Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay

Summary

We present a three-dimensional (3D) in vitro differentiation protocol generating neurospheres of reproducible size to produce cranial neural crest cells from mouse embryonic stem cells. We show that this methodology reduces variability compared to previous protocols and how it can be used for multiplexed assay to study cranial neural crest cell development.

Abstract

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest in studying the mechanisms regulating cell fate decisions and plasticity. Originating in the dorsal neuroepithelium, this cell population is transient and relatively rare in the developing embryo - making functional tests, genomic screens, and biochemistry assays challenging to perform in vivo. To overcome these limitations, several methods have been developed to model CNCC development in vitro. Neurosphere (NS) based culturing methods provide a complex microenvironment that recapitulates the developing anterior neuroepithelium in 3D. These systems allow the growth of many NS in the same plate to generate a large amount of CNCC, but the produced NS present a high variability in shape, size, and number of CNCC formed - making quantitative assays difficult to perform. This protocol outlines a reproducible method for generating NS from mouse embryonic stem cells (mESC) in a 96-well format. NS generated in 96-well plates produce cranial neural crest cells (CNCC), which can be further cultured. By controlling the number of starting cells, this approach reduces variability in the size and shape between NS and increases reproducibility across experiments. Finally, this culture system is adaptable to several applications and offers a higher degree of flexibility, making it highly customizable and suitable for multiplexing experimental conditions.

Introduction

Cranial neural crest cells (CNCC) are a stem-like cell population that arises in the anteriormost part of the developing embryo, at the border between the neural plate and the surface ectoderm¹. CNCC then undergo an epithelial-to-mesenchymal transition (EMT), delaminate from the neuroepithelium, and migrate dorsoventrally towards various locations in the embryo where they differentiate into a wide variety of cell types². Studying this cell population is of great interest as it possesses a remarkable plasticity³ and the unique ability to differentiate into both ectodermal and mesenchymal derivative....

Protocol

1. Generation of a single-cell suspension from mouse ESC colonies

NOTE: This protocol is adapted to the use of CK35 mESC (an mESC line competent for germ line transmission, to have then the option to develop in vivo models¹⁵) grown on inactivated feeders in a gelatin-coated TC-treated 6-well plate. One well of a TC-treated 6-well plate should yield approximately 1.5 × 10⁶ mESC, which is sufficient for the rest of the protocol. This can be scaled up if necessary. Adjust the initial steps in accordance with the chosen ESC strain and maintenance culture method, as well as the prop....

Results

Following the protocol, mESC colonies were dissociated, and 3000 cells were seeded in non-TC treated U-bottom 96-well plates. On day 2, aggregated NS were transferred into non-TC treated flat-bottom 96-well plates to allow them to attach. A simplified visualization of the NS aggregation protocol is provided in Figure 1A. NS were cultured until day 9 and then processed for immunofluorescence staining. Cells that migrated from the NS onto the plate were transferred to coverslips for imaging an.......

Discussion

In vitro 3D differentiation models allow analyzing complex cell interactions that could be difficult - or could not - be observed in 2D cell culture. Several models have been developed to study CNCC development in vitro. These are generally directly derived from ESC colonies^7,21 or tissue explants^22,23. Although these systems have proven efficient for generating neural crest cells, such .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm syringe filters	ClearLine	146560
15 mL High-Clarity Polypropylene Conical Tube	Falcon	352096
200 µL ClearLine Plus Low Binding Filter Tips	Dutscher	713263
40 µm filters	Falcon	352340
5 mL Serological pipette	Starstedt	86.1253.001
50 mL High-Clarity Polypropylene Conical Tube	Falcon	352070
Accutase	Merck-Sigma	A6964
Alexa Fluor 488 donkey anti rabbit IgG (H+L)	Thermofisher Scientific	A21206
Alexa Fluor 594 donkey anti mouse IgG (H+L)	Thermofisher Scientific	A21203
Alexa Fluor 647 donkey anti goat IgG (H+L)	Thermofisher Scientific	A31571
Antibiotic-antimycotic solution	Merck-Sigma	A5955
B27 PLUS supplement	Thermofisher Scientific	17504044
Bovine serum albumin (BSA)	Merck-Sigma	A9418
Chloroform	Carlo Erba	438601
Collagenase Type IV	Thermofisher Scientific, Gibco	17104019
Costar 6 well clear TC-treated multiple well plates	Corning	3516
Cover glasses, round	VWR	630-2113
DMEM KnockOut	Thermofisher Scientific	10829018
DMEM/F12+Glutamax	Thermofisher Scientific	10565018
DMEM high glucose	Merck-Sigma	D0822
DNA LoBind Tubes, 2 mL	Eppendorf	30108078
DNase/RNase-Free Distilled Water	Thermofisher Scientific	10977-035
Dulbecco’s Phosphate Buffered Saline (PBS)	Thermofisher Scientific	14190144
Eppendorf Safe-Lock Tubes, 0.5 mL	Eppendorf	30121023
Eppendorf Safe-Lock Tubes, 2 mL	Eppendorf	30120094
ESGRO mLIF Medium Supplement	Merck-Sigma	ESG1107
Ethanol 70%	Carlo Erba	528170
Fetal Bovine Serum	Merck-Sigma	F7524
Fibronectin	Merck-Sigma	F085-2MG
Fluoromount-G	Invitrogen	00-4958-02
Gelatin solution	Merck-Sigma	ES-006-B
GlutaMAX	Thermofisher Scientific	35050061
Human EGF	Peprotech	AF-100-15-500UG
Human FGF-basic	Peprotech	100-18B
Human SOX9 Antibody	R&Dsystems	AF3075
Insulin from bovine pancreas	Merck-Sigma	I6634
iScript cDNA Synthesis Kit	Biorad	1708891
Mouse Anti-Human AP-2 alpha Monoclonal Antibody, Unconjugated	DSHB	3B5
Mouse Anti-Human PAX7 Monoclonal Antibody, Unconjugated	DSHB	PAX7
N2 supplement	Thermofisher Scientific	17502048
Neurobasal Medium	Thermofisher Scientific	21103049
Non-Tissue culture treated plate, 96 well, Flat bottom	Falcon	351172
Non-Tissue culture treated plate, 96 well, U-bottom	Falcon	351177
Paraformaldehyde 16% solution, em grade	Electron Microscopy Sciences	15710
Propan-2-ol	Carlo Erba	415154
Purified anti-Tubulin β 3 (TUJ1) Antibody	Biolegend	MMS-435P
RapiClear 1.47	Sunjin Lab	RC147001
RapiClear 1.52	Sunjin Lab	RC152001
Scotch Double Sided 12.7 mm × 22.8 m			Clear fibreless double sided tape
SensiFAST SYBR No-ROX Kit	Meridian Bioscience	BIO-98020
Sterile Disposable Surgical Scalpels	Swann-Morton	05XX
Superfrost Plus Adhesion Microscope Slides	Epredia	J1800AMNZ
Triton X-100	Thermofisher Scientific	A16046.AP
TRIzol Reagent	FisherScientific	15596026
Trypsine-EDTA (0.05%)	Thermofisher Scientific	25300054
Tween-20	Fisher Scientific	10113103
TWIST1 Rabbit mAb (IF Formulated)	Cell signaling technology	E7E2G
β-mercaptoethanol	Thermofisher Scientific	31350010