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.”
Attachments
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 0.5μg/ml 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”).
