Equine Embryonic Stem Cell-Derived Tenocytes are Insensitive to a Combination of Inflammatory Cytokines and Have Distinct Molecular Responses Compared to Primary Tenocytes.

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-1ß 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.

Effects of resveratrol and its analogues on the cell cycle of equine mesenchymal stem/stromal cells.

Resveratrol (RSV; trans-3,5,4'-trihydroxystilbene) strongly activates sirtuin 1, and it and its analogue V29 enhance the proliferation of mesenchymal stem/stromal cells (MSCs).Although culture medium containing 5-azacytydine and RSV inhibits senescence of adipose tissue-derived MSCs isolated from horses with metabolic syndrome, few studies have reported the effects of RSV on equine bone marrow-derived MSCs (eBMMSCs) isolated from horses without metabolic syndrome. The aim of this study was to investigate the effects of RSV and V29 on the cell cycle of eBMMSCs. Following treatment with 5 µM RSV or 10 µM V29, the cell proliferation capacity of eBMMSCs derived from seven horses was evaluated by EdU (5-ethynyl-2'-deoxyuridine) and Ki-67 antibody assays. Brightfield images of cells and immunofluorescent images of EdU, Ki-67, and DAPI staining were recorded by fluorescence microscopy, and the number of cells positive for each was quantified and compared by Friedman's test at P<0.05. The growth fraction of eBMMSCs was significantly increased by RSV and V29 as measured by the EdU assay (control 28.1% ± 13.8%, V29 31.8% ± 14.6%, RSV 32.0% ± 10.8%; mean ± SD; P<0.05) but not as measured by the Ki-67 antibody assay (control 27.0% ± 11.2%, V29 27.4% ± 10.8%, RSV 27.7% ± 6.8%). RSV and V29 promoted progression of the cell cycle of eBMMSCs into the S phase and may be useful for eBMMSC expansion.

Exploration of Mediators Associated with Myocardial Remodelling in Feline Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) affects both humans and cats and exhibits considerable interspecies similarities that are exemplified by underlying pathological processes and clinical presentation to the extent that developments in the human field may have direct relevance to the feline disease. Characteristic changes on histological examination include cardiomyocyte hypertrophy and interstitial and replacement fibrosis. Clinically, HCM is characterised by significant diastolic dysfunction due to a reduction in ventricular compliance and relaxation associated with extracellular matrix (ECM) remodelling and the development of ventricular hypertrophy. Studies in rodent models and human HCM patients have identified key protein mediators implicated in these pathological changes, including lumican, lysyl oxidase and TGF-ß isoforms. We therefore sought to quantify and describe the cellular location of these mediators in the left ventricular myocardium of cats with HCM and investigate their relationship with the quantity and structural composition of the ECM. We identified increased myocardial content of lumican, LOX and TGF-ß2 mainly attributed to their increased expression within cardiomyocytes in HCM cats compared to control cats. Furthermore, we found strong correlations between the expressions of these mediators that is compatible with their role as important components of cellular pathways promoting remodelling of the left ventricular myocardium. Fibrosis and hypertrophy are important pathological changes in feline HCM, and a greater understanding of the mechanisms driving this pathology may facilitate the identification of potential therapies.

Technical note: Cartilage imaging with sub-cellular resolution using a laboratory-based phase-contrast x-ray microscope.

BACKGROUND: Microscopic imaging of cartilage is a key tool for the study and development of treatments for osteoarthritis. When cellular and sub-cellular resolution is required, histology remains the gold standard approach, albeit limited by the lack of volumetric information as well as by processing artifacts. Cartilage imaging with the sub-cellular resolution has only been demonstrated in the synchrotron environment. PURPOSE: To provide a proof-of-concept demonstration of the capability of a laboratory-based x-ray phase-contrast microscope to resolve sub-cellular features in a cartilage sample. METHODS: This work is based on a laboratory-based x-ray microscope using intensity-modulation masks. The structured nature of the beam, resulting from the mask apertures, allows the retrieval of three contrast channels, namely, transmission, refraction and dark-field, with resolution depending only on the mask aperture width. An ex vivo equine cartilage sample was imaged with the x-ray microscope and results were validated with synchrotron tomography and histology. RESULTS: Individual chondrocytes, that is, cells responsible for cartilage formation, could be detected with the laboratory-based microscope. The complementarity of the three retrieved contrast channels allowed the detection of sub-cellular features in the chondrocytes. CONCLUSIONS: We provide the first proof-of-concept of imaging cartilage tissue with sub-cellular resolution using a laboratory-based x-ray microscope.

