Young-Pearse Lab Molecular Mechanisms of Neurodevelopment and Neurodegeneration
Young-Pearse Lab

Ngn2 induced Neurons

Protocol for differentiation of excitatory cortical projection neurons from transduced human ES and iPS cells with NGR viruses

 

Based on:

Rapid single-step induction of functional neurons from human pluripotent stem cells. Zhang Y, Pak C, Han Y, Ahlenius H, Zhang Z, Chanda S, Marro S, Patzke C, Acuna C, Covy J, Xu W, Yang N, Danko T, Chen L, Wernig M, Südhof TC. Neuron. 2013 Jun 5;78(5):785-98. doi: 10.1016/j.neuron.2013.05.029. PMID: 23764284

modified by Eggan lab

modified by TYP lab):

 

Making NGR lines from iPSCs:

Differentiating NGRs to inducible neuron (iN) fate:

Viral Transduction of iPS Cells with -TetO-Ngn2- Puro, TetO-GFP, Ubq-rtTA to make NGR lines:

When doing viral infections, be sure to wear sleeves to avoid contamination, dispose of tips, tubes etc. in bleach and to wipe down surface used with bleach.

Day 0

  1. Plate iPS cells in 1 well of 12 well plate with 380,000 cells per well in MeTSR media + Rocki

Day 1

  1. Collect lentivirus from -80C freezer and keep on ice. (Val’s shelf in Hera)
  1. Make-up master mix with following concentrations:

Per 12 well, in 1 ml of MeTSR media (concentrations based on our ultrapure titre >109):

I have tried using Stemflex to infect cells and it has failed! Be sure to use mTeSR media!

(FOR NGRs – do not need to use CAMKII)

 

titre

concentration

ul per 12well (380,000 cells)

pTet-O-NGN2-puro lentivrirus

2.73X10^9

0.1 ul/ 50K cells

0.7

Tet-O-FUW-eGFP lentivirus

2.05×10^9

0.05ul/ 50K cells

0.35

Fudelta GW-rtTA lentivirus

1.37×10^9

0.11ul/50K cells

0.77

CAMKII lentivirus

1.78×10^9

0.05ul/ 50K cells

0.35

Day 2

  1. Split each 12 wp to 1×10 cm plate

Days 3-7

Feed with mTeSR media everyday. When cells look confluent in 10cm dish (usually around D6 or 7), split for maintenance/expansion. Count cells! Usually should expand 1à10

NOTES: Try to freeze down cells at earlier passage #, cannot do induction past P10.

 

Protocol for generating iNs (induced neurons) from NGRs (iPSCs transduced with virus):

1- Maintain NGR cells at ~100,000 cells/cm2 in mTeSR (we use 10 cm dishes);                             

-Split cells every ~7 days

            -Feed 10 cm dishes everyday with 7 mls mTeSR media 

2- d0: Split cells and plate ~200,000 cells/cm2 with mTeSR+ 1:1000 ROCKi

            ** About 4E6 cells per 10 cm plate for differentiation plates

** If only keeping cells for maintenance, plate 2E6 and continue feeding regularly

            ** You may want to make D0 falls on Thursday or Friday so that D4 will never end          up on a weekend. 

3- On d1, feed with:

KSR media + Doxycyclin(dox) at 1:10k to induce Ngn2 and GFP expression. 

4- On d2, feed with:

1:1 ratio of KSR: N2B + Puromycin (puro) at 1:2000 to select for transduced cells, and Dox (1:10k)

** All cells not expressing NGN2/puro resistance gene should die off at this point

5- On d3, feed with:

 N2B + B27 (1:100)+ Puro (1:2000) + dox (1:10k)

6- on d4, freeze neurons!

