Growth Properties and Pluripotency Marker Expression of Spontaneously Formed Three-dimensional Aggregates of Human Adipose-derived Stem Cells

BACKGROUND AND OBJECTIVES: Recent findings suggest that therapeutic potential of mesenchymal stem cells (MSCs) could be increased through aggregation into three-dimensional (3D) bodies, and different culture methods have been employed to obtain 3D spheroids of MSCs. In the current study we report accidentally encountered spontaneous formation of adipose-derived stem cell (ASC) bodies in standard ASC culture of a single donor. METHODS AND RESULTS: Human ASCs from passages 1 to 3, cultured in a medium containing 5% autologous serum (AS), spontaneously clustered and formed floating 3D bodies. After a transfer of floating ASC bodies onto new adherent plastic dish, they attached to the surface and gradual migration of spindle-shaped ASCs out of the bodies was detected. A substitution of AS with allogeneic sera did not hinder this ability, but commercial medium containing fetal bovine serum delayed the process. Substantial part of ASCs surrounding transferred ASC bodies showed alkaline phosphatase (AP) activity, while ASC aggregates were AP negative. Similar 3D bodies formed when ASCs were grown on an uncoated glass surface. These ASC aggregates as well as clusters of ASCs, where formation of the 3D bodies is initiated, expressed pluripotency marker NANOG, but the expression of OCT4A was not detected. CONCLUSIONS: Obtained results suggest that spontaneously formed ASC aggregates may represent a more primitive cell subpopulation within the individual ASC culture. The ability to form 3D aggregates, the expression of NANOG, and the lack of the AP activity may be used to enrich ASC cultures with potentially more primitive cells serving as an excellent basis for therapeutic applications.

Simvastatin inhibits expression of pluripotent markers Oct3/4, Nanog, and Sox-2 in human breast cancer MCF-7 Cells / 中国肿瘤临床

Objective:To investigate the effect of simvastatin on expression of pluripotent markers Oct3/4, Nanog, and Sox-2 in human breast cancer MCF-7 cells. Methods:Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunofluo-rescent staining, flow cytometry, and Western blot were used to detect the expression of pluripotency markers Oct3/4, Nanog, and Sox-2 in human breast cancer MCF-7 cells treated with different doses of simvastatin. Results:qRT-PCR revealed the more signifi-cant inhibition of gene expressions of Oct3/4, Nanog, and Sox-2 in human breast cancer MCF-7 cells when subjected to high doses of simvastatin (10, 50, and 100 μmol/L) compared with the control group (P0.05). The inhibitory effect of simvastatin on the gene expressions of Oct3/4 and Nanog was more significantly apparent at 50 and 100 μmol/L dosages than at 10 μmol/L (P0.05). Between the two higher-dose treatments (50 and 100 μmol/L), no significant difference in the inhibitory expressions of Oct3/4, Nanog, and Sox-2 in MCF-7 cells was found. Meanwhile, in the 10 μmol/L simvastatin treatment, immunoflurescent staining showed a marked reduction in the protein expression of all three pluripotent markers in MCF-7 cells, and flow cytometry demonstrated a decrease of Oct3/4-, Nanog-, and Sox-2-positive cells (P<0.05). Western blot further revealed that the protein expression of Oct3/4, Nanog, and Sox-2 in MCF-7 cells was significantly declined by the same simvastatin dose (P<0.05). Conclusion: Simvastatin can inhibit the expression of pluripotent markers Oct3/4, Nanog, and Sox-2 in human breast cancer MCF-7 cells, proving the anti-cancer properties of simvastatin.