Jan 22, 2025
JAX_DPC: Protocol to produce human dorsal forebrain organoids
Forked from a private protocol
- anahita.amiri1,
- Cole Lorig1,
- Patrick Fleming1,
- Juliana Alcoforado Diniz1,
- Flora Vaccarino2,
- Paul Robson1,3
- 1The Jackson Laboratory for Genomic Medicine;
- 2Yale University;
- 3UCONN Health
- MorPhiC Consortium
Protocol Citation: anahita.amiri, Cole Lorig, Patrick Fleming, Juliana Alcoforado Diniz, Flora Vaccarino, Paul Robson 2025. JAX_DPC: Protocol to produce human dorsal forebrain organoids. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxw1ddv8j/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 16, 2025
Last Modified: January 22, 2025
Protocol Integer ID: 118580
Funders Acknowledgements:
JAX MorPhiC DPC
Grant ID: 1 UM1 HG012651-01
Abstract
Brain organoid models have enormous potential for investigating normal fetal brain development such as cell fate mapping, lineage analysis, and evolution as well as disease modeling, drug screening and are potential source for regenerative medicine. Here we describe a detail protocol for generating human dorsal forebrain organoids. This protocol has been modified from Rigamonti et al 2016.
Materials
MATERIALS
mTeSR plus (STEMCELL technologies)
KnockOut Serum Replacement (ThermoFisher Scientific, 10828028)
KnockOut DMEM (Thermo
Fisher Scientific, 10829018)
Neurobasal Medium (Gibco, 21103049)
L-glutamine (Gibco, 25030081)
MEM non-essential amino acids (NEAA) (Gibco),
penicillin-streptomycin (Gibco),
b-mercaptoethanol (Gibco, 21985023)
N2 supplement (100X) (ThermoFisher, 17502048)
B27 supplement without vitamin A 5OX (ThermoFisher, 12587010)
D-(+)-Glucose solution (Sigma, G8644)
Glutamax (100x) (Gibco, 35050-061)
HEPES pH7.4 (Fisher, 15630080)
BDNF (R&D Systems, 248-BDB-010)
GDNF (R&D Systems, 212-GD-010)
Accutase (StemCell Technologies, 7922)
Reagents and Consumables:
Reagents and Consumables:
mTeSR plus (STEMCELL Technologies 100-0276
KnockOut‱ Serum Replacement (ThermoFisher Scientific, 10828028)
KnockOut‱ DMEM (Thermo Fisher Scientific, 10829018)
MEM non-essential amino acids (Gibco, 11140-050)
L-glutamine (Gibco, 25030081)
b-mercaptoethanol (Gibco, 21985023)
penicillin-streptomycin (ThermoFisher, 15070063 )
DMEM/F12 (ThermoFisher, 11330032)
N2 supplement (100X) (ThermoFisher, 17502048)
B27 supplement without vitamin A 5OX (ThermoFisher, 12587010)
Glutamax (100x) (Gibco, 35050-061)
D-(+)-Glucose solution (Sigma, G8644)
Neurobasal‱ Medium (Gibco, 21103049)
HEPES pH7.4 (Fisher, 15630080)
Accutase (StemCell Technologies, 7922)
Y-27632 (Stemcell Technologies, 72307)
SB 431542 (Tocris, 1614)
LDN193189 (Stemcell Technologies, 72149)
XAV939 (Selleck, S1180)
FGF2 (R&D Systems, 233-FB)
EGF (R&D Systems, 236-EG-01M
BDNF (R&D Systems, 248-BDB-010)
GDNF (R&D Systems, 212-GD-010)
Media preparation
Media preparation
mTeSR plus:
Add mTeSR supplement to mTeSR basal media.
Store in 40 mL aliquots at -20C.
Keep active aliquots in 4C.
KSR medium (50ml):
KnockOut DMEM (41 ml)
KnockOut Serum Replacement (7.5 ml-final 15%)
MEM non-essential amino acids (0.5 ml)
L-glutamine (0.5 ml)
b-mercaptoethanol (50 ul-final 55uM)
Penicillin/Streptomycin Solution (0.5 ml)
Neural induction (NIM) medium (50 ml):
DMEM/F12 (46.25 ml)
N2 supplement (0.5 ml-final 1%)
B27 supplement without vitamin A (1 ml-final 2%)
Glutamax (0.5 ml-final 1%)
MEM non-essential amino acids (NEAA) (0.5 ml-final 1%)
D-(+)-Glucose solution (0.75 ml-final 0.15%)
Penicillin/Streptomycin Solution (0.5 ml)
Terminal differentiation (TD) medium (50 ml):
Neurobasal Medium (46.75 ml)
N2 supplement (0.5 ml-final 1%)
B27 supplement without vitamin A (1 ml-final 2%)
Glutamax (0.5 ml-final 1%)
MEM non-essential amino acids (NEAA) (0.5 ml-final 1%)
HEPES (0.75 ml-final 15mM)
b-mercaptoethanol (50 ul-final 55uM)
Penicillin/Streptomycin Solution (0.5 ml-final 1%)
Procedure:
Procedure:
Grow hiPSCs in a well of a 6-well plate. Start when they are about 80% confluent.
Maintain healthy hiPSCs on matrigel-coated plates. Passage iPSCs using dispase. Remove any spontaneous differentiated cells.
Lifting iPSCs (day 1)
Prepare 5 ml of mTeSR plus medium supplemented with 5μM Y-27632.
Wash iPSCs (70-80% confluent) with PBS.
Add 1 mL pre-warmed PBS:Acc (1:1) to each well of a 6-well plate. Incubate at37°C for 10 minutes.
Add 1 ml mTeSR plus to dilute accutase. Transfer cells to a 15mL conical tube. Use 1 mL, DPBS to wash/collect remaining cells from plate into conical tube.
Centrifuge at 250x g for 5 minutes. Aspirate supernatant.
Resuspend cell pellet in 1mL mTeSR+5μM Y-27632. Pipette 4-5x.
Pipetting aggressively would damage iPSCs.
Count cells. Add 3-4x106 cells/well and add up to 4mL mTeSR+5μM Y-27632.
Place the plate on an orbital shaker (95rpm) in a humidified tissue
culture incubator at 37 °C and 5% CO2.
iPSCs in suspension (day2-3)
Day 3- Media change with mTeSR plus (no Y-27632).
Neural induction (day 4-10)
Day 4: media change with mTeSR plus supplemented with 10μM SB431542, 1μM LDN193189.
Day 5-7: media change with 100% KSR medium supplemented with 10μM SB431542, 1μM LDN193189 and 2μM XAV939.
Day 8: media change with 75% KSR and 25% NIM (neural induction medium) supplemented with 10μM SB431542, 1μM LDN193189.
Day 10: media change with 50% KSR and 50% NIM supplemented with 10μM SB431542, 1μM LDN193189.
Day 12: media change with 25% KSR and 75% NIM medium
NPC (neural progenitor cell) proliferation
Day 14-19: media change with 100% NIM medium supplemented with FGF2 (10 ng/ml) and EGF (10 ng/ml). media change every other day.
Terminal differentiation (TD)
Day 20-120: media change with TD media supplemented with 10 ng/ml BDNF and 10 ng/ml GDNF. Change media every 3-4 days.