Granéli Cecilia, Thorfve Anna, Ruetschi Ulla, Brisby Helena, Thomsen Peter, Lindahl Anders, Karlsson Camilla
Department of Biomaterials, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden; BIOMATCELL, VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden.
Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at The University of Gothenburg, Sweden.
Stem Cell Res. 2014 Jan;12(1):153-65. doi: 10.1016/j.scr.2013.09.009. Epub 2013 Sep 27.
Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.
如今，体外评估骨髓间充质干细胞（BMSCs）成骨分化最常用的工具是证明多种相关标志物的表达，如碱性磷酸酶（ALP）、RUNX2和骨钙素（OCN）。然而，迄今为止，尚无单一的表面标志物或标志物组合能够明确界定向成骨谱系分化的人骨髓间充质干细胞（hBMSCs）。因此，本研究的目的是探讨这个问题。利用基于细胞培养中氨基酸稳定同位素标记（SILAC）的定量蛋白质组学技术，研究成骨分化和未分化的hBMSCs中差异表达的表面标志物。通过质谱（MS）分析标记的膜蛋白，鉴定出成骨分化细胞和未分化细胞之间表达比大于2的52种蛋白质。随后，通过流式细胞术和酶联免疫吸附测定（ELISA）对其中一些蛋白质的SILAC表达比进行验证，并对三种候选标志物的谱系特异性进行研究。表面标志物CD10和CD92在向成骨和脂肪生成谱系分化的hBMSCs中表达显著增加。此外，在软骨生成分化过程中，CD10表达略有增加。此外，细胞内蛋白质结晶αB（CRYaB）仅在成骨分化的hBMSCs中表达显著增加，在向软骨生成或脂肪生成谱系分化过程中不受影响。从目前的结果可以得出结论，CD10和CD92是成骨和脂肪生成分化的潜在标志物，而CRYaB是体外hBMSCs成骨分化过程中特异性表达的潜在新型成骨标志物。
