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

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 ....

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

List of materials used in this article

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