Expression of Stem Cell Markers in ERM by Epigenetic Agents

Objectives: Epigenetic mechanisms regulate multiple aspects of normal development including stem cell maintenance and differentiation. Fibroblasts can be reprogrammed to pluripotency by the stimulation with the DNA demethylated agent, 5–Azacytidine (5–Aza) (Nature. 2008). One of the stem cell markers, Sox2, was localized in dental lamina but not in Epithelial cell rests of Malassez (ERM) (Eur J Oral Sci. 2013). We examined the expression of stem cell markers in ERM cells treated with epigenetic agents.
Methods: Porcine ERM were cultured in ES cell medium containing under the following conditions: no additions (control); 0.1, 1 or 10 μM 5–Aza; 2 mM Valproate (Vpa) as a HDAC inhibitor; both 5–Aza and Vpa. The cultures were sampled on the 7th and 14th days. The expression levels of Nanog, Oct3/4, Sox2 Klf4, Keratin8, Keratin19, Amelogenin, Ameloblastin, Enamelin, DMP1, COL1A2, Osteopontin, Osteocalcin and PCNA mRNA were observed by quantitative RT–PCR. For quantitative real-time RT-PCR, a reaction mixture was prepared using KAPA SYBR Fast qPCR Kit (NIPPON Genetics, Tokyo, Japan), primers and RT products. Real-time PCR was performed using Light Cycler^® Nano (Roche Diagnostics, Tokyo, Japan). The relative expression of each mRNA was calculated using the ΔΔCt method. The cells were stained with Alkaline Phosphatase (ALP) and were immunocytochemically stained with Nanog and Oct3/4. The significance of the differences was analyzed using the Kruskal-Wallis test.
Results: Expression levels of Nanog, Oct3/4, Sox2, Klf4, Amelogenin and Ameloblastin mRNA in ERM treated with 1 μM 5–Aza alone or both 1 μM 5–Aza and Vpa were significantly higher than in controls (p<0.05). The cells were positively stained with ALP, Nanog and Oct3/4.
Conclusions: The results indicate that epigenetic agents may induce dedifferentiation of ERM.