  • Wash 10cm plate and add 2.5mls accutase per plate (1:2 accutase:PBS + Rocki)
  • Count cells!
  • Freeze down 1-2E6 cells per cryovial with 1:1 media: Freezing media
  • Media: NBM + B27 (1:50) and BDNF, CNTF and GDNF (1:1000) + ROCKi (1:1000) + Dox (1:10k) + Puro (1:2000)
  • Freezing media: 20% DMSO in FBS
  • Cells should only be in 200ul total per cryovial
  • Put in -80 for 1 night and then transfer to LN2

Important notes:

  • Passage NGR maintenance cells with accutase (Gibco, A11105) to maintain- we usually passage cells every 7 days
  • Use Accutase diluted 1:2 in PBS + 1:1000rocki
    • Accutase should never be warmed up at 37°C, this will inactivate it
  • If co-culture with glia is desired, add glia on d4 or d5 at ~70,000 cells/cm2.
    • Usually if you are looking to study synaptic expression or E-phys/MEAs
  • Further optimization of puromycin on D4 and beyond may be needed for different lines

Medias/ Product Numbers:

Reagent

Vendor

Cat no.

 [Stock]

 [Final]

Dilution

BDNF

Peprotech

450-02

10 ug/ml in 0.1% BSA/PBS

10 ng/ml

1:1000

CNTF

Peprotech

450-13

10 ug/ml in 0.1% BSA/PBS

10 ng/ml

1:1000

GDNF

Peprotech

 450-10

10 ug/ml in 0.1% BSA/PBS

10 ng/ml

1:1000

Rocki

(Y-27632) 

Stemcell Technologies

72304

5 mg

10 mM

1:1000

Other Reagents:

Reagent

Vendor

Cat no.

 [Stock]

Dilution

  

B27

Life technologies

17504-044

10mL

1:50 or 1:100

  

PBS

Invitrogen

14190-250

500 mL

    

Puromycin

Life Technologies

A11138-03

10 mg/ml

1ug/mL- 10ug/mL (each line required optimization)

  

DNASE1

Biolabs

MO30L

2000ul

    
  • CNTF/BDNF/GDNF: 10 ug vial
    • Centrifuge vial
    • Make 10 ug/ml (1000x stock) by adding 1 ml 0.1% BSA/PBS to vial. Pipette to mix. Aliquot in 100 ul volumes and store at -20oC
  • Rocki (Y-27632)
    • Stock is 10mM solution in DMSO (may vary depending on MW of batch)
    • Use at 1:1000
  • Puromycin:
    • stock is 10 mg/ml in HEPES (at -20oC in 1 ml aliquots)
    • Use at 1-10 ug/ml (=1:10,000-1:1000)
  • Doxycycline hyclate: (Sigma D9891 5g, 4oC)
    • Make 20 mg/ml stock (10000x) in sterile water
    • Aliquot in 200 ul – 1000 ul volumes and store at -20oC
    • Use at 2 ug/ml
  • 20% Dextrose
    • Make 100 ml using milliQ water + 20g D-(+)-glucose. Stir.
    • Sterile filter at store at 4oC.
  • N2 supplement B: at -20oC (100x)
  • Freezing media:
    • 8 mls FBS
    • 2 mls DMSO
    • store at -4oC
    • use 1:1 with media for freezing (usually 200 ul total per cryovial)

 

mTeSR media (1 L: 2x500mL kits)

mTeSR 800 mls (Stemcell Technologies, 05857)

mTeSR supplement 200 mls

10 mls PenStrep

Sterile filter and store in 4 oC

Human KSR media (500 mL)

415 mL KO DMEM (Gibco)

75 mL Knockout Replacement Serum (KSR Invitrogen 10828-028)

5 mL  MEM NEAA (Invitrogen, 11140-050)

0.5 mL  beta-mercaptoethanol (Invitrogen, 21985-023)

5 mL   Glutamax (Gibco, 35050)

Sterile Filter, cover in aluminum foil and store in 4 oC

N2B media (500 mL)

500 mL DMEM/F-12 (life technologies, 11330057)

5mL Glutamax (Gibco, 35050)

7.5mL 20% Dextrose

5 mL N2 supplement B (add after filtration step) (Stemcell Technologies 07156)

Sterile filter and store in 4 oC in foil

NBM (500 mL)

485 mL Neurobasal Medium (NBM) (Gibco, 21103)

