Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses
Introduction
Equine multipotent mesenchymal stem cells (MSC) have been isolated from numerous tissues including, but not limited to, bone marrow (BM) (1., 2., 3., 4.), adipose tissue (AT) (3,5,6), umbilical cord tissue (UCT) (3,7) and umbilical cord blood (UCB) (8., 9., 10.). Tissue selection for stem cell harvest depends on a number of factors. Bone marrow contains moderate numbers of MSC but harvesting BM can be invasive and MSC numbers decrease in older animals (11). Adipose tissue contains high numbers of MSC (5); however, collection of AT is invasive and can be difficult because of the small amount of accessible fat in highly trained athletic horses. Collection of placental tissues is non-invasive and both UCT and UCB are ready sources of equine MSC (3,8., 9., 10.,12). Data suggest that MSC from placental tissues may have higher proliferation potential and broader differentiation capacity (10,11,13). Nonetheless, to date there have been no prospective clinical trials comparing the efficacy of MSC derived from equine AT, BM or placental tissues for tissue regeneration or immunomodulation. Adult-derived MSC may have diverse therapeutic applications, including tissue regeneration, angiogenesis and immunomodulation. In equine medicine, MSC are widely used to treat orthopedic injuries involving tendons, ligaments, cartilage and bone (14,15). For therapeutic use, MSC can be administered intra-articularly, intralesionally, intravenously or regionally. For osteoarthritis, ligament, menisci, cartilage and other joint lesions, MSC (or other regenerative medicine products such as adipose-derived stromal vascular fraction and platelet-rich plasma) are often injected directly into the joint (intra-articular administration) (16., 17., 18., 19.). Intra-articular MSC have been shown to home to affected tissues and help with tissue regeneration (16,19).
Typically, MSC are obtained from ex vivo culture of the patient's own tissues (autologous). However, the use of autologous cells for patient treatment has inherent limitations. The treatment of acute lesions is not feasible as MSC expansion from primary tissues via tissue culture takes time (10–21 days) and there are marked variations in MSC recovery and growth kinetics that make administration of standard, appropriately timed MSC doses difficult. These obstacles are not unique to equine MSC therapy and also present challenges in human stem cell therapeutic applications. As such, BM-derived ‘third party’ (allogeneic) MSC therapy is currently in phase III clinical trials for selected human disease conditions, including Crohn's disease and steroid-refractory acute graft-versus-host disease (www.osiristx.com [accessed on: March, 2010]). There are currently no published data assessing the in vitro or in vivo immunomodulatory capacity of equine MSC. However, work with MSC from human beings, rodents and other animal species (dogs, pigs and non-human primates) suggests that the use of allogeneic, unmatched MSC is possible as MSC, regardless of tissue origin, are reported to have inherently low immunogenicity in vitro (20., 21., 22.).
MSC express leukocyte antigen major histocompatibility complex (MHC) class I molecules but do not express MHC class II or T-cell co-stimulatory molecules (22,23), and MSC do not elicit a proliferative response from allogeneic lymphocytes (24., 25., 26.). A few of the proposed mechanisms of MSC-induced immunosuppression include decreasing T-cell proliferation, inducing a shift towards regulatory T cells (23,24,27,28), secreting soluble factors, including prostaglandin E2 and interleukin (IL)-10 (29., 30., 31.), and decreasing production of inflammatory cytokines by various immune cell populations (32). Placentally derived multipotent cells also exhibit immunosuppressive properties (25,33., 34., 35.). Most studies using animal models suggest that MSC also display immunosuppressive capacities in vivo; however, some in vivo studies have demonstrated that allogeneic MSC can elicit an immune response in the host (36,37). BM-derived allogeneic MSC have been shown to be as effective as autologous MSC in animal models of myocardial infarction (38,39), acute kidney injury (40) and bone formation (41). In the dog, allogeneic MSC have proved beneficial in a bone defect model without the use of immunosuppressive drugs (42), whereas in mice and pigs the immunomodulatory behavior of allogeneic cells in vivo is debatable (36,37,43). In a preliminary study in horses, there were no visible, adverse responses to a single injection of allogeneic BM-derived MSC into an experimentally induced equine tendon lesion (44). Together, these preliminary findings suggest that allogeneic MSC may be tolerated for therapeutic applications in equine patients but additional studies in the horse are clearly needed.
Our group has isolated, characterized, differentiated and expanded equine MSC derived from UCB and UCT (3,10,12). However, the efficacy and safety of these cells when administered back into horses (autologous or allogeneic application) has not yet been demonstrated. The collection, processing and storage of UCT and UCB from individual horses can be cost-prohibitive. As such, it is imperative to know whether the use of autologous MSC will be mandatory or if MSC from related or unrelated donor horses can be used to develop an allogeneic tissue bank. MSC derived from both UCT and UCB are of foal origin. For equine orthopedic therapeutics, the ability to use placentally derived MSC from the foal for treatment of related individuals (sire, dam and half-sibling) would increase the potential utility and benefit of these cells to horse owners, breeders and trainers. Our long-term objective is to determine whether stored, expanded allogeneic (unrelated ‘third-party’) equine MSC can be used for equine tissue repair. The objectives of this study were to determine the effects of a single injection of autologous, related allogeneic and unrelated allogeneic placentally derived MSC (UCT and UCB) on physical health, gait and synovial fluid parameters when injected into healthy equine joints. We hypothesized that the injection of unrelated allogeneic placentally derived MSC would not induce significant alterations compared with the injection of autologous or related allogeneic MSC in the contralateral joint. The benefits of this model are that equine joints are accessible, particularly susceptible to inflammation, and can be assessed using minimally invasive techniques. Our work with horses provides a unique translational opportunity to bring novel cell-based therapies to the veterinary market while also modeling allogeneic UCT- and UCB-derived MSC as a treatment for orthopedic injuries in humans, and exploring the feasibility of banking autologous and allogeneic MSC for musculoskeletal regenerative medicine applications.
Section snippets
Horses
Sixteen healthy horses, divided into three groups, were used in this study. Group 1 consisted of Quarterhorse foals (n = 6, two female, four male) with an age range of 6–8 months. Group 2 consisted of their dams (n = 5), with an age range of 6–20 years. Group 3 consisted of half-siblings to the group 1 foals (n = 5, four female, one male) with an age range of 2–4 years. Three of the horses in the half-sibling group were Quarterhorses and two were Belgian crosses. All of the horses were housed at
MSC characterization
MSC derived from equine UCB and UCT MSC were spindle-shaped, highly proliferative and morphologically similar to those described previously for equine MSC (1,3,8,10). Our UCB and UCT MSC were capable of in vitro osteogenic, chondrogenic and adipogenic differentiation, exactly as described previously (data not shown) (3,10). Immunocytochemical phenotyping of our UCB-derived MSC has been published previously (10). UCT-derived MSC were uniformly positive for vimentin (Figure 1A), variably positive
Discussion
An allogeneic ‘off-the-shelf’ MSC product offers the advantage of near-immediate cellular therapy for the treatment of acute orthopedic lesions without the inherent lag period associated with isolation and expansion of autologous MSC. Our data suggest that, while a single intra-articular injection of placentally derived MSC does elicit inflammation in a non-injured joint, there is no difference in the type or degree of inflammation elicited by self, related or non-self cells. In addition, the
Acknowledgments
This project was supported in part by Thermo Genesis Corp., Rancho Cordova, CA, USA.
Financial support was also provided by the Center for Equine Health, including the Harriet Pfleger Foundation, and a gift from Mr Dick and Carolyn Randall. The authors thank Dr Jamie A. Textor, the staff of the Center for Equine Health and the staff in the William R. Pritchard Veterinary Teaching Hospital Hematology Lab, School of Veterinary Medicine, University of California, Davis, for assistance in sample
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