ST2 mesenchymal stem cell differentiation to adipocyte and osteoblast

Time course ID: mouse_ST2_adipocytes, mouse_ST2_ osteoblasts
Sample provider: Yasushi Okazaki, Yutaka Nakachi and Yosuke Mizuno

Introduction

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.

Sample

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).

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Figure 3. Sampling points for ST2 time-course CAGE data

Quality control

Marker gene expression:

Adipocyte differentation, hCAGE:

Figure 3. Gene expression (TPM) of key regulators (Cebpb, Erg2, Pparg) and differentiation markers (Lpl, Fabp4, adipoq).

Osteoblast differentiation, hCAGE:

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

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.

Beginning of non-public section

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Zenbu configurations and status

ST2 cell series

Expression profiles

Gene and CAGE cluster expression for the adipocyte and osteocyte differentiation series
- https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/mouse_ST2_adipocytes/expression_tables/
- https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/mouse_ST2_osteocytes/expression_tables/

MARA based network results

Related samples

Other ST2 data (available)
- microarray time-course data: Another time-course data, not same samples as FANTOM5 CAGE, but ST2 was used. CBX90

Other ST2 data (not published)
- Tilingarray data: PPARγ ChIP (adipo 2day, 6day) and RUNX2 ChIP (osteo 3day, 6day)
- microarray time-course data: These data is derived from same RNAs at same time-point as helicos CAGE on FANTOM5, and more frequent time-point sampling. please see Nakachi's presentation)

Quality control

Marker gene expression

Media: Mouse ST2 osteocytes.png

Media: mouse_ST2_osteocytes.png