5 mL Glutamax (Gibco, 35050)

7.5 mL 20% Dextrose

2.5mL MEM NEAA (Invitrogen, 11140-050)

Sterile filter and store in 4 oC

                       

Day 4 IN Plating Protocol from Thaw

  • For best results, coat Poly-O-Laminin night before and regular matrigel, day of plating for at least 1 hour.
  • Warm up re-suspension media:  NBM + 1:100 B27 + 1:1000 rocki  
  • Warm up D4 plating media: NBM + 1:50 B27 + 1:1000 rocki  + 1:1000 BDNF/ GDNF/CNTF + 1:2000 puromycin + 1:10K dox
  1. Take cells from liquid nitrogen and immediately add to beads.
  2. Take vial out of beads once ‘just-thawed’ (only about 1-2 minutes max).
  3. Recover cells, adding 800 ul NBM + 1:100 B27 + 1:1000 rocki per vial
      1. Transfer to 1 x 15 ml tube
      2. Add another 2-3 mls of NBM + 1:100 B27 + 1:1000 rocki  
      3. Take 15 ul from tube after triturating to count

% viability after thawing D4 neurons should be at least 80%.

  1. Count/spin down: 200g for 5 min.
  2. Re-suspend cells in D4 plating media

            – If plating MEAs or co-cultures, omit the puro!

  1. Usually plate at 50K density in 96wp, see chart below to plan accordingly:

Well #

Size (cm2)

5K cells / well in 96wp

12K  cells / well in 96wp

25K  cells / well in 96wp

50K  cells / well in 96wp

100K  cells / well in 96wp

96

0.30

5.01E+03

1.20E+04

2.50E+04

5.01E+04

9.99E+04

48

0.70

1.17E+04

2.80E+04

5.83E+04

1.17E+05

2.33E+05

24

2.00

3.34E+04

8.00E+04

1.67E+05

3.34E+05

6.66E+05

12

4.00

6.68E+04

1.60E+05

3.33E+05

6.68E+05

1.33E+06

6

10.00

1.67E+05

4.00E+05

8.33E+05

1.67E+06

3.33E+06

For RNA seq: plate cells at 50K cells/96wp density à usually 3.35e5 in a 24wp

For proteomics: plate cells at 1E6 in one well of a 6wp

  1. For larger wells, I have started plating cells, change media on d5 and then feed every 4/3 days (Mon/Thurs or Tues/Friday) I think this helps mitigate cells un- attaching or pulling back from wells.

Plating MEAs

First- thaw rodent astrocytes into 1xT75 flask (cells located box E6) – see plating instructions on drive.

  • Coat MEA plates with poly-o-laminin (POL) for at least 3 hours (I usually do this the night before)
  • The next day I remove the POL and add 100ul matrigel to each well.
    1. **Notes: (Corning cat #354234) we usually aliquot matrigel into 0.5 mg aliquots (based on protein concentration of vial), resuspend in 6 mls of cold, filtered DMEM/F12 and add 5 ul per well ofMEA  
  • Let matrigel sit for 1 hour. 
  • When you are then ready to plate cells, remove matrigel and wash once with PBS. 
  • We plate cells in a very low volume of media (for a 48wp – plate neurons in 30ul of media) 
  • I usually plate iNs first and then astrocytes immediately after in another 30 ul of media. I have attached our plating densities. **Note: Recently we have tried plating at an even lower density and may think that lower density is better.
    1. For a 48wp, plate 24K iNs, 24K primary rodent astrocytes  
  • After 3-4 hours (after cells have attached) I flood wells with media and bring it up to 100ul per well of 96wp or additional for larger wells. **Note: I have had contamination issues if I use too much media because of water vapor on top of lid)
  • I usually feed with Neurobasal media for the first 2 weeks (without dox and puro) and then I switch over to Brain Phys (Stemcell technologies)
    1. BrainPhys + 1x50x supplement of SM1 (do not need to add any more growth factors in
    2. Note: You could probably feed with Brainphys from the beginning but I we wait a little because it is cheaper