Materials and Reagents

1.5 mL tube
Wide bore filtered 1,000 µL pipette tips (Thermo Fisher Scientific, catalog number: 2079G)
35 mm tissue culture-treated dish (SPL Life Sciences, catalog number: 20060)
Sterile Cell Strainer, 40 µm Nylon Mesh (Thermo Fisher Scientific, catalog number: 22363547)
Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023)
Cell-dissociation enzyme, TrypLE Express (Thermo Fisher Scientific, catalog number: 12605028)
Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061)
Advanced DMEM (Thermo Fisher Scientific, catalog number: 12491015)
Ham’s F-12 Nutrient Mix (Thermo Fisher Scientific, catalog number: 11765054)
Penicillin-Streptomycin, 100× (Thermo Fisher Scientific, catalog number: 15140122)
Fetal bovine serum (FBS)-US source, heat inactivated (Denville Scientific, catalog number: FB5001-H)
Fibronectin, human protein, plasma (Thermo Fisher Scientific, catalog number: 33016015)
Bouin’s fixative (Polysciences, catalog number: 16045)
Ammonium hydroxide solution, 28–30% (Sigma-Aldrich, catalog number: 221228)
Alcian blue, 8GX (Sigma-Aldrich, catalog number: A3157)
Acetic acid, glacial (Sigma-Aldrich, catalog number: 695092-2.5L)
Guanidine hydrochloride (Sigma-Aldrich, catalog number: G3272)
Fibronectin stock solution (1 mg/mL) (see Recipes)
100 µg/mL fibronectin in 10% FBS/Advanced DMEM (see Recipes)
10 µg/mL fibronectin in 10% FBS/Advanced DMEM (see Recipes)
4% FBS in Advanced DMEM/Ham’s F-12 (see Recipes)
Solution A for alcian blue staining (see Recipes)
Solution B for alcian blue staining (see Recipes)
1 mg/mL Alcian blue solution (see Recipes)
6M guanidine hydrochloride (see Recipes)

Equipment

Dumont #5 tweezer, tip size 0.10 × 0.06 mm (Roboz, catalog number: RS-5045), sterilized by autoclave
Dumont #7 tweezer, tip size 0.17 × 0.10 mm (Roboz, catalog number: RS-5047), sterilized by autoclave
Micro dissecting scissors, 3.5” straight (Roboz, catalog number: RS-5910), sterilized by autoclave
Stereo microscope, any brand (e.g., Leica M165-FC)
Centrifuge (Thermo Sorval Legend Micro 21)
Class II A/B3 Biological safety cabinet
CO₂ incubator, NAPCO SERIES 8000DH
Varioskan Flash Spectral Scanning Multimode Reader (Thermo Scientific)

Procedure

Cell isolation
1. Pour 12 mL of PBS in a 10 cm dish on ice.
2. Pre-warm TrypLE to 37°C.
3. Euthanize a pregnant mouse (E10.5) via methods approved by the Institutional Animal Care and Use Committee (IACUC), such as inhalation of CO₂followed by thoracotomy.
4. Harvest the uterus and place it in ice-cold PBS (Figure 1).
5. Harvest embryos from the uterus and keep them in ice-cold PBS (Figure 1, Video 1).
  
  Figure 1. Harvest embryos from a pregnant mouse. (A–C). A pregnant mouse was euthanized with CO₂. The skin was cut with a pair of scissors, and a uterus with embryos (arrowheads) was exposed by pushing the digestive tract upward anteriorly. (D). The uterus was cut with a pair of scissors, by following the dotted arrows (top to bottom). (E). A tweezer was inserted as per the arrow, to separate the uterine wall from the embryo. (F–I). Embryos were carefully removed from the placenta, the yolk sac, and the amnion. Scale bars: 1 mm.
  
  Video 1. Harvest an E10.5 embryo from the uterus.
  An embryo is located at the light red area of the isolated uterus (Figure 1E, upper part), and the placenta is located at the dark red area (Figure 1E, lower part). A tweezer will be carefully inserted from one of the cut sites of the separated uterus. The uterine wall will be carefully removed to harvest an embryo with yolk sac. Subsequently, the yolk sac and the amnion will be removed as shown in Figure 1H–I.
6. Isolate the BA1 (Figure 2, Video 2), and place them into 1.5 mL tubes with 100 µL of ice-cold PBS (one BA1 to a tube).
  
  Figure 2. Harvest the first branchial arch from embryo. (A). Embryos were divided into the head and the trunk. A tweezer was inserted between BA1 and BA2, as following the arrow. (B, C). The BA1 was divided from the MA with a tweezer, as following the arrow in B, and the isolated BA1 was transferred to a 1.5 ml tube. MA; maxillary arch, BA1; first branchial arch, BA2; second branchial arch. Scale bars: 1 mm.
  
