Low-dose xenogeneic mesenchymal stem cells target canine osteoarthritis through systemic immunomodulation and homing.

BACKGROUND: As current therapies for canine osteoarthritis (OA) provide mainly symptomatic improvement and fail to address the complex pathology of the disease, mesenchymal stem cells (MSCs) offer a promising biological approach to address both aspects of OA through their immunomodulatory properties. METHODS: This study aimed to investigate the safety and efficacy of xenogeneic MSCs in dogs with OA at different dose levels after intravenous injection. OA was surgically induced in the right stifle joint. Thirty-two male and female dogs were divided into three treatment groups and a control group. Regular general physical examinations; lameness, joint, radiographic, and animal caretaker assessments; pressure plate analyses; and blood analyses were performed over 42 days. At study end, joint tissues were evaluated regarding gross pathology, histopathology, and immunohistochemistry. In a follow-up study, the biodistribution of intravenously injected 99mTc-labeled equine peripheral blood-derived MSCs was evaluated over 24h in three dogs after the cruciate ligament section. RESULTS: The dose determination study showed the systemic administration of ePB-MSCs in a canine OA model resulted in an analgesic, anti-inflammatory, and joint tissue protective effect associated with improved clinical signs and improved cartilage structure, as well as a good safety profile. Furthermore, a clear dose effect was found with 0.3 × 106 ePB-MSCs as the most effective dose. In addition, this treatment was demonstrated to home specifically towards the injury zone in a biodistribution study. CONCLUSION: This model-based study is the first to confirm the efficacy and safety of systemically administered xenogeneic MSCs in dogs with OA. The systemic administration of a low dose of xenogeneic MSCs could offer a widely accessible, safe, and efficacious treatment to address the complex pathology of canine OA and potentially slow down the disease progression by its joint tissue protective effect.

Intravenous Injection of Equine Mesenchymal Stem Cells in Dogs with Articular Pain and Lameness: A Feasibility Study.

Osteoarthritis is a frequently occurring joint disorder in veterinary practice. Current treatments are focused on pain and inflammation; however, these are not able to reverse the pathological condition. Mesenchymal stem cells (MSCs) could provide an interesting alternative because of their immunomodulatory properties. The objective of this study was to evaluate the potential of a single intravenous (IV) injection of xenogeneic equine peripheral blood-derived MSCs (epbMSCs) as treatment for articular pain and lameness. Patients with chronic articular pain were injected intravenously with epbMSCs. They were evaluated at three time points (baseline and two follow-ups) by a veterinarian based on an orthopedic joint assessment and an owner canine brief pain inventory scoring. Thirty-five dogs were included in the safety and efficacy evaluation of the study. Results showed that the epbMSC therapy was well tolerated, with no treatment-related adverse events and no increase in articular heat or pain. A significant improvement in lameness, range of motion, joint effusion, pain severity, and interference scores was found 6 weeks post-treatment compared with baseline. This study demonstrates that future research on IV administration of epbMSCs is warranted to further explore its possible beneficial effects in dogs with chronic articular pain and lameness. Clinical Trial gov ID: EC_2018_002.

Immunogenicity analysis of BPV-1 positive equine sarcoid-derived cultured fibroblasts.

Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovine papilloma-virus (BPV)- 1 infected fibroblasts using a flow cytometric crossmatch assay. The presence of antibodies against exogenous bovine serum albumin (BSA) and fibroblast-like mesenchymal stromal cells (MSCs) was also evaluated by ELISA and flow cytometry, respectively. The viral load in the sarcoid explants, the corresponding cultured sarcoid fibroblasts, and matched peripheral blood mononuclear cells (PBMCs) from affected horses were determined by quantitative BPV-1/- 2 PCR analysis. Antibodies against autologous sarcoid cells were present in six out of twelve sarcoid-affected horses. Serum from all horses showed cross reactivity with allogeneic sarcoid cells, while only a part reacted with BSA or MSCs. Screening of host PBMCs demonstrated the absence of BPV E1 nucleic acids. Statistical analysis revealed a significantly higher mean viral load in the parental sarcoid tissue compared to the low passage fibroblasts (P < 0.001). These results support the hypothesis that sarcoid-affected horses may develop antibodies recognizing tumour-specific antigens. In contrast to sarcoid explants, equine PBMCs do not seem to contain complete BPV genomes. These results provide a basis for future investigations on the clinical relevance of these antibodies.

