Cell line: ST2 cells were obtained from RIKEN BioResource Center (BRC, Tsukuba, Japan). These cell line is bone marrow-derived stromal cell line. ST2 differentiated most efficiently into both osteoblasts and adipocytes [3]. Cell culture: ST2 cells were cultured according to the protocols supplied by BRC (RPMI1640 supplemented with 10% fetal bovine serum) [4]. Differentiation induction: Adipocyte differentation: Adipogenic differentiation was induced by changing the medium to differentiation medium supplemented with 10% fetal bovine serum (FBS), 0.5 mM 3-isobutyl-1-methlxanthine, 0.25 mM dexamethasone, and insulin-transferrin-selenium-X supplement containing 5 mg/ml of insulin (Invitrogen, Carlsbad, CA) and 1 mM rosiglitazone. After 48 hr, the differentiation medium was replaced with conditional culture medium supplemented with 10% FBS [3]. Pre-conditioned medium which was originally same as culure medium, was carried by parallel culturing of ST2, to avoid the perturbation by "medium-change" shock as soon as possible.
 Figure 1. Histological staining of ST2 cells using Nile Red staining during adipocyte differentiation. The number of Nile Red stained lipid droplets increased in ST2 cell (4 days after adipocyte induction). Bar: 100 μm.
Osteoblast differentiation: Osteogenic differentiation was induced by changing the medium every three days to culture medium supplemented with 100 ng/ml of bone morphogenetic protein 4 (BMP4, R&D Systems, Mineapolis, MN) [3]. Pre-conditioned medium which was originally same as culure medium, was carried by parallel culturing of ST2, to avoid the perturbation by "medium-change" shock as soon as possible.
   Figure 2. ALP staining of ST2 cells for 0, 6 and 20 days after osteoblast induction. The ALP activity of ST2 cells (right, 20 days) were more prominently increased in the presence of BMP4 than 0 day (left).
Sampling: ST2 cells are sampled during adipocyte or osteoblast differentiation (15min, 30min, 1-3hr, 6,12,18,24,36,48hr, 3-6day) and non-treatment control (2 points; 0,6day).
 Figure 3. Sampling points for ST2 time-course CAGE data
References: 3. Tokuzawa Y, Yagi K, Yamashita Y, Nakachi Y, Nikaido I, et al. Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation. PLoS Genet (2010) 6(7):e1001019. PMID:20628571 4. Mizuno Y, Yagi K, Tokuzawa Y, Kanesaki-Yatsuka Y, Suda T, et al. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun (2008) 368(2):267-272. PMID:18230348.
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