Low levels of H₂S enhance hepatic differentiation of pulp stem-cells

Objectives: We have recently established the protocol for differentiating human tooth pulp stem cells (HTPC) to hepatocyte like cells using serum free medium. On the other hand, we reported the effect of close to physiological levels of hydrogen sulfide, to promote osteoclast differentiation in osteoblasts. The objective of the present study is to determine the effect of hydrogen sulfide on the hepatic differentiation. Methods: Cell cultures were isolated from deciduous teeth (n=6), and then the CD117 positive cell fraction was obtained using magnetic beads antibody separator. Cells were grown for 5 days in Dulbecco's modified eagle medium supplemented with 50ng/mL insulin-transferrin-selenium-x (ITS-x), 100μg/mL Embryo-trophyc factor (ETF) and 20ng/mL hepatocyte growth factor (HGF) for hepatic commitment. For hepatic differentiation the cells were cultured in Iscove's modified dulbecco's medium (IMDM) supplemented with 50ng/mL ITS-x, 100μg/mL ETF, 10ng/mL oncostatin, 20ng/mL HGF and 10nM dexamethasone (15 days) with hydrogen sulfide at 0.1 ng/mL in 5% CO₂/Air. Cells were characterized for expressing the hepatic markers alpha feto-protein, albumin and carbamoyl phosphate synthetase. Also urea concentrations and glycogen synthesis were determined. Results: After hepatic induction cell types changed from spindle shaped, fibroblast like to polygonal, hepatocyte-like morphology in both control and test groups. The hepatic markers were expressed more in test group exposed to hydrogen sulfide than in control during the differentiating procedure, determined by the intensity of the fluorescent staining. Urea and glycogen production were also increased, especially glycogen was dramatically increased more than 5 times compared to control (p<0.01) Conclusions: The present results demonstrated that our protocol for hepatic differentiation can be employed as a hepatic differentiation model. Furthermore, the present results suggested that hydrogen sulfide at physiological concentration in human blood increased the ability of hepatic differentiation of HTPC.