7-Ketocholesterol Effects on Osteogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells

Abstract

Some oxysterols were shown to promote osteogenic differentiation of mesenchymal stem cells (MSCs). Little is known about the effects of 7-ketocholesterol (7-KC) in this process. We describe its impact on human adipose tissue-derived MSC (ATMSC) osteogenic differentiation. ATMSCs were incubated with 7-KC in osteogenic or adipogenic media. Osteogenic and adipogenic differentiation was evaluated by Alizarin red and Oil Red O staining, respectively. Osteogenic (ALPL, RUNX2, BGLAP) and adipogenic markers (PPARƔ, C/EBPα) were determined by RT-PCR. Differentiation signaling pathways (SHh, Smo, Gli-3, β-catenin) were determined by indirect immunofluorescence. ATMSCs treated with 7-KC in osteogenic media stained positively for Alizarin Red. 7-KC in adipogenic media decreased the number of adipocytes. 7-KC increased ALPL and RUNX2 but not BGLAP expressions. 7-KC decreased expression of PPARƔ and C/EBPα, did not change SHh, Smo, and Gli-3 expression, and increased the expression of β-catenin. In conclusion, 7-KC favors osteogenic differentiation of ATMSCs through the expression of early osteogenic genes (matrix maturation phase) by activating the Wnt/β-catenin signaling pathway, while inhibiting adipogenic differentiation. This knowledge can be potentially useful in regenerative medicine, in treatments for bone diseases.

Keywords: 7-KC; Wnt/β-catenin; adipose tissue; mesenchymal stem cell; osteogenic differentiation; oxysterol.

Figure 1

Osteogenic and adipogenic differentiation of ATMSCs. Effect of 7-KC. Osteogenic differentiation: (A): Control, ATMSCs cultivated for 21 days with osteogenic specific medium alone. (B,C): Osteogenic medium with 3 and 5 µM 7-KC, respectively. Staining with Alizarin Red. Bar scale: 12.5 µm. Adipogenic differentiation: (D): Control, ATMSCs cultivated for 14 days with adipogenic specific medium. (E,F): Adipogenic medium with 3 and 5 µM 7-KC M, respectively. Staining with Oil Red O. White bar scale: 200 µm, black bar scale: 50 µm.

Figure 2

Expression of osteogenic differentiation markers in ATMSCs as measured by RT-PCR. ATMSCs were incubated with osteogenic specific medium with or without 10 µM 7-KC for different periods of time. (A): RUNX2. (B): ALPL. (C): BGLAP. Data are mean ± SD from three independent experiments in duplicate. Results are shown in log scale. * p < 0.05; ** p < 0.01; *** p < 0.0001.

Figure 3

Expression of adipogenic differentiation markers in ATMSCs as measured by RT-PCR. ATMSCs were incubated with osteogenic specific medium with or without 10 µM 7-KC for different periods of time. (A): PPARƔ expression. (B): C/EBPα expression. ATMSCs were incubated with osteogenic or adipogenic specific medium with or without 10 µM 7-KC for 21 days. (C): PPARƔ expression. (D): C/EBPα expression. Data are mean ± SD from three independent experiments in duplicate. Results are expressed in log scale. * p < 0.05; ** p < 0.01; *** p < 0.0001.

Figure 4

Osteogenic signaling pathways measured by immunofluorescence. Effect of 7-KC and osteogenic and adipogenic media. (A): Mean fluorescence intensity of SHh expression. (B,C): Mean fluorescence intensity of Smo expression in membrane/cytoplasm and nucleus, respectively. (D,E): Mean fluorescence intensity of Gli-3 expression in membrane/cytoplasm and nucleus, respectively. (F): Percentage of cells expressing β-catenin in membrane/cytoplasm. (G): Mean fluorescence intensity of β-catenin expression in membrane/cytoplasm. (H): Percentage of cells expressing β-catenin in the nucleus. (I): Mean fluorescence intensity of β-catenin expression in the nucleus. Data are mean ± SD from three independent experiments in duplicate. * p < 0.05; ** p < 0.01.