Assessing the functional properties of tenogenic primed mesenchymal stem cells in ex vivo equine tendon and ligament explants: A preliminary study.

Injuries to equine tendons and ligaments are career-compromising, causing reduced performance and premature retirement. Promising treatment alternatives have been investigated in the field of mesenchymal stem cells (MSCs). In this study, the tissue adherence and protein expression of tenogenic primed mesenchymal stem cells (tpMSCs) after administration to ex vivo tendon and ligament explants is investigated. First, collagen type I (COL I) and smooth muscle actin (SMA) expression was assessed in cytospins prepared from native MSCs and tpMSCs. Second, equine superficial digital flexor tendon and suspensory ligament explants were cultivated, and a lesion was treated with both cell types. Subsequently, cell adhesion to the explants and the amount of COL I and SMA positive cells was evaluated. The cytospins revealed a significantly higher COL I and lower SMA expression in tpMSCs compared to native MSCs. In the explants, tpMSCs showed a significantly higher tendon and ligament adherence. Furthermore, a significantly higher percentage of COL I positive and a lower percentage of SMA positive cells were observed in the lesions treated with tpMSCs. The results of these explant co-cultures may demonstrate at least a part of the mechanism of action and functional properties of tpMSCs in restoring function to tendons and ligaments.

Repeated intra-articular administration of equine allogeneic peripheral blood-derived mesenchymal stem cells does not induce a cellular and humoral immune response in horses.

OBJECTIVE: The use of mesenchymal stem cells (MSCs) for the treatment of equine joint disease is widely investigated because of their regenerative and immunomodulatory potential. Allogeneic MSCs provide a promising alternative to autologous MSCs, since the former are immediately available and enable a thorough donor screening. However, questions have been raised concerning the immunogenic potential of allogeneic MSCs, especially after repeated administration. METHODS: Current retrospective study assessed the cellular and humoral immunogenicity of ten jumping and dressage horses with naturally occurring degenerative joint disease which were treated 3 times intra-articularly with a 1 mL stem cell suspension containing 1.4-2.5 million chondrogenic induced equine allogeneic peripheral blood-derived MSCs (ciMSCs) combined with 1 mL equine allogeneic plasma. Stem cells from 2 donor horses were used. Horses were clinically evaluated for joint effusion, presence of pain to palpation and skin surface temperature at the local injection site, joint range of motion, occurrence of adverse events and the presence of ectopic tissue. The cellular immune response was analyzed using a modified mixed lymphocyte reaction and the humoral immune response was investigated using a flow cytometric crossmatch assay by which the presence of alloantibodies against the ciMSCs was evaluated. Presence of anti-bovine serum albumin antibodies was detected via ELISA. RESULTS: Clinical evaluation of the horses revealed no serious adverse effects or suspected adverse drug reactions and no ectopic tissue formation at the local injection site or in other areas of the body. Generally, repeated administration led to a decrease of horses with joint effusion of the affected joint. Pain to palpation, skin surface temperature and joint range of motion did not increase or even decreased after treatment administration. Allogeneic ciMSCs did not induce a cellular immune response and no alloantibodies were detected in the recipients' serum, regardless the presence of BSA antibodies in 70 % of the horses. CONCLUSION: Repeated intra-articular injections with allogeneic equine ciMSCs did not elicit clinically relevant adverse events. Furthermore, current study indicates the absence of a cellular or a humoral immune response following repeated intra-articular injections.

Scintigraphic tracking of 99mTechnetium-labelled equine peripheral blood-derived mesenchymal stem cells after intravenous, intramuscular, and subcutaneous injection in healthy dogs.

