狗代表了用于基于转化细胞的研究的优秀的大型动物模型。重要的是,犬多能基质细胞(cMSCs)的特性和特异性转化研究的理想组织来源尚未建立。这项研究的目的是表征源自滑膜的cMSCs,骨髓,和脂肪组织使用供体匹配的研究设计和一系列全面的体外表征,分化,和免疫调节试验。
从五只患有颅交叉韧带断裂的狗中分离犬MSCs。使用集落形成单位(CFU)测定对所有15种cMSC制剂进行评估,流式细胞术分析,多能性相关基因的RT-PCR,增殖试验,三系分化试验,和免疫调节试验。数据报告为平均值±标准偏差,并使用重复测量方差分析和Tukey事后检验进行比较。在p<0.05时确立显著性。
所有组织样本都产生了塑料粘附剂,cMSCs的纺锤形制剂。细胞对于CD34、CD45和STRO-1是阴性的,并且对于CD9、CD44和CD90是阳性的,而细胞对于CD105的阳性程度是可变的,这取决于起源的组织。细胞多能性相关基因NANOG呈阳性,OCT4和SOX2。考虑供体和组织来源,CFU电位有显著差异,增殖率,三系分化,和免疫调节反应。滑膜和骨髓cMSCs表现出优越的早期成骨活性,但是在评估晚期成骨时,未检测到显着差异。有趣的是,骨形态发生蛋白-2(BMP-2)补充是早期和晚期成骨分化所必需的,这一发现与其他犬类研究一致。此外,滑膜和脂肪cMSCs增殖更快,显示更高的CFU潜力,在软骨形成试验中形成了更大的聚集体,尽管与滑膜和骨髓颗粒相比,脂肪颗粒中的蛋白聚糖和II型胶原染色主观上降低了。最后,源自所有三种组织来源的cMSC在脂多糖刺激的共培养测定中调节鼠巨噬细胞TNF-α和IL-6水平。
而来自滑膜的cMSCs,骨髓,和脂肪组织有许多相似之处,增殖和三系分化存在重要差异,在选择cMSCs进行翻译研究时应予以考虑。这些结果和相关方法将被证明对涉及犬模型的未来转化研究有用。
The dog represents an excellent large animal model for translational cell-based studies. Importantly, the properties of canine multipotent stromal cells (cMSCs) and the ideal tissue source for specific translational studies have yet to be established. The aim of this
study was to characterize cMSCs derived from synovium, bone marrow, and adipose tissue using a donor-matched
study design and a comprehensive series of in-vitro characterization, differentiation, and immunomodulation assays.
Canine MSCs were isolated from five dogs with cranial cruciate ligament rupture. All 15 cMSC preparations were evaluated using colony forming unit (CFU) assays, flow cytometry analysis, RT-PCR for pluripotency-associated genes, proliferation assays, trilineage differentiation assays, and immunomodulation assays. Data were reported as mean ± standard deviation and compared using repeated-measures analysis of variance and Tukey post-hoc test. Significance was established at p < 0.05.
All tissue samples produced plastic adherent, spindle-shaped preparations of cMSCs. Cells were negative for CD34, CD45, and STRO-1 and positive for CD9, CD44, and CD90, whereas the degree to which cells were positive for CD105 was variable depending on tissue of origin. Cells were positive for the pluripotency-associated genes NANOG, OCT4, and SOX2. Accounting for donor and tissue sources, there were significant differences in CFU potential, rate of proliferation, trilineage differentiation, and immunomodulatory response. Synovium and marrow cMSCs exhibited superior early osteogenic activity, but when assessing late-stage osteogenesis no significant differences were detected. Interestingly, bone morphogenic protein-2 (BMP-2) supplementation was necessary for early-stage and late-stage osteogenic differentiation, a finding consistent with other canine studies. Additionally, synovium and adipose cMSCs proliferated more rapidly, displayed higher CFU potential, and formed larger aggregates in chondrogenic assays, although proteoglycan and collagen type II staining were subjectively decreased in adipose pellets as compared to synovial and marrow pellets. Lastly, cMSCs derived from all three tissue sources modulated murine macrophage TNF-α and IL-6 levels in a lipopolysaccharide-stimulated coculture assay.
While cMSCs from synovium, marrow, and adipose tissue share a number of similarities, important differences in proliferation and trilineage differentiation exist and should be considered when selecting cMSCs for translational studies. These results and associated methods will prove useful for future translational studies involving the canine model.