Tenogenic induction from induced pluripotent stem cells unveils the trajectory towards tenocyte differentiation.

The musculoskeletal system is integrated by tendons that are characterized by expression of a functionally important transcription factor Scleraxis (Scx). To date, there has been no solid culture system for tenogenic differentiation. Here we developed the tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence that reflects a series of dynamic differentiation process. Among several developmentally relevant factors, TGF-?2 was the most potent inducer for differentiation of tenocytes expressing Tenomodulin, a mature differentiation marker. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters including mature tenocyte population and tenogenic differentiation trajectory, which well recapitulates in vivo developmental process. Dataset of scRNA-seq and transcriptome of Scx deficient tendon enabled identification of Scx-related genes in which tendon-related genes are enriched. Collectively, this study leads to a new opportunity for tendon research and provides further insight into mechanistic understanding of the differentiation pathway to a tenogenic fate. Overall design: Single cell-based RNA-sequencing during tenogenic induction from iPSCs and profiling of Achilles tendon mRNA of 14-day old wild type (Wt) andScx -/- (KO) mice.