Characterising embryonic stem cell-derived tenocytes and determining the changing role of scleraxis during tendon development

Tendon injuries occur commonly in equine athletes. Adult tendons undergo poor natural regeneration, resulting in scar-tissue which is prone to re-injury. Fetal tendons however are capable of completely scar-less regeneration, a property which is intrinsic to the fetal cells themselves. Novel cell therapies should therefore try to recapitulate this scar-less fetal tendon regeneration. This thesis builds on previous research into the use of horse embryonic stem cells (ESCs) to aid tendon regeneration. The aim of this thesis was to determine if tendon cells derived from ESCs were more similar to fetal or adult tendon cells, as well as try to understand if scleraxis (SCX), an essential gene in tendon formation, has different roles at different stages of tendon development. Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture was compared using RNA-sequencing. No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome wide transcriptomic analysis using RNA- sequencing revealed that ESC-derived tenocytes transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this thesis adds to the growing evidence that monolayer cultured cells gene expression profiles converge, with adult and fetal tenocytes having only 10 differentially expressed (DE) genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in three- dimensional culture, large distinctions in gene expression between these two developmental stages were found, with 542 genes being DE. The effects of knocking down the expression of SCX on gene expression in adult, fetal and ESC-derived tenocytes was then determined using RNA-sequencing and qPCR. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes effected in adult tenocytes, indicating that scleraxis- dependent processes differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, novel genes that SCX differentially interacts with in adult and fetal tenocytes were identified. SCX knockdown in ESCs resulted in upregulation of cartilage markers, a result which still needs to be confirmed in further biological replicates. In summary, the data presented in this thesis provides an unprecedented insight into some of the differences between fetal regenerative and adult reparative tenocytes. It also indicated that ESC-derived tenocytes are more similar to fetal rather than adult tenocytes, highlighting their potential as a therapeutic cell source. The results presented also indicate a role for SCX in modulating ECM synthesis and breakdown and provides a useful dataset for further study into SCX gene regulation. Taken together this data is likely to be important for the future development of novel cellular or pharmacological therapeutics