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The relationship between orientation and mobility (O&M) performance and residual vision is not clearly understood. Intuitively, good vision should result in good O&M, and little or no vision should result in poor O&M. However, the literature suggests a more complicated picture. The authors propose that certain psychological variables, such as particular personality traits and intelligence, may account for some of the complexity. This article reviews previous work in this area and outlines directions for future research. [ABSTRACT FROM AUTHOR]

The suspensory ligament (SL) is a key component of the elaborate and highly adapted suspensory apparatus in the horse. In addition to contributing to stabilisation of the metacarpophalangeal joint, the SL has a spring like function to reduce the energetic cost of locomotion. Although the SL is highly prone to injury in horses of all ages and competing in a wide range of disciplines, knowledge regarding fundamental structure-function relationships in the SL is lacking, particularly compared with other injury-prone tendinous structures such as the superficial digital flexor tendon. In this review, we discuss current knowledge of SL composition, structure and mechanical properties and describe the epidemiology, aetiology and pathophysiology of injuries. We evaluate different diagnostic approaches and treatment modalities and identify key areas for future research., (© 2024 The Author(s). Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Hypertrophic cardiomyopathy (HCM) is the most common heart disease in domestic cats, often leading to congestive heart failure and death, with current treatment strategies unable to reverse or prevent progression of the disease. The underlying pathological processes driving HCM remain unclear, which hinders novel drug discovery. The aim of this study was to generate a cellular model of the feline HCM-causing MYBPC3 mutation R820W. Using CRISPR/Cas9 gene editing we introduced the R820W mutation into a human induced pluripotent stem cell (iPSC) line. We differentiated both homozygous mutant clones and isogenic control clones to cardiomyocytes (iPSC-CMs). Protein quantification indicated that haploinsufficiency is not the disease mechanism of the mutation. Homozygous mutant iPSC-CMs had a larger cell area than isogenic controls, with the sarcomere structure and incorporation of cMyBP-C appearing similar between mutant and control iPSC-CMs. Contraction kinetic analysis indicated that homozygous iPSC-CMs have impaired relaxation and are hypocontractile compared to isogenic control iPSC-CMs. In summary, we demonstrate successful generation of an iPSC model of a feline MYBPC3 mutation, with the cellular model recapitulating aspects of HCM including cellular hypertrophy and impaired relaxation kinetics. We anticipate that further study of this model will lead to improved understanding of the disease-causing molecular mechanism, ultimately leading to novel drug discovery., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dutton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1β stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing., (© 2024. The Author(s).)

We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF- K B) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF- K B inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF- K B-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1β increased NF- K B-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF- K B signaling. Administering direct pharmacological inhibitors of NF- K B did reduce NF- K B-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1β. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1β which can only be rescued by blockade of interleukin 1 receptor, but not NF- K B, signalling., (© 2023. The Author(s).)

Fractures caused by bone overloading are a leading cause of euthanasia in Thoroughbred racehorses. The risk of fatal fracture has been shown to be influenced by both environmental and genetic factors but, to date, no specific genetic mechanisms underpinning fractures have been identified. In this study, we utilised a genome-wide polygenic risk score to establish an in vitro cell system to study bone gene regulation in horses at high and low genetic risk of fracture. Candidate gene expression analysis revealed differential expression of COL3A1 and STAT1 genes in osteoblasts derived from high- and low-risk horses. Whole-genome sequencing of two fracture cases and two control horses revealed a single-nucleotide polymorphism (SNP) upstream of COL3A1 that was confirmed in a larger cohort to be significantly associated with fractures. Bioinformatics tools predicted that this SNP may impact the binding of the transcription factor SOX11. Gene modulation demonstrated SOX11 is upstream of COL3A1 , and the region binds to nuclear proteins. Furthermore, luciferase assays demonstrated that the region containing the SNP has promoter activity. However, the specific effect of the SNP depends on the broader genetic background of the cells and suggests other factors may also be involved in regulating COL3A1 expression. In conclusion, we have identified a novel SNP that is significantly associated with fracture risk and provide new insights into the regulation of the COL3A1 gene.

