Development and performance of a targeted whole exome sequencing enrichment kit for the dog (Canis Familiaris Build 3.1)

Whole exome sequencing is a technique that aims to selectively sequence all exons of protein-coding genes. A canine whole exome sequencing enrichment kit was designed based on the latest canine reference genome (build 3.1.72). Its performance was tested by sequencing 2 exome captures, each consisting of 4 pre-capture pooled, barcoded Illumina libraries on an Illumina HiSeq 2500. At an average sequencing depth of 102x, 83 to 86% of the target regions were completely sequenced with a minimum coverage of five and 90% of the reads mapped on the target regions. Additionally, it is shown that the reproducibility within and between captures is high and that pooling four samples per capture is a valid option. Overall, we have demonstrated the strong performance of this WES enrichment kit and are confident it will be a valuable tool in future disease association studies

The use of equine chondrogenic‐induced mesenchymal stem cells as a treatment for osteoarthritis : a randomised, double‐blinded, placebo‐controlled proof‐of‐concept study

Background: There is a need to improve therapies for osteoarthritis in horses. Objectives To assess the efficacy of equine allogeneic chondrogenic-induced mesenchymal stem cells combined with equine allogeneic plasma as a novel therapy for osteoarthritis in horses. Study design: Randomised, double-blinded, placebo-controlled experiment. Methods: In 12 healthy horses, osteoarthritis was induced in the metacarpophalangeal joint using an osteochondral fragment-groove model. Five weeks after surgery, horses were randomly assigned to either an intra-articular injection with chondrogenic-induced mesenchymal stem cells + equine allogeneic plasma (= intervention) or with 0.9% saline solution (= control). From surgery until the study end, horses underwent a weekly joint and lameness assessment. Synovial fluid was collected for cytology and biomarker analysis before surgery and at Weeks 5, 5 + 1d, 7, 9 and 11. At Week 11, horses were subjected to euthanasia, and the metacarpophalangeal joints were evaluated macroscopically and histologically. Results: No serious adverse events or suspected adverse drug reactions occurred during the study. A significant improvement in visual and objective lameness was seen with the intervention compared with the control. Synovial fluid displayed a significantly higher viscosity and a significantly lower glycosaminoglycan concentration in the intervention group. Other biomarkers or cytology parameters were not significantly different between the treatment groups. Significantly less wear lines and synovial hyperaemia were present in the intervention group. The amount of cartilage oligomeric matrix protein, collagen type II and glycosaminoglycans were significantly higher in the articular cartilage of the intervention group. Main limitations: This study assessed the short-term effect of the intervention on a limited number of horses, using an osteoarthritis model. This study also included multiple statistical tests, increasing the risk of type 1 error. Conclusions: Equine allogeneic chondrogenic-induced mesenchymal stem cells combined with equine allogeneic plasma may be a promising treatment for osteoarthritis in horses

Regenerative skin wound healing in mammals : state-of-the-art on growth factor and stem cell based treatments

Mammal skin has a crucial function in several life-preserving processes such as hydration, protection against chemicals and pathogens, initialization of vitamin D synthesis, excretion and heat regulation. Severe damage of the skin may therefore be life-threatening. Skin wound repair is a multiphased, yet well-orchestrated process including the interaction of various cell types, growth factors and cytokines aiming at closure of the skin and preferably resulting in tissue repair. Regardless various therapeutic modalities targeting at enhancing wound healing, the development of novel approaches for this pathology remains a clinical challenge. The time-consuming conservative wound management is mainly restricted to wound repair rather than restitution of the tissue integrity (the so-called “restitutio ad integrum”). Therefore, there is a continued search towards more efficacious wound therapies to reduce health care burden, provide patients with long-term relief and ultimately scarless wound healing. Recent in vivo and in vitro studies on the use of skin wound regenerative therapies provide encouraging results, but more protracted studies will have to determine whether the effect of observed effects are clinically significant and whether regeneration rather than repair can be achieved. For all the aforementioned reasons, this article reviews the emerging field of regenerative skin wound healing in mammals with particular emphasis on growth factor- and stem cell-based therapies

Tenogenically induced allogeneic mesenchymal stem cells for the treatment of proximal suspensory ligament desmitis in a horse

