A unique mineralizing pool of Gli1+ stem cells builds the tendon enthesis and demonstrates therapeutic potential

Summary The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for the transfer of force from muscle to bone to produce joint motion. The enthesis is prone to injury due to mechanical demands, and it cannot regenerate. A better understanding of how the enthesis develops will lead to more effective therapies to prevent pathology and promote regeneration. Here, we used single-cell RNA sequencing to define the development transcriptome of the entheses over postnatal stages. Six resident cell types, including enthesis progenitors and mineralizing chondrocytes, were identified along with their transcription factor regulons and temporal regulation. Following our prior discovery of the necessity of Gli1-lineage cells for enthesis development and healing, we then examined their transcriptomes at single cell resolution and demonstrated their clonogenicity and multipotency. Transplantation of these Gli1- lineage cells to enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration. Highlights The transcriptome and differentiation trajectory of enthesis stem cells during postnatal development are defined at single cell resolution. Transcription factor regulons drive enthesis stem cell differentiation. Gli1-lineage enthesis stem cells demonstrate in vivo and in vitro clonogenicity and multipotency. Transplantation of Gli1-lineage enthesis stem cells to enthesis injuries improves healing.