Abstract
BackgroundTendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic.MethodsEquine 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 were compared using RNA sequencing.ResultsNo 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 study adds to the growing evidence that monolayer cultured cells’ gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed.ConclusionThe information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.
Highlights
Tendon injuries occur frequently in human and equine athletes
Fetal and embryonic tenocytes cultured in 3D reveals no quantitative or qualitative differences We have previously demonstrated that equine Embryonic stem cells (ESCs) can differentiate into tenocytes in both 2D and 3D culture, showing increased differentiation in 3D [50]. 3D constructs cultured for 14 days showed no significant differences in the degree of contraction between cell types
There are many differences in gene expression between ESC-derived, fetal and adult tenocytes, this study shows that of the 26,991 genes mapped, ESCtenocytes have fewer differentially expressed (DE) genes when compared to fetal tenocytes (2708 genes) as opposed to adult tenocytes (2940 genes)
Summary
Tendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. AT injuries in humans have remarkable similarities with superficial digital flexor tendon (SDFT) injuries in horses [2, 3] Both the AT and SDFT serve to connect skeletal muscle to bone, and provide a means of energystoring to facilitate high-speed locomotion, a function for which no other animal model possesses [3]. Given the less demanding regulatory framework for biological treatments in animals and relative ease of access to equine tissue, the horse is one of the most scientifically sound animal models for studying such injuries [3]. Healing in both human and equine tendon is often prolonged, with injuries undergoing poor natural regeneration and instead healing via fibrotic scar tissue formation. This fetal regenerative response has been observed in other tissues and may be controlled at the cellular level [7,8,9,10,11,12,13]
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