Suspensory ligament injuries are a common injury in sport horses, especially in competing dressage horses. Because of the poor healing of chronic recalcitrant tendon injuries, this represents a major problem in the rehabilitation of sport horses and often compromises the return to the initial performance level. Stem cells are considered as a novel treatment for different pathologies in horses and humans. Autologous mesenchymal stem cells (MSCs) are well known for their use in the treatment of tendinopathies; however, recent studies report a safe use of allogeneic MSCs for different orthopedic applications in horses. Moreover, it has been reported that pre-differentiation of MSCs prior to injection might result in improved clinical outcomes. For all these reasons, the present case report describes the use of allogeneic tenogenically induced peripheral blood-derived MSCs for the treatment of a proximal suspensory ligament injury. During conservative management for 4 months, the horse demonstrated no improvement of a right front lameness with a Grade 2/5 on the American Association of Equine Practitioners (AAEP) scale and a clear hypo-echoic area detectable in 30% of the cross sectional area. From 4 weeks after treatment, the lameness reduced to an AAEP Grade 1/5 and a clear filling of the lesion could be noticed on ultrasound. At 12 weeks (T-4) after the first injection, a second intra-lesional injection with allogeneic tenogenically induced MSCs and platelet rich plasma was given and at 4 weeks after the second injection (T-5), the horse trotted sound under all circumstances with a close to total fiber alignment. The horse went back to previous performance level at 32 weeks after the first regenerative therapy and is currently still doing so (i.e., 20 weeks later or 1 year after the first stem cell treatment). In conclusion, the present case report demonstrated a positive evolution of proximal suspensory ligament desmitis after treatment with allogeneic tenogenically induced MSCs

Treatment of equine degenerative joint disease with autologous peripheral blood-derived mesenchymal stem cells: a case report

A 5-year-old German Warmblood stallion with chronic lameness, attributable to degenerative joint disease (DJD) of the pastern joint unresponsive to medical treatments, was treated with autologous mesenchymal stem cells (MSC). These MSC were isolated from the peripheral blood (PB) of the patient and injected into the pastern joint, at a concentration of 2.5x10(6) cells, twice with an 8-week interval. The positive response to this stem cell treatment was documented by visual gait evaluation as well as objective pressure plate analyses. This paper is the first to describe the use of autologous PB-derived MSC to treat a horse suffering from chronic DJD. The favorable outcome of this single case may stimulate further research on the use of equine peripheral blood as a source of autologous MSC in equine regenerative medicine

Equine epidermis : a source of epithelial-like stem/progenitor cells with in vitro and in vivo regenerative capacities

Besides the presence of somatic stem cells in hair follicles and dermis, the epidermis also contains a subpopulation of stem cells, reflecting its high regenerative capacity. However, only limited information concerning epidermis-derived epithelial-like stem/progenitor cells (EpSCs) is available to date. Nonetheless, this stem cell type could prove itself useful in skin reconstitution after injury. After harvesting from equine epidermis, the purified cells were characterized as EpSCs by means of positive expression for CD29, CD44, CD49f, CD90, Casein Kinase 2, p63, and Ki67, low expression for cytokeratin (CK)14 and negative expression for CD105, CK18, Wide CK, and Pan CK. Furthermore, their self-renewal capacity was assessed in adhesion as well as in suspension. Moreover, the isolated cells were differentiated toward keratinocytes and adipocytes. To assess the regenerative capacities of EpSCs, six full-thickness skin wounds were made: three were treated with EpSCs and platelet-rich-plasma (EpSC/PRP-treated), while the remaining three were administered carrier fluid alone (PRP-treated). The dermis of EpSC/PRP-treated wounds was significantly thinner and exhibited more restricted granulation tissue than did the PRP-treated wounds. The EpSC/PRP-treated wounds also exhibited increases in EpSCs, vascularization, elastin content, and follicle-like structures. In addition, combining EpSCs with a PRP treatment enhanced tissue repair after clinical application

A feasibility study on the use of equine chondrogenic induced mesenchymal stem cells as a treatment for natural occurring osteoarthritis in dogs

Conventional treatments of osteoarthritis (OA) reduce pain and the inflammatory response but do not repair the damaged cartilage. Xenogeneic peripheral blood-derived equine chondrogenically induced mesenchymal stem cells (ciMSC) could thus provide an interesting alternative. Six client-owned dogs with confirmed elbow OA were subjected to a baseline orthopedic examination, pressure plate analysis, general clinical examination, hematological analysis, synovial fluid sampling, and radiographic examination, and their owners completed two surveys. After all examinations, a 0.9% saline solution (placebo control product=CP) was administered intra-articularly. After 6 weeks, all examinations were repeated, owners again completed two surveys, and equine ciMSCs were administered in the same joint. After another 6 weeks, dogs were returned for a final follow-up. No serious adverse events or suspected adverse drug reactions were present during this study. No significant differences in blood analysis were noted between the CP and ciMSC treatment. Two adverse events were observed, both in the same dog, one after CP treatment and one after ciMSC treatment. The owner surveys revealed significantly less pain and lameness after ciMSC treatment compared to after CP treatment. There was no significant difference in the orthopedic examination parameters, the radiographic examination, synovial fluid sampling, and pressure plate analysis between CP treatment and ciMSC treatment. A single intra-articular administration of equine ciMSCs proved to be a well-tolerated treatment, which reduced lameness and pain according to the owner's evaluations compared to a placebo treatment

