The characteristics of bone marrow and epiphysis-derived mesenchymal stem cells from canine body donation after euthanasia
Date Issued
2014
Date
2014
Author(s)
Hsu, Tzu-Yu
Abstract
Compared to embryonic stem cells, mesenchymal stem cells （MSCs） are more suitable for clinical cell therapy due to the low immunogenicity, low tumorigenic risk and better accessibility. In this study, two kinds of MSCs with different anatomical origins from canine body donations after euthanasia were investigated, aiming to establish a procedure to effectively expand and maintain the stem cell inventory for the potential clinical applications.
The cells from aspirated bone marrow were separated by density gradient centrifugation. Mononuclear cells were harvested from plasma interface and seeded in culture dish, while non-attached cells were removed by changing medium. Epiphysis-derived mesenchymal stem cells （EMSCs） can be harvested from epiphysis by directly culturing in dish without enzymatic digestion. In confirming that the cells isolated from the body donors are MSCs, the isolated cells were successfully induced differentiation into three lineages: osteocytes, adipocytes and chondrocytes. The flow cytometry of the surface markers showed consistent profiles as MSCs markers （CD44+, CD90+, CD34-, CD45R-）. To elucidate the influence of age and anatomical origins on the harvest of MSCs, we collected MSCs from both humerus and femur with different ages. No difference in the numbers of colony-forming units （CFUs） as well as population doubling time between two anatomical locations and two age groups indicated that these factors do not affect the harvest efficiency of BMMSCs. Several tri-lineage marker genes were investigated by qPCR to assess the tri-lineage differentiation between age groups and no significant difference is detected. However, in old group, the number of CFUs in P12 is significantly decreased compared to P1 and P4 implying the clonogenicity is losing though passage. Among the surface markers, a higher percentage of positive cells of CD44, which was associated with cell transformation, can be observed in passage 12 compared to passage 4 implying the higher tumorigenic risk in P12. To evaluate the potential tumor promoting effects on endogenous tumors in the recipients, the expression of chemokine ligand 5 （CCL5） was detected by qPCR, and there is no difference in the expression of CCL5 between age groups and various passages.
In summary, we successfully purified BMMSCs and EMSCs from canine after euthanasia in animal hospital and shelter. Our results indicated that age and anatomical origin do not affect the isolation efficiency, cell proliferations and potential of tri-lineage differentiation in MSCs. For clinical application, more studies such as nude mice inoculation are needed to confirm the tumorigenic risk of MSCs in the future. Furthermore, pathogen screening methods and profiles will be established as part of the cell donation protocol.
Subjects
安樂死
犬
狗
骨髓間葉幹細胞
骨骺間葉幹細胞
SDGs
Type
thesis
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