Influence of Rho/ROCK inhibitor Y-27632 on proliferation of equine mesenchymal stromal cells.

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-ß3 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-ß3. This included a higher proliferation with TGF-ß3 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-ß3 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.

Transdermal drug delivery in horses: An in vitro comparison of skin structure and permeation of two model drugs at various anatomical sites.

BACKGROUND: Oral and parenteral drug delivery in horses can be difficult. Equine-specific transdermal drug formulations offer improved ease of treatment; development of such formulations requires a deeper understanding of the structural and chemical tissue barrier of horse skin. HYPOTHESIS/OBJECTIVES: To compare the structural composition and barrier properties of equine skin. ANIMALS: Six warmblood horses (two males, four females) with no skin diseases. MATERIALS AND METHODS: Routine histological and microscopic analyses were carried out with image analysis for skin from six different anatomical locations. In vitro drug permeation was analysed using a standard Franz diffusion cell protocol coupled with reversed phase-high-performance liquid chromatography detailing flux, lag times and tissue partitioning ratios of two model drug compounds. RESULTS: Epidermal and dermal thicknesses varied between sites. The dermal and epidermal thicknesses of the croup were 1764 ± 115 µm and 36 ± 3.6 µm, respectively, and were significantly different (p < 0.05) from the inner thigh thicknesses which were 824 ± 35 µm and 49 ± 3.6 µm. Follicular density and size also varied. The highest flux for the model hydrophilic molecule (caffeine) was for the flank (3.22 ± 0.36 µg/cm2 /h), while that for the lipophilic molecule (ibuprofen) was for the inner thigh (0.12 ± 0.02 µg/cm2 /h). CONCLUSIONS AND CLINICAL RELEVANCE: Anatomical location differences in equine skin structure and small molecule permeability were demonstrated. These results can aid in the development of transdermal therapies for horses.

Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell-derived factors.

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-1ß, 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-1ß 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-1ß 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.

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations.

Introduction: The interfascicular matrix (IFM; also known as the endotenon) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Methods: Immunolabelling of equine superficial digital flexor tendon was used to identify the interfascicular matrix niche, localising expression patterns of CD31 (endothelial cells), Desmin (smooth muscle cells and pericytes), CD146 (interfascicular matrix cells) and LAMA4 (interfascicular matrix basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. Results: Labelling for CD146 using standard histological and 3D imaging of large intact 3D segments revealed an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms extensive, interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralisation (osteogenesis) and lipid production (adipogenesis). Discussion: This study demonstrates that the interfascicular matrix is a unique tendon cell niche, containing a vascular-rich network of basement membrane, CD31+ endothelial cells, Desmin+ mural cells, and CD146+ cell populations that are likely essential to tendon structure and/or function. Contrary to our hypothesis, interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes. Instead, our results indicate CD146 as a pan-vascular marker within the tendon interfascicular matrix. Together with previous work demonstrating that endogenous tendon CD146+ cells migrate to sites of injury, our data suggest that their mobilisation to promote intrinsic repair involves changes in their relationships with local interfascicular matrix vascular and basement membrane constituents.

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4.

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.

Large Animal Models in Regenerative Medicine and Tissue Engineering: To Do or Not to Do.

Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using in vitro and ex vivo experiments as well as in silico modeling to minimize the need for in vivo studies for screening and early development studies, followed by large animal models which more closely resemble human disease. Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.

Histological evaluation of cellular response to a multifilament electrospun suture for tendon repair.

BACKGROUND: Rotator cuff tendon repair in humans is a commonly performed procedure aimed at restoring the tendon-bone interface. Despite significant innovation of surgical techniques and suture anchor implants, only 60% of repairs heal successfully. One strategy to enhance repair is the use of bioactive sutures that provide the native tendon with biophysical cues for healing. We investigated the tissue response to a multifilament electrospun polydioxanone (PDO) suture in a sheep tendon injury model characterised by a natural history of failure of healing. METHODOLOGY AND RESULTS: Eight skeletally mature English Mule sheep underwent repair with electrospun sutures. Monofilament sutures were used as a control. Three months after surgery, all tendon repairs healed, without systemic features of inflammation, signs of tumour or infection at necropsy. A mild local inflammatory reaction was seen. On histology the electrospun sutures were densely infiltrated with predominantly tendon fibroblast-like cells. In comparison, no cellular infiltration was observed in the control suture. Neovascularisation was observed within the electrospun suture, whilst none was seen in the control. Foreign body giant cells were rarely seen with either sutures. CONCLUSION: This study demonstrates that a tissue response can be induced in tendon with a multifilament electrospun suture with no safety concerns.