BACKGROUND: Mesenchymal stem cell treatments in dogs have been investigated as a potential innovative alternative to current conventional therapies for a variety of conditions. So far, the precise mode of action of the MSCs has yet to be determined. The aim of this study was to gain more insights into the pharmacokinetics of MSCs by evaluating their biodistribution in healthy dogs after different injection routes. METHODS: Three different studies were performed in healthy dogs to evaluate the biodistribution pattern of radiolabelled equine peripheral blood-derived mesenchymal stem cells following intravenous, intramuscular and subcutaneous administration in comparison with free 99mTechnetium. The labelling of the equine peripheral blood-derived mesenchymal stem cells was performed using stannous chloride as a reducing agent. Whole-body scans were obtained using a gamma camera during a 24-h follow-up. RESULTS: The labelling efficiency ranged between 59.58 and 83.82%. Free 99mTechnetium accumulation was predominantly observed in the stomach, thyroid, bladder and salivary glands, while following intravenous injection, the 99mTechnetium-labelled equine peripheral blood-derived mesenchymal stem cells majorly accumulated in the liver throughout the follow-up period. After intramuscular and subcutaneous injection, the injected dose percentage remained very high at the injection site. CONCLUSIONS: A distinct difference was noted in the biodistribution pattern of the radiolabelled equine peripheral blood-derived mesenchymal stem cells compared to free 99mTechnetium indicating equine peripheral blood-derived mesenchymal stem cells have a specific pharmacokinetic pattern after systemic administration in healthy dogs. Furthermore, the biodistribution pattern of the used xenogeneic equine peripheral blood-derived mesenchymal stem cells appeared to be different from previously reported experiments using different sources of mesenchymal stem cells.

Safety and immunomodulatory properties of equine peripheral blood-derived mesenchymal stem cells in healthy cats.

OBJECTIVE: Due to the immunomodulatory properties of mesenchymal stem cells (MSCs) through stimulation of endogenous immune cells by paracrine signals and cell contact, they have been proposed as alternative treatment option for many inflammatory and immune-mediated diseases in veterinary medicine. However, the long-term cultivation possibilities of feline MSCs are currently compromised due to a restricted proliferation capacity. Therefore, the xenogeneic use of equine peripheral blood-derived MSCs (ePB-MSCs) would present an interesting alternative thanks to their superior cultivation properties. To the authors' knowledge, there are currently no safety reports concerning the xenogeneic use of ePB-MSCs in cats. Therefore, the overall goal of this preliminary study was to investigate if ePB-MSCs can safely be administered in healthy cats and by extension evaluating their immunogenic and immunomodulatory properties. METHODS: Ten healthy cats were intravenously (i.v.) injected with 3 × 105 ePB-MSCs at three time points (T0, T1, T2). All cats were daily inspected by the caretaker and underwent a physical examination with hematological and biochemical analysis at day 0 (T0), week 2 (T1), week 4 (T2) and week 6 (T3) by a veterinarian. Furthermore, a modified mixed lymphocyte reaction (MLR) was performed at T0 and T3 for each cat in order to evaluate immunogenic and immunomodulatory properties of the ePB-MSCs RESULTS: No adverse clinical effects could be detected following repeated i.v. administration of ePB-MSCs in all cats. Significant lower protein (T1: P-value = 0.002; T2: P-value > 0.001; T3: P-value = 0.004) and albumin levels (T1: P-value = 0.003; T2: P-value = 0.001) were seen after repeated administration of ePB-MSCs, compared to T0. However, all biochemical and hematological parameters stayed within clinical acceptance level. In addition, the repeated injections did not induce a cellular immune response before and after repeated ePB-MSCs administration. Furthermore, convincing immunomodulatory properties of ePB-MSCs on feline peripheral blood mononuclear cells were confirmed in the MLR-assay CONCLUSION: This preliminary study demonstrates that ePB-MSCs can safely be administered in healthy cats and provide a promising alternative for the treatment of various inflammatory diseases in cats.