Materials and Reagents

Cell strainer 40 µm Nylon (Falcon, catalog number: 352340)
Ultra-Low Attachment Surface 24-well plate (Corning, catalog number: 3473)
Dulbecco’s Phosphate-Buffered Saline without calcium & magnesium (DPBS; Corning, catalog number: 21-031-CV), 2 years shelf life at room temperature
0.2% collagenase A (Roche, catalog number: 11088793001), freshly reconstituted from lyophilized stock in PBS
10 mM HEPES (1 M buffer solution; Gibco, catalog number: 15630-080), 24 months shelf-life at 4°C
Penicillin-Streptomycin (Gibco, catalog number: 15140-122), 12 months shelf life at -20°C
100 µg/mL transferrin (Sigma, catalog number: T8158) at -20°C
20 nM progesterone (Sigma, catalog number: P8783) at -20°C
30 nM Sodium selenite (Sigma, catalog number: S8295) at -20°C
60 nM Putrescine dihydrochloride (Sigma, catalog number: P5780), 4 years shelf life at -20°C
EGF (Mouse EGF Recombinant Protein; Gibco, catalog number: PMG8041), 1 year shelf life at -20°C
bFGF (mouse FGF-basic Recombinant Protein; Gibco, catalog number: PMG0035), 1 year shelf life at -20°C
B27 supplement (Gibco, catalog number: 17504044,), 1 year shelf life at -20°C
Insulin (Sigma, catalog number: I1882) at -20°C
0.25% trypsin (Gibco, catalog number: 25200-056), 2 years shelf life at -20°C
Soybean trypsin inhibitor (SBTI; Sigma, catalog number: T9128) at -20°C
Digestion Buffer (see Recipes)
Sphere Culture Media (see Recipes)
2× SBTI Solution (see Recipes)

Equipment

Shaker (Thermo Scientific, MaxQtm 4000 Benchtop Orbital Shakers)
Centrifuge (Thermo Scientific, Sorvall ST 16R Centrifuge)
Incubator (Thermo Forma, Series II Water Jacketed CO₂ Incubator)

Procedure

Isolation of mesenchymal cells from suture mesenchyme
1. Dissect a 2 mm wide portion of calvarial tissue containing the sagittal (SAG) suture and its adjacent parietal bones from mouse calvarium (Figure 1).
  
  Figure 1. Schematic of mouse calvarial bones and sutures. F, frontal bone; P, parietal bone; IP, interparietal bone.
2. Hint: It is easy to remove the calvarium from the meninges. The cutout size should be as small as possible, to limit the amounts of contaminant cells from the bone marrow and periosteum.
3. Grab both sides of the parietal bones with two forceps and gently pull them apart (Figure 2).
  
  Figure 2. Schematic diagrams illustrating the separation of bone pieces to release the cells from the suture mesenchyme.
4. Attention: The dissected calvarial tissue containing two small parietal bone pieces must be separated in the midline to expose the suture mesenchyme. Otherwise, the number of skeletal stem cells would be significantly reduced.
5. Hint: To avoid contamination of stem cells from the bone marrow, do not cut the separated calvarial tissue into pieces (this process is performed for calvarial cell isolation). Do not keep the dissected tissue on ice, rather quickly incubate it with Digestion Buffer as described in step 6.
6. Submerge the separated pieces in 40 mL of Digestion Buffer and incubate in a shaker at 200 × g (MaxQtm 4000 Benchtop Orbital Shakers) and 37°C for 1 h.
7. Filter the dissociated cells through a 40 µm strainer.
8. Spindown the cells by centrifugation at 400 × g and 4°C for 7 min.
9. Remove the supernatant to collect the cell pellets.
10. Hint: About 5 × 10⁴ cells can be obtained from one sagittal suture
11. Hint: Suture mesenchymal cells have been successfully isolated from the calvaria of mice at postnatal day 10 (P10) and P28.
Sphere culture
1. Resuspend the cell pellets with Sphere Culture Media and count the cell number.
2. Seed the cells in 2 mL of sphere culture media at 10⁴–10⁵ cells per well on an Ultra-Low Attachment Surface 24-well plate (Figure 3A).
3. Carefully change half the amount of the culture media in the culture every other day, without disturbance of cell settling.
4. Hint: Sphere formation will be visible within 10 to 14 days (Figure 3B and Maruyama et al., 2021).
  