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon.

Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000 fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis.

Muscles are anchored to bones through specialized tissues called tendons. Made of bundles of fibers (or fascicles) linked together by an 'interfascicular' matrix, healthy tendons are required for organisms to move properly. Yet, these structures are constantly exposed to damage: the interfascicular matrix, in particular, is highly susceptible to injury as it allows the fascicles to slide on each other. One way to avoid damage could be for the body to continually replace proteins in tendons before they become too impaired. However, the way proteins are renewed in these structures is currently not well understood ­ indeed, it has long been assumed that almost no protein turnover occurs in tendons. In particular, it is unknown whether proteins in the interfascicular matrix have a higher turn over than those in the fascicles. To investigate, Choi, Simpson et al. fed rats on water carrying a molecular label that becomes integrated into new proteins. Analysis of individual proteins from the rats' tendons showed great variation in protein turnover, with some replaced every few days and others only over several years. This suggests that protein turnover is actually an important part of tendon health. In particular, the results show that turnover is higher in the interfascicular matrix, where damage is expected to be more likely. Protein turnover also plays a part in conditions such as cancer, heart disease and kidney disease. Using approaches like the one developed by Choi, Simpson et al. could help to understand how individual proteins are renewed in a range of diseases, and how to design new treatments.

Development of a Cartilage Oligomeric Matrix Protein Neo-Epitope Assay for the Detection of Intra-Thecal Tendon Disease.

The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH2-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (n = 7) versus 58.8 ± 64.3 µg/mL (n = 13); p = 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant > 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (n = 6) versus 1029.8 ± 2188.8 ng/ml (n = 14); p = 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use.

Histopathological and immunohistochemical evaluation of cellular response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch for tendon repair.

We investigated endogenous tissue response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch intended for tendon repair. A sheep tendon injury model characterised by a natural history of consistent failure of healing was chosen to assess the biological potential of woven and aligned electrospun fibres to induce a reparative response. Patches were implanted into 8 female adult English Mule sheep. Significant infiltration of tendon fibroblasts was observed within the electrospun component of the patch but not within the woven component. The cellular infiltrate into the electrospun fibres was accompanied by an extensive network of new blood vessel formation. Tendon fibroblasts were the most abundant scaffold-populating cell type. CD45+, CD4+ and CD14+ cells were also present, with few foreign body giant cells. There were no local or systemic signs of excessive inflammation with normal hematology and serology for inflammatory markers three months after scaffold implantation. In conclusion, we demonstrate that an endogenous healing response can be safely induced in tendon by means of biophysical cues using a woven and electrospun patch.

Evaluation of the Effects of Synovial Multipotent Cells on Deep Digital Flexor Tendon Repair in a Large Animal Model of Intra-Synovial Tendinopathy.

Intra-synovial tendon injuries are a common orthopedic problem with limited treatment options. The synovium is a specialized connective tissue forming the inner encapsulating lining of diarthrodial joints and intra-synovial tendons. It contains multipotent mesenchymal stromal cells that render it a viable source of progenitors for tendon repair. This study evaluated the effects of autologous implantation of cells derived from normal synovium (synovial membrane cells [SMCs]) in augmenting repair in an ovine model of intra-synovial tendon injury. For this purpose, synovial biopsies were taken from the right digital flexor tendon sheath following creation of a defect to the lateral deep digital flexor tendon. Mononuclear cells were isolated by partial enzymatic digestion and assessed for MSC characteristics. Cell tracking and tendon repair were assessed by implanting 5 × 106 cells into the digital flexor tendon sheath under ultrasound guidance with the effects evaluated using magnetic resonance imaging and histopathology. Synovial biopsies yielded an average 4.0 × 105 ± 2.7 × 105 SMCs that exhibited a fibroblastic morphology, variable osteogenic, and adipogenic responses but were ubiquitously strongly chondrogenic. SMCs displayed high expression of CD29 with CD271NEGATIVE and MHC-IILOW cell-surface marker profiles, and variable expression of CD73, CD90, CD105, CD166, and MHC-I. Implanted SMCs demonstrated engraftment within the synovium, though a lack of repair of the tendon lesion over 24 weeks was observed. We conclude healthy synovium is a viable source of multipotent cells, but that the heterogeneity of synovium underlies the variability between different SMC populations, which while capable of engraftment and persistence within the synovium exhibit limited capacity of influencing tendon repair. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:128-138, 2020.

