A mineralizing pool of Gli1-expressing progenitors builds the tendon enthesis and demonstrates therapeutic potential.

The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for force transfer 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 developmental transcriptome of the mouse 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 the prior discovery of the necessity of Gli1-lineage cells for mouse enthesis development and healing, we then examined their transcriptomes at single-cell resolution and demonstrated clonogenicity and multipotency of the Gli1-expressing progenitors. Transplantation of Gli1-lineage cells to mouse enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration.