Background

Conventional methods have shown successful isolation of mesenchymal stromal cells (MSCs) from bone marrow and other tissues (Friedenstein et al., 1974; da Silva Meirelles et al., 2006). MSCs contain skeletal stem cells (SSCs) with self-renewing and skeletogenic differentiating abilities (Sacchetti et al., 2007). However, only ~10–20% of them are genuine SSCs (Robey et al., 2014). The difficulties in engraftment, survival, and differentiation of the transplanted MSCs have also been well documented (Caplan and Correa, 2011; Zeitouni et al., 2012). Furthermore, the cellular source of the endogenous MSC remains unknown. Here, we describe a protocol for the isolation of SSCs from the calvarial suture mesenchyme. These SuSCs have been demonstrated to generate bone at a single cell level upon kidney capsule transplantation, which can faithfully assess SuSC properties in vivo (Maruyama et al., 2016). It is also important to develop a protocol capable of maintaining their stemness in vitro. The successful establishment of the skeletal stem cell culture protocol permits further assessments of stem cell characteristics, e.g., self-renewal, proliferation, fate determination, and differentiation in an ex vivo setting. The preservation of stem cell stemness in culture is also critical for bone tissue engineering and opens the possibility of exploring next-generation therapeutics. We also describe an ex vivo protocol to culture SuSCs for an extended period. The cultured SuSCs can generate bones upon implantation to the ectopic site (Maruyama et al., 2021). We demonstrate that this culture method can determine the label-retaining ability and asymmetric division of SuSCs. It also provides an outstanding system to further our examination of additional stem cell characteristics, e.g., cell fate determination, generation of skeletal progenitors, and skeletogenic differentiation, as well as studies at the transcriptome (RNA-seq), epigenetics (ATAC-seq and ChIP-seq), and single-cell levels (scRNA-seq). Using these new tools, we can reconstruct lineage relationships between cells within craniofacial and skeletal tissues—a long-standing challenge in biology. These are extremely important advancements in stem cell-based therapy for bone regeneration and repair.