Persistent inflammation is associated with the poor regeneration of musculoskeletal tissues. Embryonic stem cells (ESCs) have an attenuated response to inflammatory cytokines, but there are mixed reports on the response of induced pluripotent stem cells (iPSCs) to inflammation. Horses provide a relevant large animal model for studying musculoskeletal tissue diseases and the testing of novel therapies. The aim of this study was to determine if equine iPSCs are responsive to the inflammatory cytokines IL-1β, TNFα and IFN-γ in their undifferentiated state, or following differentiation into tendon and cartilage-like cells. We demonstrated that in undifferentiated iPSCs, the cytokines induce NF-κB P65 and STAT1 nuclear translocation which leads to cell death, decreased OCT4 expression and increased expression of inflammatory genes. Following differentiation towards cartilage-like cells exposure to the cytokines resulted in STAT1 nuclear translocation, changes in cartilage gene expression and increased expression of matrix metalloproteinases (MMPs) and inflammatory genes. Exposure of iPSC-derived tendon-like cells to the cytokines resulted nuclear translocation of NF-κB P65 and STAT1, altered tendon gene expression, increased MMP expression and increased expression of inflammatory genes. Equine iPSCs are therefore capable of responding to inflammatory stimulation and this may have relevance for their future clinical application., (© 2023. The Author(s).)

Mesenchymal stromal cells (MSC) isolated form bone marrow and adipose tissue are the most common cells used for cell therapy of orthopedic diseases. MSC derived from different tissues show differences in terms of their proliferation, differentiation potential and viability in prolonged cell culture. This suggests that there may be subtle differences in intracellular signaling pathways that modulate these cellular characteristics. The Rho/ROCK signaling pathway is essential for many cellular functions. Targeting of this pathway by the ROCK inhibitor Y-27632 has been shown to be beneficial for cell viability and proliferation of different cell types. The aim of this study was to investigate the effects of Rho/ROCK inhibition on equine MSC proliferation using bone marrow-derived MSC (BMSC) and adipose-derived MSC (ASC). Primary ASC and BMSC were stimulated with or without 10 ng/mL TGF-β3 or 10 μM Y-27632, as well as both in combination. Etoposide at 10 μM was used as a positive control for inhibition of cell proliferation. After 48 h of stimulation, cell morphology, proliferation activity and gene expression of cell senescence markers p53 and p21 were assessed. ASC showed a trend for higher basal proliferation than BMSC, which was sustained following stimulation with TGF-β3. This included a higher proliferation with TGF-β3 stimulation compared to Y-27632 stimulation ( p  < 0.01), but not significantly different to the no treatment control when used in combination. Expression of p21 and p53 was not altered by stimulation with TGF-β3 and/or Y-27632 in either cell type. In summary, the Rho/ROCK inhibitor Y-27632 had no effect on proliferation activity and did not induce cell senescence in equine ASC and BMSC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Melzer, Burk, Guest and Dudhia.)

Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1β, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells' ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1β using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1β stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB., (© 2022. The Author(s).)

Competing Interests: JI is employed by CVS Group plc. With the University of Liverpool, JI is the joint license holder for the LOAD client-reported outcomes measure. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may 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, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation., Competing Interests: Declaration of competing interest None., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Background: 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. 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., Methods: 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 were compared using RNA sequencing., Results: 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 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., Conclusion: The 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.

Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo environment, in this study, we have utilised a novel and affordable, custom-made bioreactor to apply a cyclical strain to tendon-like constructs generated in three-dimensional (3D) culture by equine tenocytes. Dynamic shear analysis (DSA), dynamic scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy were used to determine the mechanical and chemical properties of the resulting tendon-like constructs. Our results demonstrate that equine tenocytes exposed to a 10% cyclical strain have an increased amount of collagen gel contraction after 7 and 8 days of culture compared with cells cultured in 3D in the absence of external strain. While all the tendon-like constructs have a very similar chemical composition to native tendon, the application of strain improves their mechanical properties. We envisage that these results will contribute towards the development of improved biomimetic artificial tendon models for the development of novel strategies for equine regenerative therapies., (© 2020 John Wiley & Sons, Ltd.)

