Uchida Hitoshi, Maruyama Takamitsu, Hsu Wei
Forsyth Institue, Cambridge MA 02142, USA.
Department of Dentistry, University of Rochester Medical Center, Rochester NY 14642, USA.
Bio Protoc. 2023 Mar 20;13(6):e4639. doi: 10.21769/BioProtoc.4639.
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.
成体干细胞不仅维持组织稳态，而且在损伤期间对组织再生也至关重要。骨骼干细胞是多能干细胞，移植到异位部位时甚至可以生成骨骼和软骨。这种组织生成过程需要基本的干细胞特性，包括自我更新、植入、增殖以及在微环境中的分化。我们的研究团队已成功从颅缝中鉴定并分离出骨骼干细胞（SSCs），即缝干细胞（SuSCs），它们负责颅面骨的发育、稳态以及损伤诱导的修复。为了评估它们的干性特征，我们展示了使用肾包膜移植进行体内克隆扩增研究。结果显示在单细胞水平上有骨形成，从而能够在异位部位准确评估干细胞数量。评估干细胞存在的敏感性使得可以利用肾包膜移植通过有限稀释法来确定干细胞频率。在此，我们描述了肾包膜移植和有限稀释法的详细方案。这些方法对于评估成骨能力和确定干细胞频率都极具价值。
