Human ES/iPS cell NCC-SMC differentiation protocol
-------------------------------------Notes:
cell density may need to be adjusted accordingly, for each cell line tested
Differentiation Protocol:
Pre differentiation
1) Matrigel your plates/flasks 1hr in advance.
a. Add 100ul/cm2 of matrigel solution to your wells/flasks.
b. Incubate at 37C.
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Pre differentiation Day -2/-1
Harvest cells by aspirating media, rinse once with 1X PBS, accutase (50ul/cm2) cells for 3min at 37C
until cells are visibly dissociated.
Resuspend cells in a small volume (1ml/well 6w harvested) of N2B27-CDM + bFGF (20ng/ml) + Roci
(10uM/1:1000). Gently mix to create an even cell suspension by pipetting up&down once on Slow.
Count using the Countess- take a 10ul aliquot of cell suspension and add to 10ul Trypan Blue. Triturate
15X for single cell suspension. Count 2X.
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Plate cells on Matrigel (reduced growth factor)-coated plate at a density of 2 x104 cells/cm2.
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Incubate cells in 37C, 5% CO2.
Neural Priming Day -2 to Day 0
Change medium with N2B27-CDM + bFGF (20ng/ml)
Neural Crest Differentiation Day 1-5:
Cells should be ~50% confluent at Day 1.
Date
Date
Date
Day 1
100:0 (KSR:N2)
50mL:0mL
Day 2
75:25 (KSR:N2)
37.5mL:12.5mL
Day 3
50:50 (KSR:N2)
25mL:25mL
Day 4
25:75 (KSR:N2)
12.5mL:37.5mL
Day 5
0:100 (KSR:N2)
0mL:50mL
Make 500mL of KSR-NCC media
Make 500mL of N2-NCC media
SMAD inhibitors SB431542 (Stock at 10mM in DMSO) and LDN-13189 (Stock 1mM in DMSO) should be
added to the medium Days 1-5.
Add SB431542 at 2000X (5uM final conc.) and LDN-13189 at 20,000X (50nM final conc.).
NC Cell Culturing
1) Coat new plate(s) with Matrigel/KO DMEM.
2) Rinse cells once with 1X PBS (no Ca2+/Mg2+)
3) Treat cells with TrypLe (50ul/cm2). Incubate for 5-10min at 37C until cells begin to lift.
4) Quench TrypLe with 5X SFM, carefully transfer cells to 15mL/50ml falcon tube
5) Pellet cells 800rpm for 3min
6) Remove supernatant, do not disturb cell pellet
7) Resuspend cells with SFM containing bFGF (20ng/mL) and EGF (20ng/mL)
8) Count using the Countess- take a 10ul aliquot of cell suspension and add to 10ul Trypan Blue. Triturate
for single cell suspension. Count 2X.
9) Seed at ~4 x 105 cells/cm2 e.g 4x 105 cells/well of an 8-well chamber slide (0.8cm2/well) and 4 x 106cells
per well of a 6-well. Expect a lot of cell death and change medium the next day to remove dead cells.
10) Change medium every 2-3 days for maintenance.
11) (Optional) Freeze cells at 4.5x106cells per vial. Use pre-cooled (40C) 10% DMSO in FBS and
‘Mr.Freeze’ to promote viability. Avoid excessive/fast pipetting of the cells once they are in DMSO.
Transfer the cells in the Mr Freeze to the -800C freezer ASAP (do not leave cells in DMSO at room
temp for longer than 5mins if possible).
Induction of SMC fate Day 6-10:
1) Day 6, Split cells onto Fibronectin/Poly-d-lysine coated plates (1:100 from 1mg/mL stock)
2) Day 6, Replace NC media with SMCM (Lonza CC-3182); change every other day
3) Day 8, Add Myocardin-Cy3 RNA probe (1:20 dilution in sterile-PBS from stock)
4) Day 9, Sort Myocardin-Cy3-positive cells and replate on Fibronectin/poly-d-lysine coated plates
~1.5X10^5 cells/well (of 6-well)
5) Day 10 onward, check cells and perform experiments (change media every other day, and split similar to
primary SMCs)
N2B27-CDM (500mL)
470mL
DMEM/F12 (Invitrogen 11330)
5mL
N2 Supplement (100X) (Invitrogen 17502048)
10mL
B27 Supplement (Invitrogen 17504)
3.5mL
7.5% BSA Fraction V (Invitrogen 15260037)
5mL
Glutamax
5mL
MEM-NEAA
1mL
-mercaptoethanol (55mM stock)
-----------------------Filter (0.22um) and add bFGF to 50mL fresh each time
50uL bFGF (Stock at 20ug/mL, final 20ng/mL)*
* do not use bFGF for differentiation days
KSR Medium (500mL)
415mL
KO DMEM
75mL
Knockout serum replacement
5mL
Glutamax
5mL
MEM-NEAA
0.5mL
-mercaptoethanol (stock 55mM)
--------Filter (0.22um), store at 4C.
N2 Medium (500mL)
492.4mL
DMEM/F12 (Invitrogen 10565)
1.55mL
Glucose (50% stock, 0.15% final)
5mL
N2 supplement
1mL
Insulin (stock at 10mg/mL; Sigma, I9278)
2.5mL
HEPES (1M stock, 5mM final)
--------Filter (0.22um), store at 4C.
SFM
500mL
KO DMEM/F12 (Invitrogen 10565)
10mL
StemPro Neural Supplement (final 2%)
5mL
Glutamax (final 1%)
--------Filter (0.22um) and store at 4C
When ready to use, add bFGF and EGF to 50mL fresh each time
50uL bFGF (Stock at 20ug/mL, final 20ng/mL)
Other Reagents
Matrigel – growth factor reduced 5mL BD 356230
To prepare Matrigel aliquots, thaw matrigel on ice at 4C overnight.
Aliquot at 500uL into cryovials.
Store at -20C, until further use.
Thaw aliquot by adding another 500uL of KO DMEM to matrigel and slowly pipette up and down.
Add entire solution to a total of 50mL KO DMEM.
Store solution at 4C.
SB431542 (TGF inhibitor) Tocris 1614, 10mg
To prepare 52, 50uL, 10mM aliquots:
MW of SB is 384.39 g/mol
Add 2.6mL of sterile DMSO
Aliquot 50uL into sterile epi tubes.
Store at -20C.
Use within one month after thawing.
LDN-193989 (BMP type I inhibitor) Stemgent 04-0074, 2mg
To prepare 180, 25uL,1mM aliquots:
MW of SB is 442.94 g/mol
Add 4.5mL of sterile DMSO
Aliquot 25uL into sterile epi tubes.
Store at -20C.
Use within one month after thawing.
bFGF Peprotech 100-18B 100ug
To prepare 20 aliquots at 250uL, stock 20ug/mL in PBS
Add 5mL of PBS.
Aliquot 250uL stocks.
Store at -20C.
Use within one month after thawing.
Roci (10mM, 1000X stock) from GSCC