Figure 2

After characterization in terms of mesenchymal stem cell surface markers expression and cell viability, the osteogenic differentiation of WJMSCs was assessed in the first- and second-passage cultures. Cells were cultured for 21 days in Osteogenic Differentiation Medium (Osteogenic BulletKit, PT-3924 & PT-4120, Lonza, Basel, Switzerland) or in DMEM-LG as a control. The ability of the cells to become mature osteoblasts was evaluated in terms of mineral matrix deposition assessed by Alizarin Red staining (AR-S, Sigma Aldrich, St Luis, MO, USA). The cells fixed in 70% ethanol for 1 h at room temperature, were washed with PBS, stained with 40 mM AR-S (pH 4.2) for 10 min at room temperature, washed five times with deionized water and incubated in PBS for 15 min to eliminate non-specific staining. The stained matrix was then observed at different magnification using a Leitz microscope. In panel A the mineralization status (+, positive or -, negative) was correlated with the basal level (at day ''0'') of RUNX-2 expression and ALP activity. RUNX-2 expression was examined by quantitative TaqMan (ABI PRISM 7700, Applied Biosystems Inc, Foster City, CA, USA) RT-PCR (Assay-on-demand, Hs00231692_m1). The data were normalized on the basis of GAPDH expression and reported as relative mRNA expression levels. ΔΔCt method was used to compare gene expression data. ALP activity was measured by the hydrolysis of p-nitrophenylphosphate (PNPP, Sigma Aldrich, St Luis, MO, USA) [22] and one unit was defined as the amount of enzyme which hydrolyzed 1 μmol/PNPP per minute. Cell protein content was determined according to the Lowry method [23]. (* = P < 0.05). B) Relationship between molecular and obstetric parameters. Basal levels of RUNX-2 expression and ALP activity were related to mother's age, weeks of pregnancy and birth weight in 20 WJMSC samples. C) The 20 samples of panel B were subdived in the two reported subgroups: premature birth (subgroup I) and full term birth (subgroup II). All examined molecular and obstetric parameters are reported. F (female), M (male), CS (Caesarian delivery), SP (spontaneous delivery). D) The ability to deposit mineralized matrix was valuated at the indicated times (0, 14, 21 days) in two representative samples of the two subgroups, by Alizarin red staining.