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MSC to osteocyte (mouse)

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Series:IN_VITRO DIFFERENTIATION SERIES
Species:Mouse (Mus musculus)
Genomic View:Zenbu
Expression table:TET
Link to TET:FILE
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



Sample Name Time point Donor/Replica
12323-130H1 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 00hr15min biol_rep1 (015mB1)
12324-130H2 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 00hr30min biol_rep1 (030mB1)
12325-130H3 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 01hr biol_rep1 (001hB1)
12326-130H4 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 02hr biol_rep1 (002hB1)
12327-130H5 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 03hr biol_rep1 (003hB1)
12328-130H6 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 06hr biol_rep1 (006hB1)
12329-130H7 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 12hr biol_rep1 (012hB1)
12330-130H8 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 18hr biol_rep1 (018hB1)
12331-130H9 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 24hr biol_rep1 (024hB1)
12332-130I1 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 36hr biol_rep1 (036hB1)
12333-130I2 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day02 biol_rep1 (048hB1)
12334-130I3 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day03 biol_rep1 (072hB1)
12335-130I4 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day04 biol_rep1 (096hB1)
12336-130I5 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day05 biol_rep1 (120hB1)
12337-130I6 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day06 biol_rep1 (144hB1)
12338-130I7 ST2 (Mesenchymal stem cells) cells, medium change (without induction) 00hr biol_rep1
12347-131A7 ST2 (Mesenchymal stem cells) cells, medium change (without induction) day06 biol_rep1
12445-132C6 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 00hr15min biol_rep2 (015mB2)
12446-132C7 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 00hr30min biol_rep2 (030mB2)
12447-132C8 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 01hr biol_rep2 (001hB2)
12448-132C9 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 02hr biol_rep2 (002hB2)
12449-132D1 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 03hr biol_rep2 (003hB2)
12450-132D2 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 06hr biol_rep2 (006hB2)
12451-132D3 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 12hr biol_rep2 (012hB2)
12452-132D4 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 18hr biol_rep2 (018hB2)
12453-132D5 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 24hr biol_rep2 (024hB2)
12454-132D6 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 36hr biol_rep2 (036hB2)
12455-132D7 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day02 biol_rep2 (048hB2)
12456-132D8 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day03 biol_rep2 (072hB2)
12457-132D9 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day04 biol_rep2 (096hB2)
12458-132E1 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day05 biol_rep2 (120hB2)
12459-132E2 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day06 biol_rep2 (144hB2)
12460-132E3 ST2 (Mesenchymal stem cells) cells, medium change (without induction) 00hr biol_rep2
12469-132F3 ST2 (Mesenchymal stem cells) cells, medium change (without induction) day06 biol_rep2
12567-133H2 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 00hr15min biol_rep3 (015mB3)
12568-133H3 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 00hr30min biol_rep3 (030mB3)
12569-133H4 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 01hr biol_rep3 (001hB3)
12570-133H5 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 02hr biol_rep3 (002hB3)
12571-133H6 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 03hr biol_rep3 (003hB3)
12572-133H7 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 06hr biol_rep3 (006hB3)
12573-133H8 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 12hr biol_rep3 (012hB3)
12574-133H9 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 18hr biol_rep3 (018hB3)
12575-133I1 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 24hr biol_rep3 (024hB3)
12576-133I2 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes 36hr biol_rep3 (036hB3)
12577-133I3 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day02 biol_rep3 (048hB3)
12578-133I4 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day03 biol_rep3 (072hB3)
12579-133I5 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day04 biol_rep3 (096hB3)
12580-133I6 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day05 biol_rep3 (120hB3)
12581-133I7 ST2 (Mesenchymal stem cells) cells, differentiation to osteocytes day06 biol_rep3 (144hB3)
12582-133I8 ST2 (Mesenchymal stem cells) cells, medium change (without induction) 00hr biol_rep3
12591-134A8 ST2 (Mesenchymal stem cells) cells, medium change (without induction) day06 biol_rep3