Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Summary

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Abstract

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

Introduction

ESCs originate from the blastocyst inner cell mass. These cells are pluripotent, i.e. they have the capacity to differentiate into any cell type of the organism of origin. ESC in vitro differentiation is of wide interest as an experimental system for investigating developmental pathways and mechanisms. It offers a potent and flexible model system to test new therapeutic approaches for correction of cell and tissue dysfunction. EBs recapitulate many aspects of cell differentiation during early embryogenesis. In particular, EBs can be used when embryonic lethality makes it difficult to determine the cellular basis of the embryonic defects^1,2. EBs can be formed either by the hanging drop or liquid suspension techniques³. The advantage of the former is the ability to generate EBs of consistent size and density, thus facilitating experimental reproducibility.

Interaction with extracellular matrix (ECM) adhesion proteins may affect the motility and survival of adherent cells. In the 2D culture system, fibronectin is often applied to increase cell adhesion to the substrate. Fibronectin is a basal lamina component recognized by 10 types of cell-surface integrin heterodimers⁴.

RA is a small lipophilic metabolite of vitamin A that induces neural differentiation^5,6. High concentrations of RA promote neural gene expression and represses mesodermal gene expression during EB formation^7,8. RA is produced by vitamin A oxidation to retinaldehyde by either alcohol or retinol dehydrogenase, followed by retinaldehyde oxidation to the final product by retinaldehyde dehydrogenase⁹. Neural differentiation requires transport of RA from the cytoplasm to the nucleus by cellular RA-binding protein 2 (CRABP2). In the nucleus, RA binds to its cognate receptor complex consisting of a RAR-RXR heterodimer¹⁰. This results in recruitment of transcriptional co-activators, and the initiation of transcription^9,11. Furthermore, RA promotes the degradation of phosphorylated (active) SMAD1, thus antagonizing BMP and SMAD signaling¹². In addition to these activities, RA increases Pax6 expression, a transcription factor that supports neural differentiation¹³. RA signaling is modulated by sirtuin-1 (Sirt1), a nuclear nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that deacetylates CRABP2, interfering with its translocation to the nucleus, and hence with RA binding to the RAR-RXR heterodimer^14,15,16.

Our goal in designing the RA-treated EB protocol described here is to optimize neural differentiation in order to facilitate in vitro analysis of the signaling pathways that regulate ESC differentiation into neuronal precursor cells. One of the advantages of this protocol is facilitation of the analysis of cell function by immunofluorescence. 3D EBs are not well penetrated by antibodies and are difficult to image. EB dissociation into a 2D monolayer at specific time points during neural differentiation facilitates immunolabeling and imaging of the cells by confocal microscopy.

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 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 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 reached 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)¹⁷, immunoblotting¹⁷, or immunofluorescence of core pluripotency transcription factors¹⁷ (Figure 2).

4. EB Formation

1. Prepare all-trans-RA stock solution at 10 mM in DMSO, and aliquot 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.

5. 2D EB Culture

1. Coat 12 mm circular glass coverslips with 30 µg/mL fibronectin for 30 min at RT. Place each coverslip in a well of a 24-well plate, and add 1 mL IMDM per well.
2. Harvest 3 day-grown hanging drop EBs one by one with 200 µL pipette tips and seed them on the fibronectin-coated coverslips, 20 EBs per well, using the same pipette tips.
   NOTE: 3-day EBs are preferable over 4-day EBs because they adhere better to the coverslips.
3. Continue culture with IMDM-15% FBS without LIF. Replace medium every 3-4 days. Collect the used medium for protein secretion analysis as needed.

6. Detection of Secreted Proteins

1. Transfer 500 µL of EB medium to 10 kDa-cutoff centrifugal filters, spin at 20,000 x g for 60 min at 4 °C.
2. Examine the medium remaining inside the centrifugal filters every 15-20 min to prevent excessive enrichment. Stop centrifugation when the volume of the remaining medium has fallen to 25 µL.
3. Collect the remaining concentrated medium after inverting the filter and spinning at 160 x g at 4 °C, following the manufacturer's instructions.
4. Detect the secreted proteins by immunoblotting with specific antibodies¹⁷.

7. 3D EB Culture

1. Collect the EBs grown for 4 days in hanging drops as described in step 5.2, and place them in 1.5 mL centrifuge tubes (30 EBs per tube).
2. Prepare collagen gel solution following the instructions of the 3D collagen culture kit (see Materials/Equipment Table). Dilute appropriate volumes of collagen solution and 5x DMEM (a kit component); add the neutralization solution (a kit component) and mix well immediately, and keep this on ice.
3. Pipette an appropriate volume of chilled collagen solution into the 1.5 mL tube containing the EBs, and gently transfer to the wells of a 6-well plate using a 1 mL pipette tip. Avoid making bubbles.
4. Immediately transfer the plate to 37 °C, 5% CO₂, for 60 min to initiate collagen polymerization.
5. Overlay the EB-containing plate with IMDM.
6. Change medium every 3-4 days.

