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PURPOSE

This study is aimed at investigating the phenotype, differentiation potential, immunomodulatory properties, and responsiveness of saphenous vein vessel wall-derived mesenchymal stromal cells (SV-MSCs) to various TLR ligands and proinflammatory cytokines, as well as comparing their features to those of their bone marrow-derived counterparts (BM-MSCs).

METHODS

SV-MSCs were isolated by enzymatic digestion of the saphenous vein vessel wall. Phenotype analysis was carried out by flow cytometry and microscopy, whereas adipogenic, chondrogenic, and osteogenic differentiation potentials were tested in assays. For comparative analysis, the expression of different stemness, proliferation, and differentiation-related genes was determined by Affymetrix gene array. To compare the immunomodulatory properties of SV-MSCs and BM-MSCs, mixed lymphocyte reaction was applied. To investigate their responses to various activating stimuli, MSCs were treated with TLR ligands (LPS, PolyI:C) or proinflammatory cytokines (TNF, IL-1, IFN), and the expression of various early innate immune response-related genes was assessed by qPCR, while secretion of selected cytokines and chemokines was measured by ELISA.

RESULTS

The isolated SV-MSCs were able to differentiate into bone, fat, and cartilage cells/direction . SV-MSCs expressed the most important MSC markers (CD29, CD44, CD73, CD90, and CD105) and shared almost identical phenotypic characteristics with BM-MSCs. Their gene expression pattern and activation pathways were close to those of BM-MSCs. SV-MSCs showed better immunosuppressive activity inhibiting phytohemagglutinin-induced T lymphocyte proliferation than BM-MSCs. Cellular responses to treatments mimicking inflammatory conditions were comparable in the bone marrow- and saphenous vein-derived MSCs. Namely, similar to BM-MSCs, SV-MSCs secreted increased amount of IL-6 and IL-8 after 12- or 24-hour treatment with LPS, PolyI:C, TNF, or IL-1, compared to untreated controls. Interestingly, a different CXCL-10/IP-10 secretion pattern could be observed under inflammatory conditions in the two types of MSCs.

CONCLUSION

Based on our results, cells isolated from saphenous vein vessel wall fulfilled the ISCT's (International Society for Cellular Therapy) criteria for multipotent mesenchymal stromal cells, and no significant differences in the phenotype, gene expression pattern, and responsiveness to inflammatory stimuli could be observed between BM-MSCs and SV-MSCs, while the latter cells have more potent immunosuppressive activity . Further functional assays have to be performed to reveal whether SV-MSCs could be useful for certain regenerative therapeutic applications or tissue engineering purposes.

目的

本研究旨在调查大隐静脉血管壁来源的间充质基质细胞（SV-MSCs）的表型、分化潜能、免疫调节特性以及对各种Toll样受体（TLR）配体和促炎细胞因子的反应性，并将其特征与其骨髓来源的对应细胞（BM-MSCs）进行比较。

方法

通过酶消化大隐静脉血管壁分离SV-MSCs。通过流式细胞术和显微镜进行表型分析，而在检测中测试其成脂、成软骨和成骨分化潜能。为了进行比较分析，通过Affymetrix基因芯片确定不同干性、增殖和分化相关基因的表达。为了比较SV-MSCs和BM-MSCs的免疫调节特性，应用混合淋巴细胞反应。为了研究它们对各种激活刺激的反应，用TLR配体（脂多糖、聚肌胞苷酸）或促炎细胞因子（肿瘤坏死因子、白细胞介素-1、干扰素）处理间充质基质细胞，并通过定量聚合酶链反应评估各种早期固有免疫反应相关基因的表达，同时通过酶联免疫吸附测定法测量所选细胞因子和趋化因子的分泌。

结果

分离出的SV-MSCs能够分化为骨、脂肪和软骨细胞。SV-MSCs表达最重要的间充质基质细胞标志物（CD29、CD44、CD73、CD90和CD105），并与BM-MSCs具有几乎相同的表型特征。它们的基因表达模式和激活途径与BM-MSCs相近。SV-MSCs在抑制植物血凝素诱导的T淋巴细胞增殖方面显示出比BM-MSCs更好的免疫抑制活性。在模拟炎症条件的处理下，骨髓来源和大隐静脉来源的间充质基质细胞的细胞反应具有可比性。具体而言，与未处理的对照相比，与BM-MSCs类似，用脂多糖、聚肌胞苷酸、肿瘤坏死因子或白细胞介素-1处理12或24小时后，SV-MSCs分泌的白细胞介素-6和白细胞介素-8量增加。有趣的是，在炎症条件下，两种类型的间充质基质细胞中可观察到不同的CXC趋化因子配体10/干扰素γ诱导蛋白10分泌模式。

结论

基于我们的结果，从大隐静脉血管壁分离的细胞符合国际细胞治疗协会（ISCT）对多能间充质基质细胞的标准，并且在BM-MSCs和SV-MSCs之间未观察到表型、基因表达模式和对炎症刺激的反应性方面的显著差异，而后者细胞具有更强的免疫抑制活性。必须进行进一步的功能检测以揭示SV-MSCs是否可用于某些再生治疗应用或组织工程目的。