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 derivatives, such as craniofacial bones and cartilages⁴. Although CNCC are relatively accessible in the embryo, they are a transient population with a low number of cells, making systemic mechanistic studies difficult to conduct in vivo. CNCC cell lines have been isolated and characterized in the last few years to overcome these limitations. In particular, the O9-1 CNCC cell line is a great tool for studying migratory and post-migratory neural crest development^5,6; however, this cell line does not allow the study of the early events prior to migration leading to neural crest induction and specification. In this regard, there have been significant developments in the development of in vitro differentiation protocols to differentiate CNCC in a dish via the use of 3D structures resembling the developing neuroepithelium called neurospheres (NS)^7,8- obtained after differentiation of embryonic stem cell (ESC) colonies. These 3D protocols robustly produce high numbers of CNCC, allowing the conduct of biochemical and genomic mechanistic studies^9,10. NS are cultured on low attachment plates in N2B27 supplemented medium, together with Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF)^10,11 to stimulate cell proliferation. These protocols are carried out in Petri dishes, cultivating numerous NS in the same plate. Within the growing NS, cells aggregate and continue to divide - reaching a diameter of 100-200 µm upon maturity. At maturity (about day 5), NS attach to the substrate and differentiate into CNCC resembling their in vivo counterparts^9,12. These CNCC then undergo EMT and delaminate onto the plate surface. Morphological differences can be observed depending on NS size, as larger spheres will appear darker in the core due to lower availability of nutrients and oxygen, leading to cells undergoing apoptosis¹³. While this type of procedure generates a large number of CNCC at the endpoint of differentiation, it presents several limitations, making the study of the various molecular dynamics occurring during the differentiation process nearly impossible. First, the use of ESC colonies - which vary in size - makes it difficult to control the starting cell number for each experiment. This results in the generation of NS of various shapes and diameters that develop differently by activating specific signaling pathways, leading to altered cell differentiation and, thus not forming a uniform sample at a given time point. Second, culturing multiple NS in the same plate often leads to them fusing together¹⁴ and potentially releasing signaling molecules that influence their neighbors' microenvironment and, thus, their development. Altogether, these procedures generate a lot of variability between samples and experiments.

Here, we present a strategy to overcome these difficulties that generate single NS - capable of producing CNCC - by aggregating mouse ESC (mESC) in non-TC treated U-bottomed 96-well plates. Starting from mESC allows studying the specification process and early stages of CNCC development compared to starting from already established neural crest cell lines. This protocol begins with the disaggregation of mESC colonies to obtain a single cell suspension, followed by the seeding of a specific number of mESC in each well of a non-TC treated U-bottomed 96-well plate. The cells are left to aggregate for two days and subsequently moved to a non-TC treated flat-bottomed 96-well plate, in which NS will be able to attach to the plate bottom. By controlling the starting cell number and the microenvironment of each NS during the differentiation process, this protocol reduces sample variability, which increases experimental reproducibility. We believe this will be a convenient platform for designing multiplexed experiments, such as testing the effect of different culture conditions or performing gene perturbation screens.

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 proper culture medium. This protocol is to be performed under sterile conditions. See the Table of Materials for details related to all the materials, reagents, and equipment used in this protocol.

1. Start from mESC at a 70%-80% confluence grown in mESC culture medium. See Table 1 for the mESC culture medium composition used in this study.
   NOTE: Do not let mESC grow over 80% confluence, as they will start to differentiate, and this will affect the aggregation process. Colonies must be compact and show a healthy morphology (no cracks or waves, distinct nuclear-cytoplasm contrast).
2. Prepare CNCC differentiation medium. See Table 1 for CNCC differentiation medium composition.
   NOTE: Once growth factors are added, the medium can be stored for up to 3 weeks at 4 °C. Ensure the medium is protected from light.
3. Prepare a fresh collagenase solution at a concentration of 2 mg/mL in the DMEM-Knockout medium.
   NOTE: Use of collagenase ensures only the colonies are detached and not the feeders, as their presence in the following steps will interfere with NS aggregation. Filter collagenase solution before use with a 0.22 µm filter.
4. Aspirate the ESC medium from mESC.
5. Gently add 1 mL of PBS to the side of the well. Rock the plate gently to ensure even washing.
6. Remove PBS and replace with 2 mL of collagenase solution. Incubate at 37 °C for 30-45 min.
   1. Check the plate under a light microscope at 10x magnification after the first 20 min and then every 5 min.
   2. When the colonies show rolled-up edges, tap vigorously on the plate side, and the colonies will detach.
7. With a 5 mL serological pipette, collect the colonies and transfer them to a 15 mL conical tube.
   NOTE: Check the plate under a light microscope for leftover colonies. These can be collected with a PBS wash.
8. Centrifugate the colonies at 16 × g for 3 min at room temperature (RT).
9. Aspirate as much medium as possible from the conical tube, being careful not to disturb the colonies at the bottom of the tube.
10. Add 1 mL of 0.05% trypsin solution and incubate the tube at 37 °C for 5 min.
11. Dissociate colonies in the tube by pipetting vigorously up and down, first with a p1000 and then with a p200 micropipette.
    NOTE: This ensures the obtention of a single-cell suspension.
12. Add 2 mL of mESC culture medium to block the trypsin, and centrifugate at 160 × g for 3 min at RT. Remove the supernatant and add 1 mL of CNCC differentiation medium.
13. Count cells using an automated cell counting device or a standardized system under the microscope, following manufacturer instructions.
14. After counting, dilute with sufficient CNCC differentiation medium to obtain a concentration of 3000 live cells per 50 µL.
15. Using a p200 micropipette, seed 50 µL of the cell suspension in each well of a non-TC treated U-bottom 96-well plate, then top up the well until 200 µL with CNCC differentiation medium.
    NOTE: While filling the plate, resuspend from time to time the single cell suspension with a p1000 micropipette to obtain a homogeneous concentration.
16. Incubate overnight in an incubator at 37 °C, 5% CO₂.

