The search for an ideal mesenchymal stromal cell donor in the horse : this dissertation is presented for partial fulfillment of the degree of Doctor of Philosophy in Veterinary Science, Massey University, Palmerston North, New Zealand, School of Veterinary Science

The search for an immune privileged allogeneic mesenchymal stromal cell (MSC) line has been an interest for many biomedical researchers. This holds true for the field of equine medicine where MSCs are frequently used in research and clinical cases for the treatment of musculoskeletal disease. An ideal allogeneic MSC suppresses the immune system of the recipient leading to decreased inflammation in the face of disease. The ideal MSC also expresses the markers of a multipotent cell, retains a high level of viability and is able to perform anabolic activities to enhance repair. Our research sought to more clearly define the expression of MSC markers harvested from different equine MSC donors. Bone marrow-derived MSCs from Thoroughbreds, Standardbreds, and a subset of universal blood donor-type Standardbreds were compared. Standardbred MSCs showed significantly less MHC class II expression at early passages as compared to Thoroughbreds. When universal blood donor Standardbreds were compared to non-blood donor Standardbreds, the only significant variation was that CD90 was expressed more highly on universal blood donor MSCs as compared to non-blood donor Standardbred MSCs. The conclusion from stage one of our research was that universal blood donor-type Standardbred horses appeared less likely to cause an MHC II driven immune reaction and had the highest levels of bone marrow-derived MSC markers expressed at passage 2-4. We then compared the MSC donor cells in an in vitro trial exploring several arms of the immune system to understand the effects of the MSCs without prior activation of the immune cells, as has been done previously. Overwhelmingly, we found that MSCs of allogeneic origin cause very little to no activation of the immune system as compared to autologous MSCs. B cell and activated T lymphocyte populations were similar between the autologous and allogeneic MSCs. Those allogeneic MSCs that expressed little MHC II prior to interaction with the immune cells (MHC II-low MSCs) had reduced activation of recipient lymphocytes and neutrophils as compared to those MSCs expressing high levels of MHC II prior to interaction with immune cells (MHC II-high MSCs). MHC II-low MSCs, both of universal blood donor and non-blood donor origin, had higher expression of the genes we studied when placed in an allogeneic environment. These include both anabolic molecules known to assist in healing and some catabolic molecules. This knowledge combined with published information that ‘activated’ MSCs can be more beneficial to healing than unactivated MSCs, support the use of the more metabolically active MHC II-low MSCs as compared to MHC II-high MSCs. Based upon a wide array of testing, allogeneic MHC II-low MSCs created a low level of immune activation and an increased level of gene anabolic gene expression as compared to autologous MSCs. In conclusion passage 2-4 MHC II-low MSCs are preferred for use in allogeneic therapy

Replication Data for: Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression

Dataset for manuscript of above title

Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression.

BackgroundAs the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors.MethodsThe bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs.FindingsThere were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18.ConclusionsUniversal blood donor- type Standardbred MSCs from passages 2-4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs