Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

Abstract

Source: Tischfield, D. J., et al Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors. J. Vis. Exp. (2017)

This video presents a method for generating cortical interneuron precursors from mouse embryonic stem cells. The protocol entails culturing the stem cells in media containing specific small molecule inhibitors, which facilitate stem cell proliferation and aggregation into embryoid bodies (EBs). Subsequently, the EBs are dissociated with enzymes and plated on culture plate wells coated with adhesion proteins to promote their differentiation into cortical interneuron precursors.

Protocol

1. Media Preparation

NOTE: Warm all media to 37 °C before use in cell culture.

1. Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
   1. Add 50 mL fetal bovine serum (FBS) to 449 mL of Dulbecco's Modified Eagle's Medium (DMEM), and filter through a 500 mL 0.22 µm pore filter unit.
   2. Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 °C for up to 1 month or aliquot and store at ≤ -20 °C.
2. N2 media (for preparing 500 mL)
   1. Add 5 mL L-alanine-L-glutamine (100x) and 500 µL 2-Mercaptoethanol (55 mM) to 489.5 mL DMEM: Nutrient Mixture F-12 (DMEM/F-12), and filter through a 500 mL 0.22 µm pore filter unit.
   2. Add 1 mL antimicrobial agent (50 mg/mL) and 5 mL N2 supplement-B after filtration. Store the media at 4 °C in the dark for up to 1 month.
3. Serum-free growth medium (KSR) (for preparing 500 mL)
   1. Add 75 mL serum-free medium supplement, 5 mL L-glutamine (100x), 5 mL MEM Non-Essential Amino Acids (MEM-NEAA) (100x), and 500 µL 2-Mercaptoethanol (55 mM) to 413.5 mL non-glutamine containing DMEM, and filter through a 500 mL 0.22 µm pore filter unit.
   2. Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 °C for up to 1 month.
4. Mouse embryonic stem cell media (for preparing 500 mL)
   1. Add 75 mL stem cell grade FBS, 5 mL MEM-NEAA (100x), 5 mL L-glutamine (100x), and 500 µL 2-Mercaptoethanol (55 mM) to 413.5 mL non-glutamine containing DMEM, and filter through a 500 mL 0.22 µm pore filter unit.
   2. Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 °C in the dark for up to 1 month or aliquot and store at ≤ -20 °C.

2. Culturing mESCs

1. Plate mitotically inactive MEF feeder cells in MEF media onto tissue culture-treated plates at 3-4 x 10⁴ cells/cm². Allow at least 12 hours for them to settle in a cell culture incubator at 37 °C with ≥ 95% relative humidity and 5% CO₂ before plating the mESCs. If not used immediately, replace the MEF media every 3 days. Dispose of MEFs if not used within 7 days of plating.
2. Add mESCs (density range from 1-4 x 10⁴ cells/cm²) to MEF plates in mESC media containing Mouse Leukemia Inhibitory Factor (mLIF) (1,000 U/mL). Incubate the cells at 37 °C with ≥ 95% relative humidity and 5% CO₂.
3. Passage mESCs using trypsin-EDTA (0.05% trypsin) (1:5-1:10) once the dish becomes ~ 70-80% confluent or if the colonies begin to touch. This typically occurs every 2 days but can take up to 4 or 5 days, depending on the initial plating density.
4. Maintain the mESCs on an MEF feeder layer in an mESC medium to keep pluripotency.
5. Before starting differentiation, passage the cells at least once onto gelatin-coated plates without MEFs to dilute out the MEFs.​
   1. Prepare gelatin-coated plates by adding 0.1% gelatin in phosphate-buffered saline (PBS) with Ca²⁺/Mg²⁺ to a tissue culture-treated dish and leaving at 37 °C for at least 1 h. Plate 1.5-2 x 10⁶ cells/10 cm plate (2.7-3.6 x 10⁴ cells/cm²) and allow the cells to expand for 2 days before beginning differentiation.