The objective of this study was to determine whether corneal stromal cells (CSCs) from the limbal and central corneal stroma in dogs have multipotent mesenchymal stem/stromal cell (MSC) properties, and whether this cell population can be differentiated into keratocyte-like cells (KDCs). Normal, donated, mesocephalic dog corneas were used to isolate CSC in vitro. Immunohistochemistry demonstrated a distinct population of CD90 expressing cells in the anterior stroma throughout the limbal and central cornea. CSC could be cultured from both the limbal and central cornea and the culture kinetics showed a progenitor cell profile. The CSC expressed stem cell markers CD90, CD73, CD105, N-cadherin, and Pax6, while CD34 was negative. Limbal and central CSC differentiated into osteoblasts, chondrocytes, and adipocytes confirming their multipotency. Coculturing allogeneic peripheral blood mononuclear cells (PBMCs) with limbal CSCs did not affect baseline PBMC proliferation indicating a degree of innate immune privilege. Limbal CSC could be differentiated into KDCs that expressed Keratocan, Lumican, and ALDH1A3 and downregulated Pax6 and N-cadherin. In conclusion, canine CSCs have multipotent MSC properties similarly described in humans and could serve as a source of cells for cell therapy and studying corneal diseases.

The natural healing process for tendon repair is associated with high upregulation of collagen type III, leading to scar tissue and tendon adhesions with functionally deficient tendons. Gene delivery systems are widely reported as potential nanotherapeutics to treat diseases, providing a promising approach to modulate collagen type III synthesis. This work investigates a proof-of-concept four-arm cationic polymer-siRNA polyplex to mediate a transient downregulation of collagen type III expression in a tendon cell culture system. The tendon culture system was first supplemented with TGF-β1 to stimulate the upregulation of collagen type III prior to silencing experiments. The four-arm poly[2-(dimethylamino) ethyl acrylate] (PDMAEA) polymer was successfully synthesized via RAFT polymerization and then mixed with siRNA to formulate the PDMAEA-siRNA polyplexes. The formation of the polyplex was optimized for the N:P ratio (10:1) and confirmed by agarose gel electrophoresis. The size and solution behavior of the polyplex were analyzed by dynamic light scattering and zeta potential, showing a hydrodynamic diameter of 155 ± 21 nm and overall positive charge of +30 mV at physiological pH. All the polyplex concentrations used had a minimal effect on the metabolic activity of cultured cells, indicating good biocompatibility. The dose and time effects of the TGF-β1 on collagen type III gene expressions were analyzed by qPCR, showing an optimal dose of 10 ng mL -1 TGF-β1 and 3-fold increase of COL3α1 expression at 48 h in cultured tenocytes. The PDMAEA - siRNA polyplex concept observed a limited yet successful and promising efficiency in silencing collagen type III at 48 h compared to PEI-siRNA. Therefore, this concept is a promising approach to reduce tissue scarring and adhesion following injuries., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

