Donor Age Effects on the Proliferative and Chondrogenic/Osteogenic Differentiation Performance of Equine Bone Marrow- and Adipose Tissue Derived Mesenchymal Stem Cells in Culture

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

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age

Background: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stem cells (MSCs) are used increasingly for autologous cell therapy in equine practice to treat musculoskeletal and other injuries. Current recommendations often call for 10–100 million MSCs per treatment, necessitating the expansion of primary cells in culture prior to therapeutic use. Of concern, human and rodent studies have shown a decline of both MSC recovery from sampled tissue and in vitro proliferative capacity with increasing donor age. This may be problematic for applications of autologous cell-based therapies in the important equine demographic of older patients. Objectives: To investigate the effect of donor age on the cellular proliferation of equine BM- and AT-MSCs. Study Design: In vitro study. Methods: BM- and AT-MSCs and dermal fibroblasts (biological control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15–17 months), adult (5–8 years), middle-aged (12–18 years), and geriatric (≥ 22 years). Proliferation of the cells was tested using an EdU incorporation assay and steady state mRNA levels measured for targeted proliferation, aging, and senescence biomarkers. Results: The cellular proliferation of equine BM- and AT-MSCs declined significantly in the geriatric cohort relative to the younger age groups. Proliferation levels in the two MSC types were equally affected by donor age. Analysis of steady state mRNA levels showed an up-regulation in tumor suppressors, apoptotic genes, and multiple growth factors in MSCs from old horses, and a down-regulation of some pro-cycling genes with a few differences between cell types. Main Limitations: Potential age-dependent differences in cell function parameters relevant to cell-therapy application were not investigated. Conclusions: The cellular proliferation of equine BM- and AT-MSCs declined at advanced donor ages. High levels of in vitro proliferation were observed in both MSC types from horses in the age groups below 18 years of age

Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis – a comparative study of isolation by tissue explant culture and enzymatic digestion

Abstract Purpose To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented adipose tissue (AT) harvested from patients with knee osteoarthritis. Methods Microfragmented abdominal AT from knee osteoarthritis patients was cryopreserved at ‐80 °C in cryoprotectant‐medium. The samples were thawed for stem cell isolation by tissue explant culture (TEC) and enzymatic digestion (ED), respectively. Viability, population doublings, and doubling time were assessed by trypan blue staining and flow cytometry. Cell type and senescence‐associated β‐galactosidase activity were analyzed by flow cytometry. Osteogenic and adipogenic differentiation was assessed quantitatively by Alizarin‐Red‐S and Oil‐Red‐O staining, respectively. Results Microfragmented AT from 7 patients was cryopreserved for a period of 46–150 days (mean (SD) 115.9 days (44.3 days)). Viable stem cells were successfully recovered and expanded from all patients using both isolation methods with no significant difference in viable population doublings or doubling time from passage 1 to 3 (p > 0.05). Low levels of senescence‐associated β‐galactosidase activity was detected for both methods with no significant difference between TEC and ED (p = 0.17). Stemness was verified by stem cell surface markers and osteogenic and adipogenic differentiation performance. Adventitial stem cells (CD31−CD34+CD45−CD90+CD146−), pericytes (CD31−CD34−CD45−CD90+CD146+), transitional pericytes (CD31−CD34+CD45−CD90+CD146+), and CD271+ stem cells (CD31−CD45−CD90+CD271+) were identified using both methods. More pericytes were present when using TEC (25% (24%)) compared to ED (3% (2%)) at passage 4 (p = 0.04). Conclusions Viable stem cells can be isolated and expanded from cryopreserved microfragmented AT using both TEC and ED. TEC provides more clinically relevant pericytes than ED

A Holocene relative sea-level database for the Baltic Sea

We present a compilation and analysis of 1099 Holocene relative shore-level (RSL) indicators locatedaround the Baltic Sea including 867 relative sea-level data points and 232 data points from the Ancylus Lake and the following transitional phase. The spatial distribution covers the Baltic Sea and near-coastal areas fairly well, but some gaps remain mainly in Sweden. RSL data follow the standardized HOLSEA format and, thus, are ready for spatially comprehensive applications in, e.g. glacial isostatic adjustment (GIA) modelling. We apply a SQL database system to store the nationally provided data sets in their individual form and to map the different input into the HOLSEA format as the information content of the individual data sets from the Baltic Sea area differs. About 80% of the RSL data is related to the last marine stage in Baltic Sea history after 8.5 ka BP (thousand years before present). These samples are grouped according to their dominant RSL tendencies into three clusters: regions with negative, positive and complex (transitional) RSL tendencies. Overall, regions with isostatic uplift driven negative tendencies dominate and show regression in the Baltic Sea basin during the last marine stage. Shifts from positive to negative tendencies in RSL data from transitional regions show a mid-Holocene highstand around 7.5 - 6.5 ka BP which is consistent with the end of the final melting of the Laurentide Ice Sheet. Comparisons of RSL data with GIA predictions including global ICE-5G and ICE-6G_C ice histories show goodfit withRSL data from the regions with negative tendencies, whereas in the transitional areas in the eastern Baltic, predictions for the mid-Holocene clearly overestimate the RSL and fail to recover the mid-Holocene RSL highstand derived from the proxy reconstructions. These results motivate improvements of ice-sheet and Earth-structure models and show the potential and benefits of the new compilation forfuture studies