Relatie tussen huisvesting en fysieke gezondheidsproblemen van paarden: een enquête over de perceptie van paardeneigenaars

The objective of this preliminary study was to investigate the relationship between the housing conditions and the health and welfare of horses. A survey, based on a questionnaire containing 36 multiple choice questions about various aspects of the housing of horses was conducted. A questionnaire was sent via email to approximately 600 horse owners in Flanders. A total of 225 horse owners completed the questionnaire. The study provides a clear picture of the risk factors that horse owners in practice recognize and the link they see between housing related diseases. Although horse owners usually are sufficiently aware of these influences, they are not taken care of in practice. According to fifty percent of the respondents, the major reason is the impracticability of the advice of the veterinarian. According to the horse owners, the main risk factors affecting the health of horses are: draft, the lack of quarantine measures and the presence of (sharp) foreign objects in the stable. As a consequence, more than 50% of the respondents report nasal discharge and coughing as common problems in their horses. Sixty-seven percent of the horse owners are satisfied with the overall management of the stable. However, there is a widespread dissatisfaction with regard to quarantine measures, in case of a disease outbreak (30% of the horse owners) and in case of the introduction of new animals into a group (36%). Fifty percent of the respondents score their own stable infrastructure 8/10 or more while about one out of four is less satisfied (7/10) about the floor and the walls of their stables. The results of this study can help owners and veterinarians to identify housing factors that may increase the risk to health and welfare problems in horses. This should lead to an improved well-being of the modern, often prolonged - housed horse

Chondrogenic priming at reduced cell density enhances cartilage adhesion of equine allogeneic MSCs : a loading sensitive phenomenon in an organ culture study with 180 explants

Background: Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain unanswered concerning mesenchymal stem cell (MSC) adhesion and incorporation into cartilage. Methods: To this end, peripheral blood (PB) MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs) for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x10(6) vs 1.0 x10(6)). In total, 180 explants of six horses (30 per horse) were divided into five groups: no lesion (i), lesion alone (ii), lesion with naive MSCs (iii), lesion with chondrogenically-induced MSCs (iv) and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v). Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14). Results: COMP expression was selectively increased by chondrogenic induction (p = 0.0488). PEMF stimulation (1mT for 10 minutes) further augmented COL II expression over induced values (p = 0.0405). On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9%) and lesion filling (3.7%) in all the different conditions (p < 0.0001). Remarkably, homogenous cell distribution was characteristic for chondrogenic induced MSCs (+/- PEMFs), whereas clump formation occurred in 39% of uninduced MSC treated cartilage explants. Finally, unloaded explants seeded with a moderately low density of MSCs exhibited greater lesion filling (p = 0.0022) and surface adherence (p = 0.0161) than explants seeded with higher densities of MSCs. In all cases, the overall amount of lesion filling decreased from day 5 to 14 (p = 0.0156). Conclusion: The present study demonstrates that primed chondrogenic induction of MSCs at a lower cell density without loading results in significantly enhanced and homogenous MSC adhesion and incorporation into equine cartilage. Copyright (C) 2015 S. Karger AG, Base

Equine allogeneic chondrogenic induced mesenchymal stem cells are an effective treatment for degenerative joint disease in horses

Degenerative joint disease is one of the main causes of equine early retirement from pleasure riding or a performance career. The disease is initially triggered by an abnormal loading of normal cartilage or a normal loading of abnormal cartilage. This primary insult is accompanied with joint inflammation, which leads to further progressive degeneration of the articular cartilage and changes in the surrounding tissues. Therefore, in search for an effective treatment, 75 adult horses with early signs of degenerative fetlock joint disease were enrolled in a randomized, multicenter, double-blinded, and placebo-controlled study. Fifty animals were injected intra-articularly with the investigational veterinary product (IVP) consisting of allogeneic chondrogenic induced mesenchymal stem cells (ciMSCs) with equine allogeneic plasma, and 25 horses were injected with 0.9% NaCl (saline) control product. From week 3 to 18 after treatment, lameness scores (P<0.001), flexion test responses (P<0.034), and joint effusion scores (P<0.001) were remarkably superior in IVP-treated horses. Besides nasal discharge in both treatment groups, no adverse events were observed during the entire study period. On long-term follow-up (1 year), significantly more investigational product-treated horses were working at training level or were returned to their previous level of work (P<0.001)