In horses and humans, tendon injuries are a significant problem. Not only can they occur in both athletes and nonathletes, they require lengthy periods of recuperation and undergo poor natural regeneration, which leads to high reinjury rates. Embryonic stem cells (ESCs) may provide a renewable source of allogeneic cells to use in clinical applications to aid tissue regeneration. Equine ESCs can undergo tenocyte differentiation in vivo and in vitro, but the immune properties of tenocytes isolated from either ESCs or tissues have not previously been characterized. Here, we demonstrate that equine tenocytes derived from fetal and adult tendon tissue and ESCs express robust levels of major histocompatibility complex (MHC) I but no MHC II in response to inflammatory cytokine interferon gamma (IFNγ). However, MHC expression does not affect their allorecognition by peripheral blood mononuclear cells in vitro. Adult and fetal tenocytes remain immune privileged and strongly immune suppressive in both the presence and absence of exogenously applied IFNγ. In contrast, ESC-derived tenocytes are immune privileged even in the presence of IFNγ, but they are only weakly immune suppressive in the presence but not in the absence of exogenously applied IFNγ. This is despite ESC-tenocytes expressing a number of genes involved in immune modulation at significantly higher levels than those expressed by adult and fetal tenocytes when in standard, nonstimulated monolayer culture. Together, this work suggests that, similar to other fibroblasts, tenocytes have immune modulatory properties, and that culture-expanded tenocytes derived from primary tissues or ESCs may be safe to use in clinical transplantations to injured tendons of unrelated animals.

Domestic cats suffer from a range of inherited genetic diseases, many of which display similarities with equivalent human conditions. Developing cellular models for these inherited diseases would enable drug discovery, benefiting feline health and welfare as well as enhancing the potential of cats as relevant animal models for translation to human medicine. Advances in our understanding of these diseases at the cellular level have come from the use of induced pluripotent stem cells (iPSCs). iPSCs can differentiate into virtually any cell type and can be derived from adult somatic cells, therefore overcoming the ethical implications of destroying embryos to obtain embryonic stem cells. No studies, however, report the generation of iPSCs from domestic cats [feline iPSCs (fiPSCs)]. Feline adipose-derived fibroblasts were infected with amphotropic retrovirus containing the coding sequences for human Oct4 , Sox2 , Klf4 , cMyc , and Nanog . Isolated iPSC clones were expanded on inactivated mouse embryonic fibroblasts in the presence of feline leukemia inhibitory factor (fLIF). Retroviral delivery of human pluripotent genes gave rise to putative fiPSC colonies within 5-7 days. These iPS-like cells required fetal bovine serum and fLIF for maintenance. Colonies were domed with refractile edges, similar to mouse iPSCs. Immunocytochemistry demonstrated positive staining for stem cell markers: alkaline phosphatase, Oct4 , Sox2 , Nanog , and SSEA1. Cells were negative for SSEA4. Expression of endogenous feline Nanog was confirmed by quantitative polymerase chain reaction. The cells were able to differentiate in vitro into cells representative of the three germ layers. These results confirm the first generation of induced pluripotent stem cells from domestic cats. These cells will provide valuable models to study genetic diseases and explore novel therapeutic strategies.

Horses, like humans, can experience bone fractures and due to their large size and the need to bear weight on all limbs during the recovery period, they can be difficult to treat. Surgical techniques to improve fracture repair are improving, but to date, regenerative medicine technologies to aid fracture healing are not commonly applied in horses. We have previously demonstrated that equine induced pluripotent stem cells (iPSCs) can be differentiated into bone forming osteoblasts in 2D culture. In this study, we report on the use of a thermoplastic, 3D-printed polymer to provide a scaffold for successful, in vitro osteoblast differentiation of equine iPSCs. The scaffold provides a transparent, cost-effective solution to allow the analysis of osteoblast differentiation using live-cell imaging, immunohistochemistry, and quantitative polymerase chain reaction. This in vitro study demonstrates the future feasibility of generating 3D bone constructs through the cell seeding of scaffolds to use in regenerative medicine strategies to improve fracture repair in a relevant, large animal model.

