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.

Synthesis and Formulation of Four-Arm PolyDMAEA-siRNA Polyplex for Transient Downregulation of Collagen Type III Gene Expression in TGF-ß1 Stimulated Tenocyte Culture.

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-ß1 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-ß1 on collagen type III gene expressions were analyzed by qPCR, showing an optimal dose of 10 ng mL-1 TGF-ß1 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.

Canine Corneal Stromal Cells Have Multipotent Mesenchymal Stromal Cell Properties In Vitro.

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.

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.

Osteoblast differentiation of equine induced pluripotent stem cells.

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.

Comparison of autologous versus allogeneic epithelial-like stem cell treatment in an in vivo equine skin wound model.

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.

Equine mesenchymal stromal cells and embryo-derived stem cells are immune privileged in vitro.

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 regeneration in human and equine athletes: Ubi Sumus-Quo Vadimus (where are we and where are we going to)?

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.

Defining the expression of marker genes in equine mesenchymal stromal cells.

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).