  Figure 3. Representative images showing cells after 1 day (A) and 8 days (B) of the sphere culture. Scale bars, 200 µm.
5. Hint: Most of the sphere is derived from a single stem cell (Maruyama et al., 2021).
6. Hint: Stem cell stemness, e.g., bone-forming ability and quiescence, has been demonstrated to be maintained in culture using the 1st and 3rd passage of the sphere by kidney capsule transplantation and label-retaining analyses (Maruyama et al., 2021).
7. Hint: Bone-forming ability is assessed by the kidney capsule transplantation assay, followed by von Kossa staining and immunostaining of osteoblast markers. Quiescence is examined by the pulse-chase assay in sphere culture.
Passaging spheres
1. Some spheres may be weakly attached to the bottom of the well. To detach the sphere, gently pipette the culture media up and down.
2. The cell suspension is collected by centrifugation at 300 × g and 4°C for 7 min.
3. Remove the supernatant and tap the tube to gently break up the pellets.
4. Add 1 mL of 0.25% Trypsin to resuspend the pellet, followed by incubation at 37°C for 5 min.
5. Add 1 mL of 2× SBTI solution to stop the activity of the trypsin.
6. Centrifuge at 300 × g and 4°C for 7 min.
7. Remove supernatant and tap the tube to gently break up the pellets.
8. Resuspend the cells in the Sphere Culture Media and count cell numbers.
9. Seed the cells at 10⁴–10⁵ cells per well.
10. Hint: This culture method has been limited to five passages.

Data analysis

Sphere measurement
1. Gently pipette the medium to detach the spheres from the culture plate.
2. Hint: No special pipet tips are required, only general ones that generate a little media flow.
3. Take images using a camera under a microscope, e.g., NIKON CB-115 (Figures 5, 6, Supplemental Figures S9, S10 of Maruyama et al., 2021).
4. Measure the sphere number and size by ImageJ (Figures 6, Supplemental Figures S9, S10 of Maruyama et al., 2021).
5. Hint: For the basic function of ImageJ: “ImageJ Basics (PDF file)” on the official page (https://imagej.nih.gov/ij/index.html) under “Documentation”-“Tutorials and Examples”.
6. Count spheres that are >20 µm in diameter.
7. Perform at least three independent experiments for statistical evaluation.
8. Examine the statistical significance of sphere number and size using a two-sided Student’s t-test.
Evaluation of bone formation after kidney capsule transplantation
1. Perform whole-mount von Kossa staining of bone in the transplanted kidney capsule (Figures 4, 5, 6, Supplemental Figures S9, S10 of Maruyama et al., 2021).
2. Image using a microscope, e.g., NIKON CB-115.
3. Measure the size of the stained area by ImageJ.
4. Perform at least three independent experiments for statistical evaluation.
5. Examine the statistical significance of sphere number and size using a two-sided Student’s t-test (Supplemental Figure S10 of Maruyama et al., 2021).

Recipes

Digestion Buffer (40 mL)
DPBS containing 0.2% collagenase A, 1% Penicillin-Streptomycin, and 10 mM HEPES
Sphere Culture Media
25 µg/mL insulin
100 µg/mL transferrin
20 nM progesterone
30 nM Sodium selenite
60 nM putrescine
20 ng/mL EGF
20 ng/ml bFGF
1× B27 supplement
1% Penicillin-Streptomycin
2× SBTI Solution
2 mg SBTI in 1 mL of DPBS

Acknowledgments

The authors thank former and current lab members for their technical and intellectual support. This work is supported by the National Institutes of Health (DE15654, DE269369) and NYSTEM (C029558) to W.H. This protocol is derived from the original research paper published in Science Translational Medicine (Maruyama et al., 2021).

Competing interests

The authors declare no competing financial interests.

Ethics

Care and use of experimental animals described in this work comply with guidelines and policies of the University Committee on Animal Resources at the University of Rochester and IACUC at the Forsyth Institute.

References

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