3. Differentiating mESCs Toward MGE-like Telencephalic Progenitors

1. Differentiation day (DD) 0 = "float cells"​
   1. After the mESCs have grown on gelatin coated plates for 2 days, aspirate the media and wash the cells once with PBS without Ca²⁺/Mg²⁺. Add enough trypsin-EDTA (0.05% trypsin) to cover the surface of the plate (typically 4 mL trypsin-EDTA for one 10 cm tissue culture dish), and place the cells back into the incubator at 37 °C with ≥95% relative humidity and 5% CO₂ for 4 min.
   2. After 4 min, quench the trypsin-EDTA using 2 times the volume with mESC media. Transfer the cells to an appropriately sized centrifuge tube and centrifuge the cells at 200 x g for 5 min. After 5 min, remove the tube and aspirate the media without disturbing the pellet. Resuspend the pellet in 1 mL KSR:N2 media (1:1) containing the BMP inhibitor LDN-193189 (250 nM) and the Wnt inhibitor XAV-939 (10 µM).
   3. Measure the cell concentration using a hemocytometer or automated cell counter. Start growing cells as embryoid bodies (EBs) by adding 75,000 cells/mL in KSR:N2 media (1:1) containing LDN-193189 (250 nM) and XAV-939 (10 µM) in non-adherent tissue culture dishes. Incubate the cells at 37 °C with ≥ 95% relative humidity and 5% CO₂.
2. On DD1, prepare for cell "landing" by coating tissue culture treated dishes with poly-L-lysine (10 µg/mL in PBS with Ca²⁺/Mg²⁺) overnight (O/N) at 37 °C with ≥ 95% relative humidity or for at least 1 h.
3. On DD2, aspirate the poly-L-lysine and coat the plates with laminin (10 µg/mL in PBS with Ca²⁺/Mg²⁺) O/N at 37 °C with ≥ 95% relative humidity.
   NOTE: While O/N is optimal, as little as 2 h may be sufficient. If plates are not used by the next day, aspirate laminin, replace with PBS without Ca²⁺/Mg²⁺, and store at 4 °C for up to 2 weeks.
4. DD3 ("land cells")
   1. Before beginning EB dissociation, aspirate the laminin and allow the plates to completely dry in a tissue culture hood. Do not use plates that appear shiny or visibly wet. Transfer the EBs with media into a 15 mL tube and centrifuge for 3-4 min at 15 x g or until the EBs have pelleted.
5. Aspirate the media and add 3 mL of cell detachment solution containing DNase (2 U/mL) and incubate at 37 °C with ≥ 95% relative humidity and 5% CO₂ for 15 min. Gently flick the tube every 3 min to aid in EB dissociation.
6. Once the EBs are no longer visible or 15 min have elapsed, add 6 mL KSR:N2 (1:1) containing DNase (1 U/mL) and centrifuge for 5 min at 200 x g.
7. Plate the cells in KSR:N2 (1:1) containing LDN-193189 (250 nM), XAV-939 (10 µM), and the ROCK inhibitor Y-27632 (10 µM) at 4.5-5 x 10⁴ cells/cm².

Materials

Mouse embryonic fibroblasts (CF-1 MEF IRR 7M)	MTI-Globalstem	GSC-6101G	1 vial of 7M MEFs is sufficient for four 10-cm TC plates.
FBS	Atlanta Biologicals	S11150H
Primocin	Invivogen	Ant-pm-2	Also known as antimicrobial agent. Do not filter with base media — add after filtration.
N2 supplement-B	Stemcell Technologies	7156	Do not filter with base media — add after filtration
Glutamax (100x)	ThermoFisher	35050061	Also known as L-alanine-L-glutamine.
KnockOut Serum Replacement (KSR)	ThermoFisher	10828028	Also known as serum-free medium supplement.
L-glutamine (100x)	ThermoFisher	25030081
MEM-NEAA (100x)	ThermoFisher	11140050
2-Mercaptoethanol	ThermoFisher	21985023
KnockOut DMEM	ThermoFisher	10829018
Hyclone FBS	VWR	82013-578	Also known as stem cell grade FBS.
Tissue culture treated dish (10cm)	BD Falcon	353003
Non-adherent sterile petri dish (10cm)	VWR	25384-342
Leukemia inhibitory factor (mLIF)	Chemicon	ESG1107	Do not freeze, store at 4'C.
DMEM/F12	ThermoFisher	11330032
0.1% Gelatin Solution	ATCC	ATCC PCS-999-027
Laminin	Sigma	L2020
Poly-L-lysine	Sigma	P6282
Trypsin-EDTA (0.05%)	ThermoFisher	25300054
Accutase	ThermoFisher	A1110501	Also known as non-trypsin containing cell dissociation reagent.
RQ1 RNase-Free DNase	Promega	M610A
LDN-193189	Stemgent	04-1974	Resuspend in DMSO and store at -80'C in single use aliquots
XAV939	Stemgent	04-1946	Resuspend in DMSO and store at -80'C in single use aliquots
ROCK inhibitor (Y-27632)	Tocris	1254	Resuspend in DMSO and store at -80'C in single use aliquots