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Expansion of plastic-adherent bone marrow-derived mesenchymal stem cells (MSCs) results in gradual loss of osteogenic potential after passage 5-6. One explanation is contamination of MSC cultures with mature cells including fibroblasts. Identification and elimination of fibroblasts from MSC cultures could improve MSC yield and differentiation potential and also prevent tumor formation after MSC transplantation. However, no specific markers currently exist that can reliably discriminate between MSCs and fibroblasts. Flow cytometry analysis demonstrated that markers currently used to define MSCs, such as CD105, CD166, CD90, CD44, CD29, CD73, and CD9, are also expressed on human skin or lung fibroblasts. However, the level of expression of CD166 was significantly higher and that of CD9 was significantly lower in MSCs than in fibroblasts. CD146 was expressed only in MSCs. Using small focused microarrays, new markers differentially expressed in MSCs and fibroblasts were identified. Real-time polymerase chain reaction confirmed that expression of CD106, integrin alpha 11, and insulin-like growth factor-2 in MSCs was at least 10-fold higher than in fibroblasts; whereas expression of matrix metalloproteinase 1 and matrix metalloproteinase 3 was almost 100-fold lower. Flow cytometry and immunostaining demonstrated that CD106 protein expression on cell surface could be upregulated in MSCs but not in fibroblasts by the treatment with tumor necrosis factor-alpha. Comparison of surface expression of commonly used and newly identified MSC markers in MSCs cultures of passage 2 and passage 6 demonstrated that CD106 (with and without tumor necrosis factor-alpha treatment), integrin alpha 11, and CD146 were downregulated in MSCs of passage 6, and CD9 was upregulated; whereas all other markers did not change. Newly identified markers that have robust differences of expression in MSCs and fibroblasts on gene and protein level could be used for quality control of MSC cultures after expansion, cryopreservation, gene transfection, and other manipulations.