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Revision as of 21:44, 3 February 2015

Series:IN_VITRO DIFFERENTIATION SERIES
Species:Mouse (Mus musculus)
Genomic View:Zenbu
Expression table:FILE
Link to TET:[{{{tet_file}}} TET]
Sample providers :Yasushi Okazaki
Germ layer:mesoderm
Primary cells or cell line:primary cells
Time span:6 days
Number of time points:16


Overview

Senile osteoporosis is the most common metabolic bone disease. This disease is often accompanied by increasing adipocytes in bone marrow tissues [1]. The ectopic adipocytes differentiation following bone loss seems to be caused by unbalanced differentiation of mesenchymal stem cells (MSCs) [2]. Although several differentiation regulators of MSCs have already been reported, little is known about the regulatory dynamics of bi-directional adipocytes/osteoblasts differentiation.
To uncover the complex mechanism of osteoporosis and metabolic disease, we performed a variety of genome-wide analyses about gene expression and regulation that could influence adipocytes/osteoblasts differentiation from mouse ST2 cells (bone marrow-derived stromal cell line) [3,4]. In particular, we focused on the differentiation-specific non-cording RNAs and antisense transcripts as the novel regulator candidates of adipocytes/osteoblasts differentiation.

References:
1. Burkhardt R, Kettner G, Bohm W, Schmidmeier M, Schlag R, et al. Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study. Bone (1987) 8(3):157-164. PMID:3606907
2. Nuttall ME, Gimble JM. Controlling the balance between osteoblastogenesis and adipogenesis and the consequent therapeutic implications. Curr Opin Pharmacol (2004) 4(3):290–294. PMID:15140422
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.

Sample description

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.

Quality control

Marker gene expression:
Adipocyte differentation, hCAGE:







Figurea 4. Gene expression (TPM) of key regulators (Hes1, Id1, Id2,Sp7) and differentiation markers (Alpl, Bglap).

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples


12323-130H1ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes00hr15minbiol_rep1 (015mB1)
12324-130H2ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes00hr30minbiol_rep1 (030mB1)
12325-130H3ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes01hrbiol_rep1 (001hB1)
12326-130H4ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes02hrbiol_rep1 (002hB1)
12327-130H5ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes03hrbiol_rep1 (003hB1)
12328-130H6ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes06hrbiol_rep1 (006hB1)
12329-130H7ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes12hrbiol_rep1 (012hB1)
12330-130H8ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes18hrbiol_rep1 (018hB1)
12331-130H9ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes24hrbiol_rep1 (024hB1)
12332-130I1ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes36hrbiol_rep1 (036hB1)
12333-130I2ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday02biol_rep1 (048hB1)
12334-130I3ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday03biol_rep1 (072hB1)
12335-130I4ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday04biol_rep1 (096hB1)
12336-130I5ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday05biol_rep1 (120hB1)
12337-130I6ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday06biol_rep1 (144hB1)
12338-130I7ST2 (Mesenchymal stem cells) cells, medium change (without induction)00hrbiol_rep1
12347-131A7ST2 (Mesenchymal stem cells) cells, medium change (without induction)day06biol_rep1
12445-132C6ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes00hr15minbiol_rep2 (015mB2)
12446-132C7ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes00hr30minbiol_rep2 (030mB2)
12447-132C8ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes01hrbiol_rep2 (001hB2)
12448-132C9ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes02hrbiol_rep2 (002hB2)
12449-132D1ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes03hrbiol_rep2 (003hB2)
12450-132D2ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes06hrbiol_rep2 (006hB2)
12451-132D3ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes12hrbiol_rep2 (012hB2)
12452-132D4ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes18hrbiol_rep2 (018hB2)
12453-132D5ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes24hrbiol_rep2 (024hB2)
12454-132D6ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes36hrbiol_rep2 (036hB2)
12455-132D7ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday02biol_rep2 (048hB2)
12456-132D8ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday03biol_rep2 (072hB2)
12457-132D9ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday04biol_rep2 (096hB2)
12458-132E1ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday05biol_rep2 (120hB2)
12459-132E2ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday06biol_rep2 (144hB2)
12460-132E3ST2 (Mesenchymal stem cells) cells, medium change (without induction)00hrbiol_rep2
12469-132F3ST2 (Mesenchymal stem cells) cells, medium change (without induction)day06biol_rep2
12567-133H2ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes00hr15minbiol_rep3 (015mB3)
12568-133H3ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes00hr30minbiol_rep3 (030mB3)
12569-133H4ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes01hrbiol_rep3 (001hB3)
12570-133H5ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes02hrbiol_rep3 (002hB3)
12571-133H6ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes03hrbiol_rep3 (003hB3)
12572-133H7ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes06hrbiol_rep3 (006hB3)
12573-133H8ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes12hrbiol_rep3 (012hB3)
12574-133H9ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes18hrbiol_rep3 (018hB3)
12575-133I1ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes24hrbiol_rep3 (024hB3)
12576-133I2ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes36hrbiol_rep3 (036hB3)
12577-133I3ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday02biol_rep3 (048hB3)
12578-133I4ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday03biol_rep3 (072hB3)
12579-133I5ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday04biol_rep3 (096hB3)
12580-133I6ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday05biol_rep3 (120hB3)
12581-133I7ST2 (Mesenchymal stem cells) cells, differentiation to osteocytesday06biol_rep3 (144hB3)
12582-133I8ST2 (Mesenchymal stem cells) cells, medium change (without induction)00hrbiol_rep3
12591-134A8ST2 (Mesenchymal stem cells) cells, medium change (without induction)day06biol_rep3
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