Bone fractures occur in horses following traumatic and non-traumatic (bone overloading) events. They can be difficult to treat due to the need for the horse to bear weight on all legs during the healing period. Regenerative medicine to improve fracture union and recovery could significantly improve horse welfare. Equine induced pluripotent stem cells (iPSCs) have previously been derived. Here we show that equine iPSCs cultured for 21 days in osteogenic induction media on an OsteoAssay surface upregulate the expression of osteoblast associated genes and proteins, including COL1A1 , SPARC , SPP1 , IBSP , RUNX2 and BGALP We also demonstrate that iPSC-osteoblasts are able to produce a mineralised matrix with both calcium and hydroxyapatite deposition. Alkaline phosphatase activity is also significantly increased during osteoblast differentiation. Although the genetic background of the iPSC donor animal affects the level of differentiation observed after 21 days of differentiation, less variation between lines of iPSCs derived from the same horse was observed. The successful, direct, differentiation of equine iPSCs into osteoblasts may provide a source of cells for future regenerative medicine strategies to improve fracture repair in horses undergoing surgery. iPSC-derived osteoblasts will also provide a potential tool to study equine bone development and disease., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Pluripotent stem cells have the capacity to grow indefinitely in culture and differentiate into derivatives of the three germ layers. These properties underpin their potential to be used in regenerative medicine. Originally derived from early embryos, pluripotent stem cells can now be derived by reprogramming an adult cell back to a pluripotent state. Companion animals such as horses, dogs, and cats suffer from many injuries and diseases for which regenerative medicine may offer new treatments. As many of the injuries and diseases are similar to conditions in humans the use of companion animals for the experimental and clinical testing of stem cell and regenerative medicine products would provide relevant animal models for the translation of therapies to the human field. In order to fully utilize companion animal pluripotent stem cells robust, standardized methods of characterization must be developed to ensure that safe and effective treatments can be delivered. In this review we discuss the methods that are available for characterizing pluripotent stem cells and the techniques that have been applied in cells from companion animals. We describe characteristics which have been described consistently across reports as well as highlighting discrepant results. Significant steps have been made to define the in vitro culture requirements and drive lineage specific differentiation of pluripotent stem cells in companion animal species. However, additional basic research to compare pluripotent stem cell types and define characteristics of pluripotency in companion animal species is still required. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

The transcription factor scleraxis is required for tendon development and is upregulated during embryonic stem cell (ESC) differentiation into tenocytes. However, its role beyond early embryonic development is not defined. We utilized a short hairpin RNA to knock down scleraxis expression in ESCs and adult and fetal tenocytes. No effect on growth or morphology was observed in two-dimensional cultures. However, scleraxis knockdown in fetal tenocytes significantly reduced COL1A1, COMP, and SOX9 gene expression. Scleraxis knockdown in adult tenocytes had no effect on the expression of these genes. Strikingly, differentiating ESCs and fetal tenocytes without scleraxis failed to reorganize a three-dimensional (3D) matrix and generate artificial tendons. This was associated with a significantly reduced survival. In contrast, there was no effect on the survival and remodeling capacity of adult tenocytes following scleraxis knockdown. Overexpression of scleraxis in fetal tenocytes rescued gene expression, cell survival in 3D, and subsequent matrix contraction. Together, these results demonstrate that scleraxis is not only essential for ESC differentiation into tenocytes but that it also has an active role in maintaining fetal tenocytes, which is then redundant in adult tenocytes.

Background: Some universities are looking to provide a more diverse range of clinical learning experiences through extended clinical placement programs. This approach will potentially have a significant impact on practitioners. The aim of this study was to conduct a national survey of optometrists to ascertain their perspectives on participating in extended clinical placement programs., Methods: Members of Optometry Australia were invited to participate in a survey conducted during June and July 2014., Results: A total of 268 practitioners participated (six per cent of registered Australian optometrists): 159 were predominantly employees or locums and 109 were owners or managers who identified as the key representative of a practice or organisation for the purpose of this survey. Almost two-thirds (65 per cent) of participants, who were employees or locums were supportive of extended clinical placement programs. Among this group, females were more likely to be supportive than males (p = 0.033). In comparison, just over one-third (34 per cent) of participants who were key decision-makers were supportive, with 30 per cent possibly supportive and 36 per cent not supportive. Among key decision-makers, males were more likely to be supportive (p = 0.009). The top three perceived advantages of supervising a student were: opportunity to mentor early career development, opportunity to give back to the profession and future recruitment. The top three perceived disadvantages were: burden on time, decrease in number of patients examined and burden on support staff. Suggested incentives for supervising students were credit for continuing professional development and financial remuneration., Conclusion: There appears to be moderate support for extended clinical placement programs; however, there are incentives that might engage a larger proportion of the profession in the future. These findings can inform the development of effective and sustainable clinical training programs for optometry students. Additionally, the findings might be used as evidence to seek Government support for clinical placement training in optometry., (© 2016 Optometry Australia.)

Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs) differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs) have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In two-dimensional differentiation assays, the iPSCs expressed tendon-associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in three-dimensional (3D) differentiation assays, the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3D in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application.

Background Aims: Several studies report beneficial effects of autologous and allogeneic stem cells on wound healing. However, no comparison between autologous versus allogeneic epithelial-like stem cells (EpSCs) has been made so far. For this reason, we first hypothesize that both EpSC types enhance wound healing in comparison to vehicle treatment and untreated controls. Second, on the basis of other studies, we hypothesized that there would be no difference between autologous and allogeneic EpSCs., Methods: Twelve full-thickness skin wounds were created in six horses. Each horse was subjected to (i) autologous EpSCs, (ii) allogeneic EpSCs, (iii) vehicle treatment or (iv) untreated control. Wound evaluation was performed at day 3, 7 and 14 through wound exudates and at week 1, 2 and 5 through biopsies., Results: Wound circumference and surface were significantly smaller in autologous EpSC-treated wounds. A significantly lower amount of total granulation tissue (overall) and higher vascularization (week 1) was observed after both EpSC treatments. Significantly more major histocompatibility complex II-positive and CD20-positive cells were noticed in EpSC-treated wounds at week 2. In autologous and allogeneic groups, the number of EpSCs in center biopsies was low after 1 week (11.7% and 6.1%), decreased to 7.6% and 1.7%, respectively (week 2), and became undetectable at week 5., Conclusions: These results confirm the first hypothesis and partially support the second hypothesis. Besides macroscopic improvements, both autologous and allogeneic EpSCs had similar effects on granulation tissue formation, vascularization and early cellular immune response., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: This study is an investigation of how Australian and New Zealand schools of optometry prepare students for culturally competent practice. The aims are: (1) to review how optometric courses and educators teach and prepare their students to work with culturally diverse patients; and (2) to determine the demographic characteristics of current optometric students and obtain their views on cultural diversity., Methods: All Australian and New Zealand schools of optometry were invited to participate in the study. Data were collected with two surveys: a curriculum survey about the content of the optometric courses in relation to cultural competency issues and a survey for second year optometry students containing questions in relation to cultural awareness, cultural sensitivity and attitudes to cultural diversity., Results: Four schools of optometry participated in the curriculum survey (Deakin University, Flinders University, University of Melbourne and University of New South Wales). Sixty-three students (22.3 per cent) from these four schools as well as the University of Auckland participated in the student survey. Cultural competency training was reported to be included in the curriculum of some schools, to varying degrees in terms of structure, content, teaching method and hours of teaching. Among second year optometry students across Australia and New Zealand, training in cultural diversity issues was the strongest predictor of cultural awareness and sensitivity after adjusting for school, age, gender, country of birth and language other than English., Conclusion: This study provides some evidence that previous cultural competency-related training is associated with better cultural awareness and sensitivity among optometric students. The variable approaches to cultural competency training reported by the schools of optometry participating in the study suggest that there may be opportunity for further development in all schools to consider best practice training in cultural competency., (© 2014 The Authors. Clinical and Experimental Optometry © 2014 Optometrists Association Australia.)

The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of equine embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared with tenogenic induction in monolayer ESC cultures. The addition of TGF-β3 to the 3D cultures further up-regulates the expression of these genes and also induces the expression of mature tenocyte markers Tenomodulin and Thrombospondin-4. Our results show that when ESCs are exposed to the intrinsic forces exerted by a 3D culture environment, they express tendon-associated genes and proteins which are indicative of tenocyte lineage differentiation and that this effect is synergistically enhanced and accelerated by the addition of TGF-β3.

