Skeletal Stem/Progenitor Cell Clonogenic Assay

Colonies were assessed as previously described^{31 (link)}. 500 freshly sorted skeletal stem/progenitor cells from pN3, CD-1 mice PF, SAG, and COR sutures and the GP were seeded into a 10 cm² plate pre-coated with 0.1% gelatin (EmbryoMax, Millipore, Burlington MA, USA) in alpha-MEM GlutaMax (supplemented with 10% fetal bovine serum 1% penicillin–streptomycin (Gibco-Life Technologies, Grand Island, NY, USA), and 0.1% ciprofloxacin HCl (bioWORLD, Dublin, OH, USA). Cells were incubated under low O₂ conditions (2% atmospheric oxygen, 7.5% CO₂) for two weeks. For evaluation of CFUs, colonies were stained with crystal violet. Cell colonies were photographed under an inverted microscope and colonies with >50 cells or more were counted. For evaluation for self-renewal ability, skeletal stem/progenitor cells were isolated by FACS and pooled from the PF, SAG, and COR sutures of pN3 CD-1 mice. 500 skeletal stem/progenitor cells were seeded into 10 cm² for two weeks as described above. After 2 weeks clones were lifted using Stem Pro Accutase (Gibco-Life Technologies, Grand Island, NY, USA) and skeletal stem/progenitor cells isolated by FACS and passaged onto 10 cm² plates as described above. For information regarding reagents used refer to Supplementary Table 3.