Effects of Small Molecules on Dental Pulp Stem Cell Properties

Objectives: Identification of the effect of small molecules (Pluripotin (SC-1), GSK-3 inhibitor IX (BIO) and Rapamycin) on the stemness and differentiation potential of human dental pulp stem cells (hDPSCs) in vitro.

Methods: Human dental pulps were extracted from teeth of healthy subjects aged 16–25 years. hDPSCs at passages 3 to 8 were first treated with various concentrations of SC1, BIO, or Rapamycin to test their cell toxicity. Cells were then treated with optimal concentrations of these small molecules for one week. hDPSCs were then induced in different differentiation medias: osteogenic, neurogenic & adipogenic. Subsequently, hDPSCs were subjected to chemical staining (Alizarin red & Oil red staining) and to qPCR for the detection and quantification of the following gene expressions: Oct4, Nanog, Sox2, P16, ALP, BSP, OCN, Runx2, Nestin, B-III Tubulin, CNPase, GFAP and NFM1.

Results: qPCR revealed increased expression of Oct4, Nanog and Sox2 and decreased expression of P16 in SC1 and rapamycin treated cells compared to the non-treated controls whereas BIO treated cells showed similar expression to controls. Chemically treated groups had decreased Alizarin red and oil red staining compared to the non-treated controls with higher ALP and BSP expression and similar OCN and Runx2 osteogenic gene expressions.  Neural markers: Nestin & B-III Tubulin were increased in chemically treated groups whereas CNPase, GFAP and NFM1 had similar gene expression when compared to the non-treated controls.

Conclusions: SC1 and rapamycin appear to enhance the stemness of hDPSCs evidenced by increased expression of pluripotent stem cell markers Oct4, Nanog and Sox2, and decreased expression of the senescent gene P16. Osteogenic and adipogenic differentiation potential was reduced in chemically treated groups evidenced by decreased chemical staining and differentiation gene expression. Mature neuronal markers expression was reduced whereas neural stem cell markers expression was increased suggesting that hDPSCs were guided to a more immature state.