
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Lab products found in correlation
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Brainphys neuronal medium, by STEMCELL (1 mentions)
Stemdiff neural induction medium smadi, by STEMCELL (1 mentions)
Accutase, by Merck Group (1 mentions)
Dibutyrl cyclicamp, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
Stemdiff smadi neural induction kit, by STEMCELL (1 mentions)
Stemdiff neural progenitor medium, by STEMCELL (1 mentions)
2 protocols using stemdiff forebrain neuron maturation kit
1
Differentiation of iPSCs into Hippocampal Neurons
2
Differentiation of iPSCs into Forebrain Neurons
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The iPSCs were differentiated into forebrain neurons using a combination of the following commercially available kits: STEMdiff SMADi Neural Induction Kit (catalog no. 08581, STEMCELL Technologies), STEMdiff Neural progenitor medium (catalog no. 05833, STEMCELL Technologies), STEMdiff Forebrain Neuron Differentiation Kit (catalog no. 08600, STEMCELL Technologies), and STEMdiff Forebrain Neuron Maturation Kit (catalog no. 08605, STEMCELL Technologies). Briefly, iPSCs were plated as single cells on Matrigel-coated plates. When the cells were at >80% confluency, the SMADi neural induction medium (prepared as per the manufacturer’s directions) was added to the single-cell culture and replenished every alternate day until day 18. The obtained NPCs were passaged on days 6, 12, and 18. Thereafter, NPCs were cultured and expanded in neural progenitor medium and used for differentiation as needed. On day 19, the NPCs were supplemented with Forebrain Neuron Differentiation medium (prepared as per the manufacturer’s directions), and the medium was replenished daily until day 25. From day 25 onward, the cells were maintained in Forebrain Neuron Maturation medium (prepared as per the manufacturer’s directions) and observed for mature neuron morphology. The mature neurons were immunostained for microtubule-associated protein 2 (MAP2) and neuronal nuclear protein (NeuN).
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