Introduction: Autologous mesenchymal stem cells (MSCs) are an attractive concept in regenerative medicine, but their mechanism of action remains poorly defined. No immune response is reported after in vivo injection of allogeneic equine MSCs or embryo-derived stem cells (ESCs) into the equine tendon, which may be due to the cells' immune-privileged properties. This study further investigates these properties to determine their potential for clinical application in other tissues., Methods: Mitomycin C-treated MSCs, ESCs, or differentiated ESCs (dESCs) were cultured with allogeneic equine peripheral blood mononuclear cells (PBMCs), and their effect on PBMC proliferation, in the presence or absence of interferon-gamma (IFN-γ) was determined. MSCs and super-antigen (sAg)-stimulated PBMCs were co-cultured directly or indirectly in transwells, and PBMC proliferation examined. Media from MSC culture were harvested and used for PBMC culture; subsequent PBMC proliferation and gene expression were evaluated and media assayed for IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 and IL-6 proteins with enzyme-linked immunosorbent assay (ELISA)., Results: Co-culture of PBMCs with ESCs or dESCs did not affect baseline proliferation, whereas co-culture with MSCs significantly suppressed baseline proliferation. Stimulation of PBMC proliferation by using super-antigens (sAgs) was also suppressed by co-culture with MSCs. Inhibition was greatest with direct contact, but significant inhibition was produced in transwell culture and by using MSC-conditioned media, suggesting that soluble factors play a role in MSC-mediated immune suppression. The MSCs constitutively secrete IL-6, even in the absence of co-culture with PBMCs. MSC-conditioned media also brought about a change in the cytokine-expression profile of sAg-stimulated PBMCs, significantly reducing PBMC expression of IL-6, IFN-γ, and TNF-α., Conclusions: Equine MSCs and ESCs possess a degree of innate immune privilege, and MSCs secrete soluble factors that suppress PBMC proliferation and alter cytokine expression. These properties may make possible the future clinical use of allogeneic stem cells to help standardize and broaden the scope of treatment of tissue injuries.

Tendon injuries are one of the most common orthopaedic problems in both human and equine athletes. When a damaged tendon heals naturally, it loses a substantial part of the original strength and elasticity. Therefore, tendons recover structurally (reparation) but not functionally (regeneration) after conservative medical or surgical treatment. Since the structure and matrix composition of human and equine tendons share many similarities, the nature of tendon injuries are also strongly comparable in both species. Therefore, the evaluation of regenerative therapies in horses may have applications for future human medicine and vice versa. The current review focuses briefly on the physiology of human and equine tendon in order to better comprehend the modus operandi of this structure under pathophysiological circumstances. In addition, the reparative effects of conservative medical and surgical interventions are discussed concisely, and an extensive overview is given on the regenerative therapies that are currently being explored. For the latter, the results of equine clinical studies might prove invaluable for gaining additional insights into the treatment of human tendinopathies, since not all of these novel regenerative therapies have been evaluated in humans yet.

