Figure 2
Sarcospheres exhibit characteristics of MSCs, express stem-cell associated markers and are tumorigenic. (A) Flow cytometry analysis of MSCs' surface markers in sarcospheres and MNNG/HOS cells. Both cells evidenced a strong positivity for CD73, CD90, CD13 and CD105. Unstained cells (grey) appear at the lower left quadrant as a negative fluorescence control. (B) Stainings demonstrating the differentiation potential of CSCs into mesenchymal lineages: osteoblasts (Alizarin Red), chondroblasts (Alcian Blue) and adipocytes (Oil Red), after incubation in osteogenic, chondrogenic or adipogenic inducing medium during 21, 16 and 14 days, respectively. (C) Western blot analysis of Oct4, Nanog, P-glycoprotein and BCRP in CSCs, SAR-OS and MNNG/HOS cells. β-actin was blotted as the loading control. The bottom panel shows the quantitative analysis of proteins (normalized to β-actin) expressed as a ratio of the levels found in MNNG/HOS cells, set as 1 for all proteins. The expression of all analyzed proteins is significantly enhanced in CSCs as compared with parental MNNG/HOS cells. The sphere-derived monolayer culture (SAR-OS) showed similar protein expression profiles to those observed in MNNG/HOS cells. Data represents mean ± standard error of the mean (SEM) of three independent experiments. *Significantly different from MNNG/HOS cells (p < 0.05). (D) Tumorigenic potential of CSCs. Balb/c nude mice were s.c injected with 1 × 105 of CSCs in the right flank and of MNNG/HOS in the left flank. Tumor growth was monitored every week. Representative image showed a CSC-derived tumor with a volume 7-fold higher to that induced by MNNG/HOS cells.