Figure 3
HDFs were efficiently converted into OC-producing osteoblasts by culturing with ALK5 i II and VitD3. (A) HDFs were cultured in osteogenic medium supplemented with ALK5 i II and the indicated supplements. After culturing for 18 days, real time RT-PCR was performed to evaluate mRNA for the indicated gene. Values are means ± S.D. n = 4. *P < 0.05 and **P < 0.01, v.s. HDFs cultured in osteogenic medium alone. #P < 0.05 and ##P < 0.01, v.s. HDFs cultured in osteogenic medium supplemented with ALK5 i II. (B and C), HDFs were cultured in osteogenic medium supplemented with ALK5 i II and VitD3 for 18 days (cOBs). The cOBs, the HDFs cultured in osteogenic medium supplemented with ALK5 i II and the HDFs cultured in complete medium as control were stained with anti-OC antibody and DAPI. Fluorescence microscopic images (magnification was x200) (B) and proportion of OC producing cells (C) are shown. Values are means ± S.D. (n = 4). **P < 0.01. v.s. control. ##P < 0.01, v.s. HDFs cultured in osteogenic medium supplemented with ALK5 i II. (D) RNA was extracted from HDFs, HDFs cultured in the osteogenic medium for 18 days, cOBs induced as in (B), osteoblasts differentiated from MSCs (MSC-OBs), and pOBs. DNA microarray analysis was performed to evaluate mRNA for the genes encoding osteoblasts-related transcription factors, signaling proteins and soluble factors. Heat map and hierarchical clustering data are shown. The genes with increased expression are colored green, whereas those with decreased expression are colored pink as indicated in the color range. The expression level of each gene was normalized to median signal intensity.
