Author ORCID Identifier

Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation


Agriculture, Food and Environment


Veterinary Science

First Advisor

Dr. James N. MacLeod


Orthopedic injuries are a major cause of lameness and morbidity in horses. Bone marrow (BM)- and adipose tissue (AT) derived mesenchymal stem cells (MSCs) have shown potential to facilitate the repair of orthopedic injuries and are being used increasingly in veterinary clinics. Presently, the use of MSCs as a therapy for equine patients is most commonly applied as autologous transplants, using BM- and AT-MSCs harvested from the patient shortly after the time of injury. Cell-based therapies are therefore delayed to enable primary cell numbers to be expanded in culture. Of concern, however, are human and rodent studies that have shown a sharp decline in MSC quantity and quality with increasing donor age. This may be problematic for the important equine demographic of older orthopedic patients due to current recommendations that often call for 10-100 million MSCs in treatment protocols.

This thesis, therefore, examines the critical gap of knowledge on the relationship between donor age and MSC parameters in horses and tests the hypothesis that increasing donor age is a major variable in equine BM- and AT-MSC proliferation and chondrogenic/osteogenic differentiation with decreasing capacities following non-linear kinetics.

To this end, BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested immediately post mortem from horses in 5 different age groups, with 4 horses in each age group. The age groups were newborn (0 days), yearling (1-2 years), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥ 22 years) horses.

In the first part of the study, the cellular proliferation of the cells was tested using an EdU incorporation assay and by targeted gene expression analysis of proliferation, aging and senescent biomarkers. The results showed that the cellular proliferation of equine MSCs declined with increasing donor age, but interestingly there were no significant difference in pairwise comparisons between age groups other than the geriatric horses. The cellular proliferation of the two MSC types was equally affected by donor age. Tumor suppressor gene expression was up-regulated with increasing donor age.

In the second part of the study, the same cells were grown in culture and stimulated separately to differentiate into both chondrocytes and osteocytes. The chondrogenic differentiation potential of the cells was compared quantitatively by measuring pellet size, matrix proteoglycan, and gene expression of articular cartilage biomarkers. The osteogenic differentiation potential of the cells was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of subchondral bone biomarkers. Overall, the data showed that the chondrogenic and osteogenic differentiation potential of equine MSCs decline with increasing donor age. The data further indicated that BM-MSCs have a larger chondrogenic pellet size and proteoglycan content, and a higher alkaline phosphatase activity compared to AT-MSCs, and that BM-MSCs calcium deposition was affected earlier by donor age. The chondrogenic and osteogenic differentiation performance of BM-MSCs declined already between newborn and yearlings. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. Gene expression of growth factors, chondrogenic and osteogenic biomarkers were down-regulated with increasing donor age.

Together, these results support the hypothesis that equine BM- and AT-MSCs proliferation and chondrogenic/osteogenic differentiation decline with increasing donor age following non-linear kinetics. Hence, the study highlights the importance of donor age considerations and MSC selection for autologous treatment of orthopedic injuries. This new knowledge has the potential to optimize autologous stem cell therapies of cartilage and bone injuries in horses, and will help advice owners on when to harvest and potentially cryopreserve the cells.

Digital Object Identifier (DOI)

Funding Information

This research was supported by the Independent Research Fund Denmark (NIH 133500133B) (2015), The University of Copenhagen (2015), Hesteafgiftsfonden (2017), KUSTOS af 1881 (2016 and 2019), and the Lourie Family Foundation (2019).