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A biological study establishing the endotoxin limit for osteoblast and adipocyte differentiation of human mesenchymal stem cells

机译：建立内毒素限制人间充质干细胞成骨细胞和脂肪细胞分化的生物学研究

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Introduction Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions, such as hypoxia or inflammation after transplantation in?vivo . Although endotoxin testing is required for evaluating the quality and safety of transplanted MSCs, no reports on their dose response to endotoxins are available to establish the limits for in?vitro MSC culture systems. In the present study, we aimed to accurately quantify the risk of endotoxin contamination in cell culture systems to establish an acceptable endotoxin limit for the differentiation of hMSC osteoblasts and adipocytes. Methods Three types of bone marrow-derived hMSCs (hMSC-1: 21-year-old, M/B; hMSC-2: 36-year-old, M/B; hMSC-3: 43-year-old, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in osteogenic or adipogenic differentiation media, respectively, from commercial kits, containing various concentrations of endotoxin (0.01–100?ng/ml). The degree of adipocyte and osteoblast differentiation was estimated by fluorescent staining of lipid droplets and hydroxyapatite, respectively. To clarify the molecular mechanism underlying the effect of endotoxin on hMSC differentiation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis. Results Although endotoxin did not effect the adipocyte differentiation of hMSCs, osteoblast differentiation was enhanced by various endotoxin concentrations: over 1?ng/ml, for hMSC-1; 10?ng/ml, for hMSC-2; and 100?ng/ml, for hMSC-3. Proteomic analysis of hMSC-1 cells revealed up-regulation of many proteins related to bone formation. These results suggested that endotoxin enhances the osteoblast differentiation of MSCs depending on the cell type. Conclusions Since endotoxins can affect various cellular functions, an endotoxin limit should be established for in?vitro MSC cultures. Its no-observed-adverse-effect level was 0.1?ng/ml based on the effect on the hMSC osteoblast differentiation, but it may not necessarily be the limit for ADSCs.
机译：简介多能干间充质干细胞（MSCs）在成年生物中广泛分布，并与组织的维护和修复，造血功能的调节以及免疫反应有关。人（h）MSC在组织工程，基于细胞的治疗和医疗设备中都有应用，但尚不清楚它们如何应对不利条件，例如体内移植后的低氧或炎症。尽管需要使用内毒素测试来评估移植的MSC的质量和安全性，但尚无关于其对内毒素剂量反应的报告来确定体外MSC培养系统的极限。在本研究中，我们旨在准确量化细胞培养系统中内毒素污染的风险，从而为hMSC成骨细胞和脂肪细胞的分化建立可接受的内毒素极限。方法三种类型的骨髓源性hMSCs（hMSC-1：21岁，M / B; hMSC-2：36岁，M / B; hMSC-3：43岁，M / B C）和脂肪干细胞（ADSC; StemPro Human）分别在市售试剂盒中的成骨或成脂分化培养基中培养，其中含有各种浓度的内毒素（0.01–100？ng / ml）。分别通过脂质滴和羟基磷灰石的荧光染色估计脂肪细胞和成骨细胞的分化程度。为了阐明内毒素对hMSC分化作用的分子机制，从培养的细胞中提取细胞蛋白，然后进行液相色谱-串联质谱shot弹枪蛋白质组学分析。结果尽管内毒素不影响hMSC的脂肪细胞分化，但成骨细胞的分化可以通过多种内毒素浓度增强：hMSC-1的浓度超过1？ng / ml; HMSC-1的浓度超过1？ng / ml。对于hMSC-2，为10？ng / ml;对于hMSC-3，为100？ng / ml。对hMSC-1细胞的蛋白质组学分析显示，许多与骨形成有关的蛋白质均上调。这些结果表明内毒素取决于细胞类型增强了MSC的成骨细胞分化。结论由于内毒素可影响多种细胞功能，因此应建立体外MSC培养的内毒素极限。根据对hMSC成骨细胞分化的影响，其未观察到的不良反应水平为0.1μng/ ml，但不一定是ADSC的极限。

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