Abstract
The musculoskeletal system is integrated by tendons that are characterized by the expression of scleraxis (Scx), a functionally important transcription factor. Here, we newly developed a tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence, which reflects a dynamic differentiation process. Among several developmentally relevant factors, transforming growth factor-beta 2 (TGF-β2) was the most potent inducer for differentiation of tenomodulin-expressing mature tenocytes. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters, including mature tenocyte population and tenogenic differentiation trajectory, which recapitulated the in vivo developmental process. Analysis of the scRNA-seq dataset highlighted the importance of retinoic acid (RA) as a regulatory pathway of tenogenic differentiation. RA signaling was shown to have inhibitory effects on entheseal chondrogenic differentiation as well as TGF-β2-dependent tenogenic/fibrochondrogenic differentiation. The collective findings provide a new opportunity for tendon research and further insight into the mechanistic understanding of the differentiation pathway to a tenogenic fate.
Highlights
Tendons and ligaments are dense regular fibrous connective tissue that connect muscle to bone and bone to bone, respectively
We examined the expression level of enhanced green fluorescent protein (EGFP) in cultured tenocytes at passage 1 (P1), mouse embryonic fibroblasts (MEFs), and tail tendons
Upon treatment with TGF-β2, the Scx+/Sox9+ cell population expanded significantly in association with the marked increase in the Scx+ cell population, while the Sox9+ cell population was diminished (Figure 4F and Supplementary Figure S5). These results provided evidence that our induction protocol can generate tenogenic and ligamentogenic progenitor populations, FIGURE 5 | Time course of in vitro tenogenic differentiation induced by treatment with TGF-β2. (A) Cell morphologies and GFP expression during in vitro differentiation from ScxGFP induced pluripotent stem cells (iPSCs). (B) EGFP expression in iPSCs and induced cells at M4, T-1, T7, and T14. n = 3. (C) The expression levels of marker genes for embryonic stem cells (ESCs) (Nanog, SRY-box 2 (Sox2)), mesoderm (T and Mixl1), sclerotome (Sox9), and tendon (Scx, Mkx, Tnmd, early growth response 1 (Egr1), and Col1a2) during tenogenic differentiation. n = 3
Summary
Tendons and ligaments are dense regular fibrous connective tissue that connect muscle to bone and bone to bone, respectively. They transmit the mechanical force generated by the muscle to the bone or stabilize the joint, and act as important components in the musculoskeletal system. We mainly used the term tendon in this study because there were no molecular markers that clearly distinguished these tissues. The major cell population of tendon is a specialized type of fibroblasts called tenocytes. They are derived from the subdomain of the sclerotome, including the syndetome in the trunk, the lateral plate
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