Inducing Pluripotency in the Domestic Cat (Felis catus).

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.

Cardiosphere-derived cells suppress allogeneic lymphocytes by production of PGE2 acting via the EP4 receptor.

Cardiosphere-derived cells (CDCs) are a cardiac progenitor cell population, which have been shown to possess cardiac regenerative properties and can improve heart function in a variety of cardiac diseases. Studies in large animal models have predominantly focussed on using autologous cells for safety, however allogeneic cell banks would allow for a practical, cost-effective and efficient use in a clinical setting. The aim of this work was to determine the immunomodulatory status of these cells using CDCs and lymphocytes from 5 dogs. CDCs expressed MHC I but not MHC II molecules and in mixed lymphocyte reactions demonstrated a lack of lymphocyte proliferation in response to MHC-mismatched CDCs. Furthermore, MHC-mismatched CDCs suppressed lymphocyte proliferation and activation in response to Concanavalin A. Transwell experiments demonstrated that this was predominantly due to direct cell-cell contact in addition to soluble mediators whereby CDCs produced high levels of PGE2 under inflammatory conditions. This led to down-regulation of CD25 expression on lymphocytes via the EP4 receptor. Blocking prostaglandin synthesis restored both, proliferation and activation (measured via CD25 expression) of stimulated lymphocytes. We demonstrated for the first time in a large animal model that CDCs inhibit proliferation in allo-reactive lymphocytes and have potent immunosuppressive activity mediated via PGE2.

Bone marrow mesenchymal stem cells do not enhance intra-synovial tendon healing despite engraftment and homing to niches within the synovium.

BACKGROUND: Intra-synovial tendon injuries display poor healing, which often results in reduced functionality and pain. A lack of effective therapeutic options has led to experimental approaches to augment natural tendon repair with autologous mesenchymal stem cells (MSCs) although the effects of the intra-synovial environment on the distribution, engraftment and functionality of implanted MSCs is not known. This study utilised a novel sheep model which, although in an anatomically different location, more accurately mimics the mechanical and synovial environment of the human rotator cuff, to determine the effects of intra-synovial implantation of MSCs. METHODS: A lesion was made in the lateral border of the lateral branch of the ovine deep digital flexor tendon within the digital sheath and 2 weeks later 5 million autologous bone marrow MSCs were injected under ultrasound guidance into the digital sheath. Tendons were recovered post mortem at 1 day, and 1-2, 4, 12 and 24 weeks after MSC injection. For the 1-day and 1-2-week groups, MSCs labelled with fluorescent-conjugated magnetic iron-oxide nanoparticles (MIONs) were tracked with MRI, histology and flow cytometry. The 4, 12 and 24-week groups were implanted with non-labelled cells and compared with saline-injected controls for healing. RESULTS: The MSCs displayed no reduced viability in vitro to an uptake of 20.0 ± 4.6 pg MIONs per cell, which was detectable by MRI at minimal density of ~ 3 × 104 cells. Treated limbs indicated cellular distribution throughout the tendon synovial sheath but restricted to the synovial tissues, with no MSCs detected in the tendon or surgical lesion. The lesion was associated with negligible morbidity with minimal inflammation post surgery. Evaluation of both treated and control lesions showed no evidence of healing of the lesion at 4, 12 and 24 weeks on gross and histological examination. CONCLUSIONS: Unlike other laboratory animal models of tendon injury, this novel model mimics the failed tendon healing seen clinically intra-synovially. Importantly, however, implanted stem cells exhibited homing to synovium niches where they survived for at least 14 days. This phenomenon could be utilised in the development of novel physical or biological approaches to enhance localisation of cells in augmenting intra-synovial tendon repair.