  Video 2. Harvest the BA1 from an embryo.
  The trunk of an embryo is gently pressed down with a tweezer, and another tweezer is inserted between the BA1 and the BA2. The head is then gently pressed down with a tweezer, and the BA1 is separated from the head.
7. Centrifuge at 100 × g and room temperature (RT) for 5 min.
8. Remove supernatant, then add pre-warmed 100 µL of TrypLE to the 1.5 mL tube.
9. Incubate at 37°C for 5–10 min. Gently pipette up and down with wide bore 1,000 µL tips.
10. Add 900 µL of 10% FBS in Advanced DMEM, then gently pipette 5–10 times.
11. Centrifuge at 100 × g and RT for 5 min, then discard supernatant.
12. Resuspend the cells with 500 µL of PBS.
13. Centrifuge at 100 × g and RT for 5 min, then discard supernatant.
14. Resuspend with 150 µL of PBS.
15. Filter the cells through a 40 µm cell strainer.
16. Take 10 µL of cell suspension into 1.5 mL tube, and mix with 10 µL of Trypan blue, to assess cell viability.
Micromass culture
1. Dilute the fibronectin stock solution with 10% FBS in Advanced DMEM, to prepare 100 µg/mL fibronectin solution.
2. Resuspend the cells with 100 µg/mL fibronectin solution (5 × 10⁵ cells/100 µL).
3. Seed 20 µL of cells onto a 35 mm dish.
4. Incubate at 37°C for 1.5 h, to allow them to attach to well.
5. Add 1 mL of Advanced DMEM including 10 µg/mL Fibronectin and 10% FBS in Advanced DMEM.
6. Incubate at 37°C for 24 h.
7. Remove media, then add 2 mL of 4% FBS in Advanced DMEM/Ham's F-12.
8. Culture for 5 days.
9. Measure chondrogenic differentiation by Alcian blue staining (Figure 3 and part C), and set up a qRT-PCR to determine the levels of chondrogenic marker genes, such as Sox9 and type 2 collagen (Yang et al., 2021).
Figure 3. Observation of primary cranial NCCs in vitro. (A). Harvested primary cranial NCCs from BA1 are shown. The morphology of the cranial NCCs from BA1 is close to O9-1 cells (Ishii et al., 2012), a neural crest stem cell line. (B). Micromass culture for cranial NCCs from control and caAcvr1; P0-Cre mice embryos was performed. Cells were stained with Alcian blue. Scale bars (A) 100 µm and (B) 1 mm.
Alcian Blue staining for micromass culture
1. Aspirate medium from micromass culture.
2. Fix with 1mL of Bouin’s fixative at RT for 15 min.
3. Wash with Solution A (see Recipes).
4. Wash with Solution B (see Recipes).
5. Stain with 2 mL of 1 mg/mL Alcian blue in 3% acetic acid (see Recipes) at RT for 1 h.
6. Wash dye off with double distilled water (ddH₂O) four times.
7. Keep in 2 mL of ddH₂O.
8. Take photos.
9. Wash dye off with ddH₂O.
10. Extract the dye by incubating cultures with 1 mL of 6 M guanidine hydrochloride (see Recipes) at RT overnight.
11. Measure the optical density at OD₅₉₅ using a spectrophotometer, such as the Thermo Scientific Varioskan Flash Spectral Scanning Multimode Reade.

Notes

To avoid cell death, embryos/cells should be kept on ice until they are seeded.
In case of cell viability less than 70–80%, consider removing dead cells by using a dead cell removal kit, such as Miltenyi dead cell removal kit (Miltenyi Biotech, Catalog number: 130-090-101).
When cells are seeded on 35 mm dishes, the drop should be kept maintaining a high-dense cell area. After culture for 1.5 h, cells should be attached on the 35 mm dish. Carefully add Advanced DMEM including Fibronectin.
The isolated cells can be used for micromass culture at the day of isolation. However, they can be passaged up to three times, with a total duration of culture of 10 days after isolation.

Recipes

Fibronectin stock solution (1 mg/mL)
1 mg fibronectin
1 mL of PBS
100 µg/mL fibronectin in 10% FBS/Advanced DMEM
60 µL of fibronectin stock solution
540 µL of 10% FBS/Advanced DMEM
10 µg/mL fibronectin in 10% FBS/Advanced DMEM
100 µL of fibronectin stock solution
1 mL of 10% FBS/Advanced DMEM
4% FBS in Advanced DMEM/Ham’s F-12
48 mL of Advanced DMEM
48 mL of Ham’s F-12
4 mL of FBS
Solution A for alcian blue staining
Add 0.5 mL of Ammonium hydroxide solution to 500 mL of 70% ethanol
Solution B for alcian blue staining
Add 25 mL of acetic acid, glacial, to 70% ethanol to make final volume 500 mL.
1 mg/mL Alcian blue solution
Add 0.6 mL of acetic acid, glacial, to ddH₂O to make final volume 20 mL. Then add 20 mg of Alcian blue 8GX.
6 M guanidine hydrochloride
Dissolve 57.3 g of guanidine hydrochloride in 100 mL of ddH₂O.

Acknowledgments

This work was supported by grant from NIH (R01DE020843 to Y.M. and R01DE025897 to Y.K.), International Fibrodysplasia Ossificans Progressiva Association (to Y.M.), and the grant-in-aid from the National Natural Science Foundation of China (31500788 to J.Y.). We thank Dr. W. Benton Swanson for critical reading. This protocol is derived from the original research paper, Yang et al., Augmented BMP signaling commits cranial neural crest cells to a chondrogenic fate by suppressing autophagic b-catenin degradation. Sci Signal 14(665): eaaz9368. (DOI: 10.1126/scisignal.aaz9368).

Competing interests

The authors declare no conflict of interest.

Ethics

All experimental procedures have been approved by the Institutional Animal Care and Use Committee at the University of Michigan (#PRO00009613) and the University of Texas (AWC-18-0137).

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