Entage of differentiated adipocytes (64 ?6 ) ARA290MedChemExpress ARA290 compared with HS (40 ?4 ), as determined by Oil Red O staining (Figure 3A). These data were confirmed by expression analysis of early (C/EBP?and C/EBP) and late (PPAR, C/EBP, LPL, and ATGL) adipocyte differentiation markers. In proliferating MSCs we detected only a minimal amount of C/EBP?and C/EBP both in cells grown with HS and with OS; there were no significant differences between the two experimental conditions. Following incubation in differentiation medium, we observed a higher increase in the expression of adipogenic markers in OS treated cultures, compared with cells incubated with HS (Figure 3B).Figure 3 Analysis of adipocyte differentiation. A) The table shows the percentage of Oil Red O positive cells treated with OS or HS and then induced to differentiate into adipocytes. The percentage of Oil Red O positive cells was calculated by counting at least 500 cells in different microscope fields. Data are expressed as mean values with standard deviations (*P <0.05). The picture shows a representative field of oil-red positive cells. B) RT-PCR expression analysis of early and late adipocyte differentiation markers in MSCs treated with OS or HS and then induced to differentiate into adipocytes. mRNA levels were normalized with respect to GAPDH, which was chosen as an internal PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28298493 control. Each experiment was repeated at least three times. The histogram shows the changes in mRNA expression levels 14 days after incubation in differentiation conditions of MSCs grown in OS (red bars) or HS (black bars). They are expressed as arbitrary units (*P <0.05). HS, healthy weight sera; MSCs, mesenchymal stem cells; OS, overweight sera.Di Bernardo et al. Stem Cell Research Therapy 2014, 5:4 http://stemcellres.com/content/5/1/Page 6 ofOsterix and osteopontin follow up in osteogenic differentiationWe examined the effects of OS on MSC differentiation into osteocytes in a similar fashion (Figure 4A, B, C, D). Alizarin red staining did not show considerable differences in the osteogenesis process of MSCs incubated with OS or HS (Figure 4D). To gain further insights into osteocyte differentiation, we performed a follow up expression analysis of osteopontin and osterix, which are involved in the osteocyte differentiation process [18,19]. In HS-treated MSCs, the differentiation marker osterix showed a typical bimodal expression profile, with a burst in expression during the first stage of differentiation (Figure 4C). This expression pattern was altered in the OS-treated MSCs. The osteopontin expression profile was also altered in OS-treated cells compared with HS samples. As expected, in HS-treated MSCs, the expression level of osteopontin, an early differentiation marker, was high in the first days of differentiation, then declined and remained stable during the entire maturation process (Figure 4B). On the contrary, in OS-treated MSCs, osteopontin expression, after an initial decrease, exhibited a progressive increase in mRNA levels during thelate differentiation phase (Figure 4B). This result suggests that osteocyte differentiation may be dysregulated in OS samples.Comparison of cytokine expression profiles in overweight and healthy weight seraAdipose tissue secretes a variety of products known as adipokines, including leptin, adiponectin, resistin, and visfatin, as well as cytokines and chemokines such as TNF-, IL-6, and monocyte chemoattractant protein-1 (MCP-1). The release of adipokines by either adipocyt.
