In-vitro characterization of canine multipotent stromal cells isolated from synovium, bone marrow, and adipose tissue: a donor-matched comparative study.
In-vitro characterization of canine multipotent stromal cells isolated from synovium, bone marrow, and adipose tissue: a donor-matched comparative study.
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DOI：
10.1186/s13287-017-0639-6
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发表时间：
2017-10-03
影响因子：
7.5
通讯作者：
Saunders WB
中科院分区：
文献类型：
--
作者：
Bearden RN;Huggins SS;Cummings KJ;Smith R;Gregory CA;Saunders WB
这只狗代表了基于细胞的翻译研究的一个极好的大型动物模型。重要的是，犬多能基质细胞(CMSCs)的特性和特定翻译研究的理想组织来源尚未建立。本研究的目的是利用供者匹配的研究设计和一系列的体外鉴定、分化和免疫调节分析来鉴定来自滑膜、骨髓和脂肪组织的cMSCs。从5只颅十字韧带断裂犬体内分离培养出犬骨髓间充质干细胞。采用集落形成单位(CFU)分析、流式细胞仪分析、多能相关基因RT-PCR、增殖实验、三系分化实验和免疫调节实验对所制备的15种cMSC进行鉴定。数据报告为平均 ± 标准差，采用重复测量方差分析和图基后检验进行比较。P <； 为0.0 5，具有显著意义。所有组织样本均产生塑料贴壁的纺锤形骨髓间充质干细胞。CD34、CD45和Stro-1阴性，CD9、CD44和CD90阳性，而CD105阳性的程度因来源不同而不同。细胞表达多能性相关基因NANOG、OCT4和SOX2。除供者和组织来源外，CFU潜能、增殖率、三系分化和免疫调节反应均有显著差异。滑膜和骨髓间充质干细胞表现出良好的早期成骨活性，但在评估晚期成骨时，没有检测到显著差异。有趣的是，补充骨形态发生蛋白-2(BMP-2)对于早期和晚期成骨分化是必要的，这一发现与其他犬类研究一致。此外，滑膜和脂肪间充质干细胞的增殖速度更快，CFU潜能更高，在软骨细胞检测中形成更大的聚集体，尽管脂肪颗粒的蛋白多糖和II型胶原染色主观上比滑膜和骨髓颗粒低。最后，在脂多糖刺激的共培养实验中，来自所有三种组织来源的cMSCs调节了小鼠巨噬细胞肿瘤坏死因子-α和IL-6的水平。虽然来自滑膜、骨髓和脂肪组织的cMSCs有许多相似之处，但在增殖和三系分化方面存在重要的差异，在选择cMSCs进行翻译研究时应该考虑到这一点。这些结果和相关方法将被证明对未来涉及犬类模型的翻译研究有用。本文的在线版本(doi：10.1186/s13287-0170639-6)包含补充材料，授权用户可以使用。
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. The online version of this article (doi:10.1186/s13287-017-0639-6) contains supplementary material, which is available to authorized users.
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影响因子：
2.6
作者：
Alves EG;Serakides R;Boeloni JN;Rosado IR;Ocarino NM;Oliveira HP;Góes AM;Rezende CM
通讯作者：
Rezende CM
影响因子：
2.2
作者：
Bockstahler, Barbara A.;Skalicky, Monika;Lorinson, Dragan
通讯作者：
Lorinson, Dragan
影响因子：
4
作者：
Braun, Julian;Kurtz, Andreas;Dong, Jun
通讯作者：
Dong, Jun
影响因子：
2.4
作者：
BERGMANN, G;SIRAKY, J;KOELBEL, R
通讯作者：
KOELBEL, R
影响因子：
2.4
作者：
Chung, Dai-Jung;Hayashi, Kei;Yellowley, Clare E.
通讯作者：
Yellowley, Clare E.