Determining Bone-forming Ability and Frequency of Skeletal Stem Cells by Kidney Capsule Transplantation and Limiting Dilution Assay

Adult stem cells not only maintain tissue homeostasis but are also critical for tissue regeneration during injury. Skeletal stem cells are multipotent stem cells that can even generate bones and cartilage upon transplantation to an ectopic site. This tissue generation process requires essential stem cell characteristics including self-renewal, engraftment, proliferation, and differentiation in the microenvironment. Our research team has successfully characterized and isolated skeletal stem cells (SSCs) from the cranial suture called suture stem cells (SuSCs), which are responsible for craniofacial bone development, homeostasis, and injury-induced repair. To assess their stemness features, we have demonstrated the use of kidney capsule transplantation for an in vivo clonal expansion study. The results show bone formation at a single-cell level, thus permitting a faithful assessment of stem cell numbers at the ectopic site. The sensitivity in assessing stem cell presence permits using kidney capsule transplantation to determine stem cell frequency by limiting dilution assay. Here, we described detailed protocols for kidney capsule transplantation and limiting dilution assay. These methods are extremely valuable both for the evaluation of skeletogenic ability and the determination of stem cell frequency.