iNeuron differentiation from human iPSCs
Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur
Abstract
We adapted a previously-described method (Boecker et al., 2020, 2021; Fernandopulle et al., 2018) for differentiating iPSCs stably expressing mNGN2 at a safe-harbor locus into human excitatory glutamatergic neurons. Pre-i 3Neuron iPSCs (human iPSCs with an integrated doxycycline-inducible mNGN2 transgene in the AAVS1 safe-harbor locus) were a gift from M. Ward (National Institutes of Health, Maryland).
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Steps
iNeuron differentiation from human iPSCs
Culture pre-iNeuron iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel in Essential 8 media, fed daily.
Passage iPSCs using warm Accutase and plate 5.5 x 106 cells onto a Matrigelcoated 15 cm dish, in Induction Media (DMEM/F12 supplemented with 1% N2- supplement [GIBCO], 1% NEAA [GIBCO], and 1% GlutaMAX [GIBCO], and containing 2μg/ml doxycycline and 10μm ROCK inhibitor).
After 24h 0m 0s, replace all media with fresh Induction Media, containing 2μg/ml doxycycline but no ROCK inhibitor.
Replace again with the same media after 24h 0m 0s (48h 0m 0s after plating).
72 hours after plating, dissociate cells with warm Accutase.
Count cells in freezing media.
Freezing media
| A | B |
|---|---|
| BrainPhys | 70% |
| FBS | 20% |
| DMSO | 10% |
| BDNF | 10 ng/mL |
| NT-3 | 10 ng/mL |
| B-27 supplement | 1x |
Freeze down cells in a Mr. Frosty container placed in a -80°C freezer .
On the following day, transfer cryopreserved neurons to liquid nitrogen storage.