Reasons for Performing Study: Injury to the superficial digital flexor tendon (SDFT) is common in racing and sport horses and poor tendon regeneration leads to high reinjury rates. Autologous mesenchymal stromal cells (MSCs) are being used clinically to improve tendon regeneration but they have some practical limitations. Embryonic stem cells (ESCs) may overcome these limitations but their fate following injection into the damaged SDFT is unknown., Objective: To inject MSCs and ESCs into distinct areas of damage in the SDFT and monitor their survival over a 3 month period., Methods: MSCs and ESCs expressing different reporter genes were injected into separate sites of mechanically induced damage in SDFTs. Cell survival and distribution were examined post mortem after 10, 30, 60 and 90 days and host immune responses determined., Results: Neither MSCs nor ESCs produced signs of cell-mediated immune response or tumour formation. ESC survival was high and numbers were maintained at a constant level over 90 days. ESCs were present at all sites of damage. In contrast, MSCs showed <5% survival at 10 days and numbers declined over the course of the experiment. MSCs were detected only at the site into which they were injected., Conclusions: ESCs survived in greater numbers than MSCs in the damaged tendon and did not induce an immune response, or form tumours at the injection sites in the 90 day time period studied. ESCs also demonstrated an ability to migrate to other areas of damage within the same tendon, whereas MSCs did not., Potential Relevance: ESCs can be used allogeneically, therefore providing a possible 'off the shelf' source of cells for therapeutic use which overcomes the practical limitations of autologous MSCs. Furthermore, MSCs and ESCs have different survival rates and migration patterns in the damaged tendon, suggesting that they may produce different functional effects. This may have clinical relevance to treating tendon injuries in the horse., (© 2010 EVJ Ltd.)

Mesenchymal stromal (MS) cells have been derived from multiple sources in the horse including bone marrow, adipose tissue and umbilical cord blood. To date these cells have been investigated for their differentiation potential and are currently being used to treat damage to horse musculoskeletal tissues. However, no work has been done in horse MS cells to examine the expression profile of proteins and cell surface antigens that are expressed in human MS cells. The identification of such profiles in the horse will allow the comparison of putative MS cells isolated from different laboratories and different tissues. At present it is difficult to ascertain whether equivalent cells are being used in different reports. Here, we report on the expression of a range of markers used to define human MS cells. Using immunocytochemistry we show that horse MS cells homogenously express collagens, alkaline phosphatase activity, CD44, CD90 and CD29. In contrast, CD14, CD79α and the embryonic stem cell markers Oct-4, SSEA (stage specific embryonic antigen) -1, -3, -4, TRA (tumor rejection antigen) -1-60 and -1-81 are not expressed. The MS cells also express MHC class I antigens but do not express class II antigens, although they are inducible by treatment with interferon gamma (IFN-γ).

Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP labelled cells located mainly within injected lesions, but with a small proportion integrated into the crimp pattern of adjacent healthy areas of tendon. Furthermore, there was no visible cell mediated immune response to allogeneic MPCs in either of the host horses.

Huntington's disease (HD) is a dominantly-inherited neurodegenerative disorder which is incurable and ultimately fatal. HD is characterised by widespread mRNA dysregulation, particularly in neurons of the forebrain, by mechanisms which are not fully understood. Such dysregulation has been demonstrated to result, in part, from aberrant nuclear localisation of the transcriptional repressor, REST. Here, we show that expression of a number of neuronal-specific microRNAs is also dysregulated in HD tissues, probably as a result of increased repression by REST. This phenomenon is observed in both murine models of HD and in the brains of human HD sufferers. MicroRNA loss is reflected in increased levels of a number of target messenger RNAs. These data are the first to demonstrate a role for microRNAs in HD, and indicate that the molecular aetiology of HD is reflected in a loss of neuronal identity, caused in part by dysregulation of both transcriptional and post-transcriptional mechanisms.

Embryonic stem-like (ES-like) cells have now been derived from the inner cell mass (ICM) of horse embryos at the blastocyst stage. Because they have been shown to express cell-surface antigens found in both human and mouse ES cells, the present study investigated gene expression patterns in day-7 horse blastocysts from which the horse ES-like cells had been derived originally. The genes studied included Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, SSEA-4, tumor rejection antigen-1-60 (TRA-1-60), TRA-1-81, and alkaline phosphatase activity, and whereas all three of the SSEA antigens were expressed in both the ICM and the trophoblast on day 7, Oct-4, TRA-1-60, TRA-1-81, and alkaline phosphatase activity were localized mostly in the ICM. Upon in vitro differentiation of the horse ES-like cells, their expression of the stem cell markers was abolished. Therefore, the species-specific expression pattern of stem cell markers in horse ES-like cells reflects gene expression in the blastocysts from which they are derived.