2. Transferring into a flat-bottom 96-well plate for CNCC differentiation

1. The next day (day 1), observe the plate under a light microscope. Ensure one small cell cluster with clear borders is visible at the bottom of each well. Put the plate back in the incubator overnight.
   NOTE: There may be some cells around the main aggregate, some dead. This will not interfere with NS aggregation.
2. On day 2, slowly remove 100 µL of medium from each well.
   1. When aspirating medium, be careful not to remove the NS. To avoid doing so, place the pipette tip close to the surface and far from the bottom.
3. Cut the tip of a p200 micropipette at around 3-4 mm from the tip. Use this to aspirate the NS with the remaining medium.
   NOTE: To facilitate picking up the NS, gently pipette up and down a couple of times before aspirating.
4. Transfer NS and the remaining medium into a non-TC treated flat-bottom 96-well plate and verify the transfer under a light microscope, then top each new well with 100 µL of prewarmed CNCC differentiation medium. Leave NS in the incubator at 37 °C, 5% CO_2, until day 4.
5. On day 4, remove 100 µL of medium from each well and replace it with 100 µL of prewarmed CNCC differentiation medium.
   NOTE: To avoid disturbing NS attachment at the bottom of the plate, slowly aspirate and replace medium.
6. On days 5 and 6, check the NS attachment under the light microscope. Ensure lighter cells - delaminating from the NS -start surrounding the main body of the NS.
7. On day 7, change the medium as described in 2.5. Change the medium with the same procedure every 2 days until the endpoint of the study.

3. CNCC passaging and maintenance

NOTE: CNCC passaging can be performed as soon as there is a sufficient quantity of cells visible around NS. This can be as early as day 7, as earlier time points do not provide a sufficient amount of CNCC.

1. Prepare CNCC Maintenance medium. See Table 1 for CNCC Maintenance medium composition.
   1. Before adding it to the medium, filter BSA after solubilization through a 0.22 μm filter. Once growth factors are added, store the medium for up to 3 weeks at 4 °C. Ensure the medium is protected from light.
2. Prepare fibronectin at 7.5 µg/mL in PBS. Mix vigorously.
3. Coat the wells of a non-TC treated 96-well plate by adding 100 μL of fibronectin solution per well. Let coat under the hood for 30 min at RT.
   NOTE: If CNCC are meant for immunofluorescence staining, place a sterile glass coverslip at the bottom of the well before coating. Ensure that the slide stays at the bottom of the well and does not float to avoid coating the opposite side to the one that will be used. Follow the instructions in section 5 for CNCC fixation and mounting.
4. In the meantime, in the non-TC treated flat-bottom 96-well plate, aspirate as much CNCC differentiation medium as possible from the wells and substitute with 50 µL accutase. Incubate at 37 °C for 5 min.
5. After incubation, add 100 µL of CNCC maintenance medium per well to quench the accutase. Remove fibronectin from the wells of the receiving non-TC treated flat-bottom 96-well plate. Filter the detached post-migratory CNCC by passing them through a 40 µm filter on top of the well of the receiving non-TC treated flat-bottom 96-well plate.
   NOTE: This will filter out cell clumps or remaining NS that have not been previously removed. CNCC grown in the same condition can be pooled in one 50 mL tube to be then seeded in the same well of a TC-treated 6-well plate. In this case, coat the well with 1 mL of fibronectin and use 1 mL of CNCC maintenance medium to quench the accutase.
6. Let post-migratory CNCC attach for 15-30 min at 37 °C. Discard medium and replace with 100 µL of CNCC maintenance medium. Change medium every 2 days.
   NOTE: Gently add medium as fast flow induces differentiation of CNCC into neural derivatives. If working in a 6-well format, use 1 mL of CNCC maintenance medium.

