FIG. 5.

Effects of hUCB-MSC CM on osteoblastic differentiation in vivo and in vitro. (A) Human UCB-MSC CM or vehicle was injected to 10-week-old female nude mice after Sham-op or OVX. After 7 days, femora and tibiae were harvested, and mRNA for osteoblast products was determined by real-time quantitative polymerase chain reaction (PCR). Data are the means±SD. *p<0.01 versus Sham-op CM (−); ^#p<0.01 versus OVX CM (−). (B, C) Human bone marrow-derived MSCs or C3H10T1/2 cells were cultured, and osteogenic differentiation was induced with an osteogenic medium (OM) containing β-glycerophosphate (10 mM) and l-ascorbic acid (50 μM) in the presence or absence of hUCB-MSC CM. Alkaline phosphatase (ALP) activities were measured after 7 days. Data are the means±SD. *p<0.01 versus OM - CM 0; ^#p<0.01 versus OM+CM 0. (D) Total RNA was extracted from C3H10T1/2 cells grown for 5 days in the presence or absence of 0.5× or 1×hUCB-MSC CM. The amount of mRNA for osteoblast products was determined by real-time quantitative PCR and expressed as the mRNA abundance relative to vehicle treatment. Data are the means±SD. *p<0.01 versus CM 0. (E–H) C310T1/2 cells were transiently transfected with TOPflash, BRE-Luc, p3TP-Lux, or −1.3OG2-Luc. Cells were then exposed to either vehicle or hUCB-MSC CM (1×). Wnt-3A CM, bone morphogenic protein (BMP)-2, or transforming growth factor (TGF)-β was added as a positive control for TOPflash, BRE-Luc, or p3TP-Lux, respectively. For−1.3OG2-Luc, expression vector for til (pcDNA3-til) was cotransfected as a positive control. Forty-eight hours after transfection, the cells were harvested and used for luminometry. Luciferase activities were normalized for transfection efficiency using Renilla activity, and all values were expressed as the fold induction relative to basal promoter activity. Data are the means±SD, and the graphical results are representative of three independent experiments, each performed in triplicate. *p<0.01 versus control. Color images available online at www.liebertpub.com/tea