The tendon is one of many minimally regenerative tissues in the mammalian body. Due to the unsatisfactory outcomes from surgical repair and the lack of pharmacologics to improve tendon healing, it is important to develop new therapies. In many cases, minimally regenerative tissues fail to recover after injury because their endogenous cell types cannot respond sufficiently to restore tissue function. As a result, scar-depositing cells are recruited and lay down fibrotic matrix. This dissertation takes a two pronged approach to solving this problem.

This first approach uses pharmacologic and genetic mouse models to probe at the signaling that is required for tenocyte recruitment. Using a model of neonatal mouse tendon regeneration, we determine the molecular basis for regeneration and identify TGFβ signaling as a major pathway. Through targeted gene deletion, small molecule inhibition, and lineage tracing, we elucidate TGFβ-dependent and –independent mechanisms underlying tendon regeneration. Importantly, functional recovery depended on TGFβ signaling and loss of function is due to impaired tenogenic cell recruitment from both Scxlin and non-Scxlin sources. We show that TGFβ signaling is required directly in neonatal tenocytes for recruitment and that TGFβ is positively regulated in tendons. Collectively, these results are the first to show a functional role for TGFβ signaling in tendon regeneration and offer new insights toward the divergent cellular activities that may lead to regenerative vs fibrotic healing.

The second approach in this dissertation for improving tendon healing utilizes developmental insights to develop a differentiation protocol from mouse embryonic stem cells to tendon cells. Manipulation of the BMP and Wnt pathways was sufficient to drive induction of somitic mesoderm, a known source of tendon progenitors in development. Subsequently, tenogenic induction was successfully achieved with Hedgehog and TGFβ pathway activation to recapitulate induction of the sclerotome and syndetome (somitic compartments and subcompartments, respectively). Single cell RNA sequencing was applied to the E14.5 tail and to the differentiation culture. We were able to derive defined novel signatures for tendon and fibrocartilage from the developing tail. Moreover, we identified populations in the culture that were highly transcriptomically similar to their in vivo counter parts. This study represents the first mouse differentiation from pluripotency and one of the only single cell sequencing datasets containing embryonic tendon. These cells can be used downstream for screening drugs, modeling disease, studying tendon development in vitro, or cell therapy.

The first project in this dissertation tests the requirement for TGFβ signaling in neonatal tendon regeneration. We hypothesize that TGFβ signaling is required for tendon regeneration, specifically tendon cell differentiation. In the second aim of this dissertation, we establish a developmentally directed differentiation protocol that leverages BMP, Wnt, SHH, FGF, and TGFβ signaling to drive mouse embryonic stem cells towards the tendon fate. In this aim, we hypothesize a strict requirement for TGFβ signaling in vitro, similar to the requirement for TGFβ in tendon development in vivo. Collectively, these approaches represent our effort to improve healing by studying the signaling that drives tendon regeneration and developing tools to improve healing should the endogenous cell types prove insufficient. These approaches are also unified by TGFβ signaling and the role this pathway plays in controlling tendon regeneration and the tendon cell fate. This dissertation underlines the importance of understanding other molecular variables which contextualize TGFβ signaling. Even within the tenocyte lineage, we show that embryonic and post-natal tenocytes respond differently to TGFβ pathway activation.