5-Aza-2'-deoxycytidine treatment induces skeletal myogenic differentiation of mouse dental pulp stem cells.
OBJECTIVE
Tissue stem cells in dental pulp are assumed to possess differentiation potentials similar to mesenchymal stem cells (MSCs). The aim of this in vitro study is to examine the differentiation potentials of mouse dental pulp stem cells (DPSCs) and develop the appropriate differentiation assay systems for skeletal myogenic differentiation of these cells.


METHODS
Dental pulps were extracted from mandible sections of C57/BL6 mice, and adherent dental pulp cells were isolated in culture. These cells were cultured in osteogenic or adipogenic induction medium to induce osteogenic and adipogenic differentiation. On the other hand, the skeletal myogenic differentiation potential of these cells was investigated using different conditions, such as serum-free medium, Myod1 overexpression, or 5-Aza-2'-deoxycytidine (5-Aza) treatment for DNA demethylation. Muscle-specific transcriptional factor expression was evaluated by RT-PCR, and myotube formation and myosin heavy chain expression were evaluated by phase-contrast microscopy and immunofluorescence staining, respectively.


RESULTS
The adherent dental pulp cells exhibited a proliferative capacity and they showed osteogenic and adipogenic differentiation as seen in previous studies. Although the expression of Myod1 mRNA and myotube formation was not detected in serum-free conditions, the forced expression of Myod1 up-regulated the expression of Myogenin and Pax7 mRNA. However, myotube formation was not confirmed. Interestingly, myosin heavy chain expression and myotube formation were observed following 5-Aza treatment of these cells.


CONCLUSIONS
These results demonstrated that mouse DPSCs possess MSC-like differentiation potential. DNA demethylation induced by 5-Aza treatment resulted in the skeletal muscle differentiation in mouse DPSCs, suggesting that DNA demethylation might trigger this differential induction of mouse DPSCs.
Nakatsuka
Ryusuke
R
Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506, Japan. nakatsur@takii.kmu.ac.jp
Nozaki
Tadashige
T
Uemura
Yasushi
Y
Matsuoka
Yoshikazu
Y
Sasaki
Yutaka
Y
Shinohara
Mitsuko
M
Ohura
Kiyoshi
K
Sonoda
Yoshiaki
Y
D050156:Adipogenesis; D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D003782:Dental Pulp / Q000166:cytology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D059630:Mesenchymal Stem Cells / Q000187:drug effects*; D051379:Mice; D008810:Mice, Inbred C57BL; D024510:Muscle Development / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D017570:MyoD Protein / Q000096:biosynthesis; D018995:Myosin Heavy Chains / Q000096:biosynthesis; D010012:Osteogenesis; D014162:Transfection
D004791:Enzyme Inhibitors; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D000077209:Decitabine; D015254:DNA Modification Methylases; D018995:Myosin Heavy Chains; D001374:Azacitidine