Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur
Abstract
We adapted a previously-described method (Pantazis et al., 2022) for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs. After stable integration of NGN2, proceed to differentiate iPSCs using protocol “iNeuron differentiation from human iPSCs.”
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549-1145.pdf
Steps
Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
Culture iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel (Corning) and feed daily with Essential 8 media (ThermoFisher).
Passage iPSCs with warm Accutase into Essential 8 media with 10micromolar (µM) ROCK inhibitor. Plate 800,000 iPSCs into one Matrigel-coated well of a 6-well plate.
3 - 6 hours after plating, cells should be healthy and attached. Perform transfection using Lipofectamine Stem and a 2:1 ratio of donor plasmid to transposase:
| A | B |
|---|---|
| OptiMEM | 200 μL |
| PB-TO-hNGN2-puro-BFP plasmid | 0.75 μg |
| EF1α-transposase plasmid | 0.37 μg |
| Lipofectamine Stem | 4 μL |
Check for transfection efficiency (BFP-labeled cells) on the next day using fluorescence microscopy.
Passage iPSCs with Accutase to a 10 cm dish when cells are confluent enough for splitting.
72h 0m 0s after transfection, select for transfected iPSCs with 0.5 puromycin.
Confirm purity of surviving transfected cells with fluorescence microscopy. When population is pure, withdraw puromycin.
Cryopreserve selected iPSCs with
| A | B |
|---|---|
| Essential 8 media | 70% |
| Knockout serum replacement | 20% |
| DMSO | 10% |
| ROCK inhibitor (Supplement) | 10 µM |
Proceed to culture and induction to neuronal fate using doxycycline (see “Protocol: iNeuron differentiation from human iPSCs”).
