Jan 29, 2024

Public workspaceCortical neuron differentiation using forced NGN2 expression

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvj3wwwlk5/v1
  • Beatrice Weykopf1,2
  • 1Stephen and Denise Adams Center for Parkinson's Disease Research of Yale School of Medicine;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Beatrice Weykopf
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvj3wwwlk5/v1
Protocol Citation: Beatrice Weykopf 2024. Cortical neuron differentiation using forced NGN2 expression. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3wwwlk5/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 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94363
Keywords: ASAPCRN, cryopreserved ngn2 neuron, ngn2 neuron, forced ngn2 expression, cortical neuron differentiation, ngn2 expression, inducible ngn2 cassette
Abstract
This is a modified protocol based on (Zhang et al.2013 and Meijer et al., 2019) to generate cryopreserved NGN2 neurons, using hPSCs that carry a doxycline-inducible NGN2 cassette.

The voluming are calculated for 10cm TC dishes and should be adapted accordingly

Materials
Media:
KSR medium
500ml250ml50mlReagent
415ml207.5ml41.5mlKO DEMEM
75ml37.5ml7.5mlKO SRM
5ml2.5ml0.5mlGlutaMax, NEAA, P/S
0.5ml0.25ml0.05mlß-mercapto ethanol
 
N2B medium
500ml250ml50mlReagent
480ml240ml48mlDMEM F12 + HEPES
5ml2.5ml0.5mlGlutaMax, N2, P/S
7.5ml3.75ml0.75ml20% Dextrose
D2 N2B w/o B27 supplement
D3 N2B 1:100 B27 supplement
NBM medium
500ml250ml50mlReagent
485ml242.5ml48.5mlNeurobasal
5ml2.5ml0.5mlGlutaMax, P/S
7.5ml3.75ml0.75ml20% Dextrose
10ml5ml1mlB27 supplement
2.5ml1.25ml0.25mlNEAA
 Consumables:             For preparation of Growth factors check Cell culture recipes sheet

ABCDEF
ReagentVendorCat no.Stock con.Working conc.Dilution
Neurobasal mediumThermofisher Scientific#21103049n.a.n.a.n.a.
Knock DMEMGibco #10829-018n.a.n.a.n.a.
KnockOut Serum Replacement Invitrogen10828-028 n.a.n.a.n.a.
MEM non-essential amino acids Invitrogen11140-050n.a.n.a.n.a.
Beta-mercaptoethanol Invitrogen21985-023n.a.n.a.n.a.
GlutaMaxInvitrogen35050061n.a.n.a.n.a.
DMEM F12 HEPESGibco11330057n.a.n.a.n.a.
N2 supplementInvitrogen17502048n.a.n.a.n.a.
20% Dextrose  20%n.a.n.a.
Penicillin/StreptomycinInvitrogen15140122100x1x1:100
TrehaloseSigma#T9531-25Gn.a.n.a.n.a.
DMSOSigma#D8418-250Mn.a.n.a.n.a.
mTeSR plusStem cell Technologies# 100-0276n.a.n.a.n.a.
Matrigel (Differentiation)Corning354234n.a.n.a.n.a.
DnaseNEB#M030n.a.n.a.n.a.
BDNFPeprotech#450-0210µg/ml10ng/ml 
GDNFPeprotech#450-1010µg/ml10ng/ml 
CNTFPeprotech# 450-1310µg/ml10ng/ml 
DAPTFisher Scientific#26-341-010mM10µM 
Doxycyclin HyclateSigma D9891-5g 2mg/ml2µg/ml 
PuromycinLife technologyA11138-0310mg/ml  
AccutaseGibco#A11105   
Y27632 ROCK Inh.Stem cell technologies#7230410mM10µM 
Cell strainer (40µm)Falcon/Corning#352340n.a.n.a.n.a.
B27 SupplementInvitrogen17504044n.a.n.a.n.a.

Troubleshooting
Overview

Differentiation overview

D0
Seed 4x106 single cells onto 1x10 cm dish (coating MG-GFR or with GF 2mg) in iPSC medium supplemented with 10 µM RI in 7ml medium
D1
Add 8ml KSR with 2µg/ml DOX
D2
9ml 1:1 KSR/N2B w/o B27 2µg/ml DOX and 5µg/ml Puro

Ensure 24h window between this media change and the previous one
Critical
D3
10ml N2B  containing 2µg/ml DOX) and 5µg/ml Puro
D4
10 ml NBM containing 2µg/ml DOX, 5µg/ml Puro, BDNF 10µg/ml , GDNF 10µg/ml , CNTF 10µg/ml
D5
10 ml NBM containing 2µg/ml DOX, 5µg/ml Puro, BDNF 10µg/ml , GDNF 10µg/ml , CNTF 10µg/ml and 10µM DAPT
D6 AraC treatment
10 ml NBM 2µg/ml DOX, 5µg/ml Puro, BDNF 10µg/ml, GDNF 10µg/ml, CNTF 10µg/ml, 10 µM DAPT
add 3 µM AraC varies between cell lines

! Discard AraC trash according to environmental health guidelines!
D7 AraC treatment
10 ml NBM 2µg/ml DOX, 5µg/ml Puro, BDNF 10µg/ml, GDNF 10µg/ml, CNTF 10µg/ml, 10 µM DAPT
add 3 µM AraC varies between cell lines

! Discard AraC trash and used medium according to environmental health guidelines!
D8
2x wash with 5ml NBM w/o GF to remove AraC completely
add 8-10 ml NBM 2µg/ml DOX, 5µg/ml Puro, BDNF 10µg/ml, GDNF 10µg/ml, CNTF 10µg/ml and
10 µM DAPT
! Discard AraC trash and used medium according to environmental health guidelines!
D9
add 8-10 ml NBM 2µg/ml DOX, 5µg/ml Puro, BDNF 10µg/ml, GDNF 10µg/ml, CNTF 10µg/ml and
10 µM DAP
D10 Cryopreservation
remove medium
1x wash with Amount4 mL l PBS

add Amount4 mL Accutase supplemented with 10µM RI for 90-120min atTemperature37 °C

After incubation time; If needed add Amount200 µL DNAse I

Add Amount3 mL NBM and dissociate using 10 ml serological pipette

Pool 3x10cm dishes and collect cells in 1x50 ml falcon after pipetting through a 40µm cell strainer on Ice

!collect all cells in a T175 TC falsk on ice!
Rinse dishes with 3 ml NBM
Take a sample from the flask to determine cell number
!Always store cells suspension on ice from now on!
Aliquot pooled cells into 15 ml falcons and centrifuge at 300g for 5min at 4˚C
Freeze cells in 1.5M or 3M aliquots and store at -80˚C overnight and transfer the next day into the liquid nitrogen tank

Freezing medium:
70% KOSRM
20% 1M Trehalose
10% DMSO