安樂死後犬骨髓與骨骺間葉幹細胞之特性分析

Abstract

由於間葉幹細胞（mesenchymal stem cells）之低免疫源性（immunogenicity）、低癌化風險（tumorigenic risk）及取得容易，因此較胚胎幹細胞（embryonic stem cells）來得適合應用於臨床細胞療法。本試驗將探討由安樂死後犬隻大體取得兩種不同來源之成體間葉幹細胞，以建立一套流程能有效率擴展並維持幹細胞庫存以供學術研究、人類臨床前研究及伴侶動物臨床應用所需。 從安樂死捐贈大體所取得之骨髓液，經由密度梯度離心後，可將骨髓液內之單核細胞群（mononuclear cells）分離出，並於培養期間利用更換新鮮培養液之方式，即可得到具貼附性之單核細胞群。骨骺間葉幹細胞之分離，則是以骨骺端海綿骨組織剪碎後，未經任何酵素作用即可直接培養於培養皿中。對本試驗所分離之單核細胞群進行定性分析，可發現此單核細胞群能行體外分化成硬骨細胞、脂肪細胞及軟骨細胞，並經由流式細胞儀分析其表面抗原呈現間葉幹細胞特性（CD44+, CD90+, CD34-, CD45R-），證實其為間葉幹細胞。 為了確認間葉幹細胞其捐贈個體之年齡與繼代次數對細胞特性是否造成影響，利用群落形成單位（colony-forming unit）代表分離效率（isolation efficiency）、細胞倍增時間（population doubling time, PDT）表示增殖能力（proliferation ability）、分化潛能（differentiation potential）代表細胞幹性（stemness）、並調查表面抗原特性是否穩定。並且檢測趨化因子配體5（Chemokine ligand 5, CCL5）表現量以判定內源性腫瘤轉移之風險。結果顯示，捐贈個體年齡（老年與幼年）與採集部位（肱骨及股骨）並不影響安樂死後犬骨髓間葉幹細胞之分離效率與增殖能力，而細胞幹性亦不受個體年齡影響。無論個體年齡差異，隨著繼代數增加則細胞增殖速率沒有明顯差異且表面抗原向性維持恆定，惟CD44陽性細胞比例於第12代（passage 12, P12）顯著較第四代（passage 4, P4）升高；已知CD44與細胞癌化有關，故此結果暗示P12之骨髓間葉幹細胞致癌率可能較高。而老年組之群落形成能力於P12相較於P4與P8則有顯著下降，暗示P12細胞轉化程度較高，因此其幹細胞特性降低。 綜合上述，以本試驗之分離法能成功取得安樂死後犬之間葉幹細胞，因此大體捐贈可成為另一項成體幹細胞供應來源，且分離效率與細胞增殖速率不受限於個體年齡及採集位置。以幹細胞特性分析，幼年個體與老年個體之骨髓間葉幹細胞其分化能力沒有差異。另一方面，隨著繼代數增加，其間葉幹細胞之癌化風險亦有可能增加。

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.