Jan 29, 2025
i3N neuronal differentiation
- 1Gandhi Lab
Protocol Citation: Alexis Penverne 2025. i3N neuronal differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrj98g5b/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 20, 2025
Last Modified: January 29, 2025
Protocol Integer ID: 118735
Abstract
This protocol is based on the protocol from the Kampmann lab (Chen et al., 2020). It describes the how to differentiate i3Ns, iPSCs with stably integrated doxycycline-inducible Ngn2
Induction - day 0
Induction - day 0
iPSCs are maintained on Geltrex in mTeSR (STEMCELL) , and passaged using 0.5 micromolar (µM) EDTA. They need to be 70-80% confluent, preferably at a low passage number when starting an induction
Coat 6-well cell culture plates or T75 flasks with Geltrex and incubate at 37 °C for 01:00:00
Prepare pre-differentiation media supplemented with 10 micromolar (µM) Rock inhibitors
Note
How to prepare 50mL Pre-differentiation media:
- 49.8 mL Knockout DMEM/F12 (Thermo Fisher)
- 500 µL 100X N2 Supplement (Thermo Fisher)
- 500 µL Non-essential amino acids (Thermo Fisher)
- 50 µL BDNF (From 10ug/mL stock)
- 50 µL NT-3 (From 10ug/mL stock)
- 50 µL Laminin (From 1mg/mL stock)
- 50 µL Doxycyclin (From 2mg/mL stock)
Wash iPSCs with PBS, add 500ul of Accutase per well and incubate at 37 °C for00:05:00 until cells have dissociated. Gently tap the plate if they are not detaching
After cells have dissociated, lift cells by adding 1mL of Knockout DMEM/F12 in well and pipetting up and down 2-3 times. Add cell suspension to a 15mL Falcon tube containing 4 mL of Knockout DMEM/F12
Spin cell suspensions at 300xG for 00:05:00
Aspirate the supernatant and resuspend the cells in 2 mL of Pre-differentiation media. Pipette gently up and down to ensure a single-cell suspension.
Count cells and prepare cell solution in pre-differentiation media. Plate 500k cells per well of a 6-well plate or 3-5 million cells in a single T75 flask.
Days 1 - 2
Days 1 - 2
Day 1: remove media and add fresh pre-differentiation media (without Rock inhibitors from this point)
Day 2: remove media and add fresh pre-differentiation media
Day 2: Prepare plates for final plating. Coat plates with PLO (100ug/mL) in Borate Buffer. Incubate plates overnight at 37 °C
Day 3
Day 3
10m
10m
Finish coating the plates: Wash 2x with ddH20. Leave to dry under the hood for 00:10:00 and coat with 100ug/mL Laminin in DMEM. Incubate at 37 °C for at least 02:00:00
10m
At this point the cells should appear more neuronal, with neurites. If cells are healthy, prepare for plating or freeze down.
Prepare neuronal media supplemented with doxycyclin (100ug/mL) and 10 micromolar (µM) Rock inhibitors
Note
How to prepare 50mL neuronal media:
- 23.4 mL DMEM/F12 (Thermo Fisher)
- 23.4 mL Neurobasal-A (Thermo Fisher)
- 500 µL Non-essential amino acids (Thermo Fisher)
- 250 µL 100X Glutamax (Thermo Fisher)
- 250 µL 100X N2 supplement (Thermo Fisher)
- 500 µL 50X B27 supplement (Thermo Fisher)
- 50 µL BDNF (From 10ug/mL stock)
- 50 µL NT-3 (From 10ug/mL stock)
- 50 µL Laminin (From 1mg/mL stock)
Wash cells with PBS and add Accutase (500 µL in 6-well plate or 4 mL in T75 flask), incubate at 37 °C for 00:05:00 until cells have dissociated
After cells have dissociated, lift cells by adding 1mL of DMEM+Neurobasal solution in well and pipetting up and down 2-3 times. Add cell suspension to a 15mL Falcon tube containing 4 mL of DMEM+Neurobasal
Spin cell suspensions at 300xG for 00:05:00
Plate cells into PLO-coated plates in neuronal media supplemented with Doxycycline and Rock inhibitors
Note
When plating, prepare a cell suspension of roughly 500k cells/mL of neuronal media. Depending on the platform used for the desired experiments (140 µL for 96-well plates, 1 mL of 12-well plates...)
Day 4 - onwards
Day 4 - onwards
Day 7: remove half of the media and add equal volume of fresh neuronal media (without Rock inhibitors and Doxycyclin from this point)
Note
The cells tend to become very peely from this point, proceed with care when handling
Day 14: remove half of the media and add equal volume of fresh neuronal media
Note
Cells are considered mature and are ready for experiments from Day 14.
Day 21: remove half of the media and add equal volume of fresh neuronal media
Day 28: remove half of the media and add equal volume of fresh neuronal media
Protocol references
Chen et al., 2020 dx.doi.org/10.17504/protocols.io.bcrjiv4n