Statistical analysis was performed using the Mann-Whitney test and with Bonferroni correction. differentiation in hASCs. The quantitative alkaline phosphatase (qALP) activity and mineralization levels were clearly enhanced in particular donor cell lines by BMP-2 stimulus. On the contrary, in other cell lines, qALP and mineralization levels were diminished and the lipid formation was enhanced. The current study also suggests that hASCs have accelerated biochemical responsiveness to BMP-2 stimulus in human serum-supplemented culture medium compared with fetal bovine serum. The production origin of the BMP-2 growth factor is also important for its response: BMP-2 produced in mammalian cells enhanced signaling and differentiation responses compared with BMP-2 produced in (Sigma-Aldrich, St. Louis, MO, https://www.sigmaaldrich.com). BMP-2 was used in concentration of 100 ng/ml unless otherwise mentioned. For the Western blot analysis, cells were cultured in 1% HS (GE Healthcare) and 1% FBS (Thermo Fisher Scientific Inc.). Real-Time Polymerase Chain Reaction Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of osteogenic and adipogenic marker genes was performed as described by Mesim?ki and coworkers [1]. Briefly, 2,000 cells per well were plated on a 6-well plate (Thermo Fisher Scientific Inc.). CHO BMP-2 was used in RT-PCR experiments (R&D TA 0910 acid-type Systems). The total mRNA was isolated at the time points of days 7 and 14 using the NucleoSpin RNA II kit (Macherey-Nagel GmbH & Co., Dren, Germany, http://www.mn-net.com). The isolated mRNA was reverse transcribed to cDNA with the High-Capacity cDNA Reverse Transcriptase Kit (Thermo Fisher Scientific Inc.). The data were normalized to the expression of housekeeping gene (human acidic ribosomal phosphoprotein P0) and the relative expression of each gene was calculated using a mathematical model described previously [19]. The primer sequences (Oligomer Oy, Helsinki, Finland, http://www.oligomer.fi) and the accession numbers are presented in Table 1. Table 1. The sequences and accession numbers of the primers used in quantitative real-time polymerase chain reaction Open in a separate window Cell Number The cell number of hASCs cultured in different conditions was analyzed at 14 and 19 days by CyQUANT Cell Proliferation Assay Kit (Thermo Fisher Scientific Inc.), according to the manufacturers protocol as described by Lindroos et al. and Tirkkonen et al. [18, 20]. Alkaline Phosphatase Activity, Mineralization, and Oil Red O-Lipid Formation Analyses of the qALP activity, mineralization, and lipid formation were conducted as previously described [18, 20]. The activity of ALP was studied quantitatively at day 14, as described in TA 0910 acid-type the Sigma TA 0910 acid-type ALP procedure (Sigma-Aldrich). The qALP activity results were normalized with the cell number from the CyQUANT analysis. TA 0910 acid-type The Alizarin red S staining of minerals was analyzed at days 14 and 19. Briefly, the cells were fixed with 70% ethanol for 1 hour (?20C) followed by staining with 2% Alizarin red S solution (pH 4.1C4.3; Sigma-Aldrich) for 10 minutes at room temperature. Finally, for the quantitative analysis, the dye staining the calcium minerals was extracted from the samples with 100 mM cetylpyridinium chloride (Sigma-Aldrich). The intensity of the dye was analyzed with Victor 1420 multiplate reader (PerkinElmer Inc., Turku, Finland, http://www.perkinelmer.com) at 540 nm and the results were normalized with the cell number from the CyQUANT analysis [18, 20]. To assess the adipogenic differentiation of hASCs at 19 days, Oil Red O staining was conducted as previously described, with slight modifications [18, 20]. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes before the last washing steps. DAPI-stained nuclei and the formation of the large lipid droplets stained with the fluorescent Oil Red O were analyzed from the microscopy images by using the ImageJ program (U.S. National Institutes of Health, Bethesda, MD, http://imagej.nih.gov/ij/). The number of the lipid TA 0910 acid-type droplets was normalized with the number of the counted nuclei. Immunocytochemical Staining For the analysis of the subcellular localization of activated SMAD1/5, mesenchymal vimentin and phosphorylated SMAD1/5 were analyzed by immunocytochemical staining after 0 minutes, 30 minutes, and 2 hours of BMP-2 stimulation (test. The resulting values were corrected with the Bonferroni multiple adjustment method based on the number of planned comparisons (supplemental online data; calculated values are listed in supplemental online Tables 2C5). All the differences between and within the groups with adjusted .05 were considered to be significant. Results BMP-2 Induces Donor Cell Line-Independent Activation of SMAD 1/5 Protein in hASCs To analyze the biological functionality of BMP-2 in MSCs, we examined whether BMP-2 activates the internal SMAD pathway. For this, five different hASC lines (HFSC 41/12, 11/12, 15/12, 6/12, and 8/12) were analyzed after BMP-2 induction at time points 24 hours and 7 days by Western blotting of levels of phosphorylated SMAD 1/5, total SMAD1, and -actin as an internal control. Quantitative results of Rabbit Polyclonal to NEDD8 the pSMAD1/5 levels revealed that SMAD1/5 was activated robustly by BMP-2 at both time points in all the hASC lines studied (Fig. 1A). Also, small hASC line-dependent.
