Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid

Protocol

1. Culture of Mouse Embryonic Fibroblasts (MEFs)

1. Prepare MEF medium, Dulbecco's modified Eagle's medium (DMEM, high-glucose), supplemented with 15% fetal bovine serum (FBS).
2. Coat 100 mm cell culture dishes with 0.5% gelatin solution for 30 min at room temperature (RT).
3. Count MEFs using a cytometer. Remove the gelatin solution and immediately pour MEF medium pre-warmed to 37 °C. Rapidly thaw vials of mitomycin C-treated MEFs in a 37 °C water bath for 2 min, then seed 2.8 x 10⁶ MEFs per 100 mm gelatin-coated dish. Adjust cell number accordingly if using dishes of other sizes. Incubate MEFs overnight at 37 °C, 5% CO₂.
4. Change the medium on the next day. Culture for 2-3 days until the MEF layer is confluent.

2. Mouse Embryonic stem cell or ESC Culture

1. Prepare ESC medium, Iscove's modified Dulbecco's medium (IMDM) supplemented with 15% FBS and 10³ U/mL leukemia inhibitory factor (LIF), 0.1 mM nonessential amino acids, 55 mM 2-mercaptoethanol, penicillin (100 U/mL), streptomycin (100 µg/mL), gentamicin (200 µg/mL), and 0.2% mycoplasma antibiotic.
2. Remove MEF medium from the dish prepared in step 1 and replace it with 37 °C pre-warmed ESC medium.
3. Defrost an ESC vial and seed cells on top of the MEF layer. Incubate at 37 °C, 5% CO₂, until ESCs reach confluence.
4. Prepare new cell culture dishes containing a confluent monolayer of MEFs. To passage the ESCs, wash once with PBS and detach with 0.25% trypsin/EDTA for 2-5 min at 37 °C, 5% CO₂. Stop the trypsinization by adding fresh IMDM with 15% FBS to the detaching cells, transfer the cell suspension to a 15 mL tube, and centrifuge at 160 x g for 5 min at RT.
5. Remove the supernatant, resuspend ESCs with fresh IMDM, and passage one-fifth of the cells to each new MEF-coated dish; incubate until cells reach confluence (typically 4-5 days).

3. Withdraw MEFs and Culture ESCs on Gelatin-coated Plates

1. Once ECSs reach confluence, detach them and MEFs with 0.25% Trypsin/EDTA as in step 2.4, resuspend the cells in fresh IMDM, transfer to a non-adhesive bacteriological petri dish, and incubate for 40 min at 37 °C, 5% CO₂.
2. Carefully transfer ESCs and MEFs-containing medium to a new gelatin-coated plate using a 5 mL pipette, avoiding repeated pipetting. The cells remaining in the petri dishes are MEFs since ESCs do not adhere.
3. Passage the ESCs every 3-4 days. Less frequent passaging may reduce ESC pluripotency. Do not exceed 60% confluence, as it may favor differentiation.
4. Repeat steps 3.2-3.3 three times or more, as required, until MEFs are no longer detectable by a cell culture microscope, using a 20X objective, to make sure MEFs are not present.
5. Verify ESC pluripotency by checking ESC culture to see if the cells form dense colonies with typical ESC polygonal morphology (Figure 1A). Test cell stemness by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunoblotting, or immunofluorescence of core pluripotency transcription factors (Figure 2).

4. EB Formation

1. Prepare an all-trans-RA stock solution at 10 mM in DMSO and aliquot it into 1.5 mL light-protected microfuge tubes. The solution is stable at -80 °C for up to two weeks. Protect it from light.
2. Trypsinize ESCs as in step 2.4; replate in fresh IMDM without LIF as a single-cell suspension.
3. Count cells using a hemocytometer, and prepare a 5 x 10⁵ cells/mL suspension in IMDM with 0.5 µM RA.
4. Plate 100 20-µL-drops per 100-mm petri dish with an 8-channel pipette and 200 µL tips, invert the dishes, and fill the inverted lid with PBS to prevent hanging drops from drying. Protect RA-containing culture media from light.
5. Culture EBs in hanging drops at 37 °C, 5% CO₂, for 4 days.

Results

Figure 1: Sprout Emergence from Syx^+/+ and Syx^-/- EBS in 3D Culture. (A) Syx^+/+ and Syx^-/- ESCs grew in clustered colonies before the induction of neural differentiation. (B and C) images of EBs formed in hanging drops with 0.5 µM RA, and then inserted into a 3D collagen matrix without RA. Images were captured on day 6 by the indicated objectives (...

Materials

Name	Company	Catalog Number	Comments
Materials
MEFs	EMD Millipore	PMEF-CF	ESC feeder layer
ESC	EMD Millipore	CMTI-2
Cell culture dish (60 mm)	Eppendorf	30701119	Cell culture
Cell culture dish (100 mm)	Falcon	353003	Cell culture
Petri dish (100 mm)	Corning	351029	Hanging drops
Dark 1.5 ml centrifuge tube	Celltreat Scientific Products	229437	RA stock solution
Microscope cover-glass	Fisherbrand	12-545-80	Circular, 12 mm diameter
Superfrost-plus microscope slides	Fisherbrand	12-550-15
Reagents
DMEM	Lonza	12-709F	MEFs culture
IMDM	Gibco	12440-046	ESCs culture
Fetal bovine serum (FBS)	EMD Millipore	ES-009-B	ESCs culture
Gelatin	Sigma-Aldrich	G2625	Dish coating
LIF	R&D Systems	8878-LF-025	To maintain ESC pluripotency
MEM Non-Essential Amino Acids Solutions	Gibco	11140050	Cell culture
2-Mercaptoethanol	Gibco	21985023	Cell culture
Penicillin-Streptomycin	Gibco	15140122	Cell culture
Gentamicin	Gibco	15750060	Cell culture
MycoZap Plus-PR	Lonza	VZA-2022	Cell culture
0.25% Trypsin-EDTA	Gibco	25200-072	Cell culture
DMSO	Sigma-Aldrich	D2650
All-trans-retinoic acid	Sigma-Aldrich	R2625-50MG	Induction of neural differentiation
PBS	Gibco	10010049
Instruments
Wide-field microscope	Nikon	Eclipse TS100	Cell culture imaging