4. NS fixation and mounting for immunofluorescence

1. At the desired time point, transfer NS from the non-TC treated flat-bottom 96-well plate into a DNA low binding 2 mL tube by cutting the tip of a p200 micropipette and gently pipet up and down to pick up the NS.
   NOTE: At later time points (day 7 onwards), NS will be big enough to be seen by the eye but also will be harder to detach.
2. Let NS settle in the tube for 3 min at RT, remove as much medium as possible, and rinse with 1 mL of cold PBS.
   NOTE: If transferring small NS from early time points (before days 3-4), spin at 16 × g for 3 min to ensure NS settle to the bottom.
3. Remove PBS and, in a chemical hood, replace with 2 mL of 4% PFA in PBS. Incubate for 20 min at RT.
   1. Invert the tube slowly after adding 4% PFA solution and let it rest during fixation on the side. The goal is to spread NS slightly so they do not stick together in this step.
4. Remove 4% PFA solution (in the chemical hood) and wash NS with 1 mL of cold PBS/0.5% Tween20. Let it settle at RT for 3 min. Repeat this 3 times in total.
   NOTE: NS will settle at the bottom.
5. Remove PBS/0.5% Tween20 and add 2 mL of PBS/0.1% Triton X-100. Invert the tube and let it rest on the side. Incubate for 1 h at RT.
6. Wash NS 3 times in cold PBS/0.5% Tween20, as indicated in step 4.4.
7. Remove PBS/0.5% Tween20 and block with 2% BSA in PBS at 4 °C for a minimum of 1 h, preferably overnight.
   NOTE: Samples can be stored in 2% BSA/PBS for up to 1 week at 4 °C. Protect from light.
8. Prepare primary antibody solution by adding the correct dilution of primary antibody to 2% BSA/PBS in a final volume of 500 µL. For the primary antibody mix used in this study, see Table 2.
9. Remove as much of the blocking solution as possible and transfer the NS to a 0.5 mL tube by cutting the tip of a p200 micropipette.
10. Add the primary antibody solution and pipette up and down slowly to resuspend the NS. Incubate overnight on a rotator at 4 °C.
11. Wash NS 3 times in cold PBS for 5 min at RT.
12. Prepare the secondary antibody solution by diluting the secondary antibodies of choice in 2% BSA/PBS and adding 1/1000 DAPI. For the secondary antibody mix used in this study, see Table 2.
    NOTE: Keep the tubes protected from light.
13. Replace PBS with 500 µL of secondary antibody solution and wrap the tube with aluminum foil to protect it from light. Incubate for 1 h on the rotator at RT.
14. Wash NS 3 times in cold PBS, as indicated in step 4.11.
15. Remove as much PBS as possible without aspirating the NS, substitute with 50 µL of clearing agent, and follow manufacturer instructions.
16. Incubate overnight at RT, protected from light.
17. Prepare mounting chambers.
    1. On a microscope slide, place three layers of double-sided tape. Use transparent, fibreless tape to avoid residues in the mounting chamber.
    2. Using a razor, cut a 3 mm × 8 mm window in the double tape.
       NOTE: Chamber dimensions depend on the sample time point. This example is suited for hosting 50 µL of mounting medium, which is optimal for 10-20 single NS at later time points (days 7-9).
18. Under a stereoscope, carefully transfer the NS in the clearing agent into the mounting chamber by cutting a low adhesion p200 micropipette tip. Use a magnification between 2x and 4x to provide a field of view of the whole chamber and enough magnification to identify the NS. If the stereoscope is outfitted for fluorescent imaging, working with a blue fluorescence filter will make identifying the NS easier, as the DAPI staining will make the otherwise transparent NS stand out.
    NOTE: Verify that the medium forms a slight convex meniscus above the chamber. This will ensure there are no bubbles in the chamber.
19. Place a coverslip on the surface and lightly press on its sides to make it adhere. Perform this step under the stereoscope and verify that the NS are not pushed outside the chamber.
20. Store at 4 °C protected from light until imaging.

5. CNCC fixation and mounting for immunofluorescence

1. Remove CNCC maintenance medium from the wells and wash them with PBS. Add PBS gently by pipetting on the side of the well.
2. Proceed with fixation, permeabilization, and staining as described in steps 4.3-4.14.
3. Mount coverslips on a microscopy glass slide by adding mounting medium onto the slide, grabbing and rotating the coverslip with the use of tweezers so that post-migratory CNCC face the glass slide, and placing it on the drop.
   1. Optional: Seal the coverslip with nail polish or other commonly employed sealing systems.
4. Store at 4 °C protected from light until imaging.

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

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