8. EB Dissociation

1. Collect the day 4 EBs (from step 4) one by one, with 200 µL pipette tips and transfer them to a non-adhesive bacteriological petri dish containing IMDM with 15% FBS; culture at 37 °C, 5% CO₂ for 4 more days. Check the EBs twice every day to make sure they do not attach to the bottom; gently shake the dish to prevent EB attachment to the bottom.
2. Centrifuge the EBs at 185 x g for 5 min at RT, in a benchtop centrifuge.
3. Remove the supernatants. The pellet should not exceed 100 µL in each tube.
4. Add to pelleted EBs 1 mL 0.25% type I collagenase per tube, supplemented with 20% FBS in PBS.
5. Incubate the EB-collagenase mixture for 1 h at 37 °C, 5% CO₂, pipetting it gently every 20 min, using 1 mL pipette tips.
6. Wash the cells gently 3x with PBS. If cell aggregates are present, use a cell strainer with a 100-µm mesh to remove them.
7. Replate the cells on a gelatin-coated 60 mm dishes in IMDM. Then incubate at 37 °C, 5% CO₂.

9. Transfection of Dissociated EBs

1. For qRT-PCR and immunoblotting, coat a 6-well plate with 0.5% gelatin.
2. For immunofluorescence, coat glass coverslips with 30 µg/mL fibronectin for 30 min at RT. Place each coverslip in a well of a 24-well plate. Add IMDM.
3. Seed 4.75 x 10⁵ or 1 x 10⁵ dissociated EB cells (steps 8.4-8.7) per well in 6- or 24-well plates, respectively.
4. Grow the cells to 70% confluence before transfection.
5. Transfect the cells by a nonliposomal lipid stem-cell-optimal reagent¹⁷ (see Materials/Equipment Table), following the manufacturer's instructions.
   NOTE: The reagent we used typically reached a transfection efficiency of 50% (see Representative Results).
6. Analyze the samples by qRT-PCR or by immunoblotting¹⁷ 2 or 3 days after transfection, respectively.

10. Analysis of EB Differentiation by Immunofluorescence

1. Wash 2D EBs or dissociated 3D EBs with PBS and fix with 4% paraformaldehyde in PBS for 30 min at RT. Wash the fixed cells 3x with PBS to remove floating cell debris.
   NOTE: Paraformaldehyde is a skin and eye irritant; use caution when handling it.
2. Add 1% triton X-100 in PBS to permeabilize the cells for 20 mins at RT, then wash 3x with PBS.
3. Remove PBS and block with 5% BSA in PBS for 30 min.
4. Prepare primary antibody dilution in 1% BSA/PBS supplemented with 0.03% triton X-100. Replace the blocking solution by the primary antibody solution. Incubate for 3 h at RT, or overnight at 4 °C, then wash cells 3x with PBS.
5. Apply secondary antibody in PBS according to the manufacturer's instructions. Incubate for 1 h at RT, then wash cells 3x with PBS.
6. Mount coverslips on glass slides with an anti-fade medium for optical microscopy.

Results

Oct4, Nanog, and SOX2 are the core transcription factors that confer ESC self-renewal and pluripotency. We applied the above protocol to compare the neural differentiation of ESCs from wild type and from a strain of genetically-modified mice where Syx, a gene coding for the RhoA-specific exchange factor Syx, is disrupted. We had implicated Syx in angiogenesis¹⁸. We noticed differences in the behaviors of EBs aggregated from Syx^+/+ and <...

Discussion

In this protocol we present a relatively simple and accessible method to study neural differentiation of murine ESCs. In previous protocols, RA was added to the medium at day 2 or day 4 of the EB hanging-drop⁸ or by suspension culture⁷, respectively, or immediately after the EB hanging drop aggregation²¹. In the protocol we devised, RA was added earlier. Despite the earlier introduction of RA to EBs formed by suspension culture, this protocol produce...

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
24-well plate	Greiner Bio-One	662160	2D EBs
6-well plate	Eppendorf	30720113	Transfection
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
3D collagen culture kit	EMD Millipore	ECM675	3D culture
Effectene Transfection Reagent	Qiagen	301427	Stem cell transfection
Microcon Centrifugal Filters (10 kDa)	EMD Millipore	MRCPRT010	Protein concentration
Name	Company	Catalog Number	Comments
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
Bovine Serum Albumin	Sigma-Aldrich	A7030-50G	Blocking and antibody dilution
Triton X-100	Sigma-Aldrich	T8787-100ML	Cell membrane permeabilization
Cell strainer	Corning	352360
Prolong Gold anti-fade reagent with DAPI	Life Tech.	P36931	Mounting reagent
16% Paraformaldehyde	Electron Microscopy Sciences	15710	Cell fixation
Fibronectin	R&D Systems	1030-FN	Dish coating
PBS	Gibco	10010049
Collagenase type I	Worthington Biochem. Corp	LS004196	EB dissociation
Name	Company	Catalog Number	Comments
Primary Antibodies
Nestin (Rat-401)	Santa Cruz Biotech	sc-33677	Detection of neural differentiation
Oct4	Santa Cruz Biotech	sc-5279	Detection of neural differentiation
Nanog	Bethyl Laboratories	A300-398A	Detection of neural differentiation
Sox2	Cell Signaling	3579	Detection of neural differentiation
Tubulin b3 (AA10)	Santa Cruz Biotech	sc-80016	Detection of neural differentiation
Name	Company	Catalog Number	Comments
Secondary Antibodies
Donkey anti-Mouse-Alexa555	Life Tech.	A31570	Immunofluorescence
Donkey anti-mouse-Alexa488	Life Tech.	A21202	Immunofluorescence
Name	Company	Catalog Number	Comments
Instruments
Wide-field microscope	Nikon	Eclipse TS100	Cell culture imaging
Confocal microscope	Nikon	C2	Immunofluorescence imaging