Effects of Nanog overexpression on the proliferation and multipotent differentiation potentiality of human dental pulp cells

Abstract

Objective To investigate the effect of overexpression Nanog on the cell proliferationand multipotent differentiation potentiality of human dental pulp cells (hDPCs) . Methods Primary human pulp cells were transfected with plasmid pSIN-EF2-Nanog-IRES-GFP-puro by lentiviral transfection and stable nanog-expressing cell line was screened under fluorescence microscope. The effect of Nanog on the proliferation of hDPCs was examined with BrdU assay. The expressions levels of pluripotency factor Oct4 and Sox2 were detected using real-time PCR and western blotting. Nanog-hDPCs were cultured in odontogenic and adipogenic induction media respectively. The odontogenic and adipogenic differentiation potentiality were evaluated by Alizarin red S staining and Oil red O staining, and the odontogenic and adipogenic markers were detected using real-time PCR. Results The recombinant lentiviral vector containing Nanog was successfully constructed and showed high efficiency during infection in hDPCs. The BrdU assay showed that the growth rate of Nanog-hDPCs was higher compared with the controls (F = 90.421, P = 0.000) . The expressions of Oct4 and Sox2 were increased in Nanog-hDPCs in comparison with the controls (FOct4 mRNA= 71.649, POct4 mRNA = 0.000; FSox2 mRNA= 106.278, PSox2 mRNA = 0.000; FOct4 = 41.632, POct4 = 0.002; FSox2 = 38.962, PSox2 = 0.002) . Furthermore, the mRNA levels of DSPP, DMP1, and OCN were increased, and the number of mineralized nodules was significantly higher in Nanog-hDPCs (FDSPP = 15.261, PDSPP < 0.05; FDMP1 = 16.235, PDMP1 < 0.05; FOCN = 17.019, POCN < 0.05) . Meanwhile, significantly higher mRNA expression levels of LPL and PPARγ2 and more lipid droplets were observed in Nanog-hDPCs compared to the controls (FLPL = 15.542, PLPL < 0.05; FPPARγ2 = 10.437, PPPARγ2 < 0.05) . Conclusion Nanog overexpression enhanced cell proliferation, upregulated the expression of pluripotent markers, and promoted the multipotent differentiation potential (odontogenic and adipogenic differentiation) of hDPCs. Key words: Dental pulp cells; Nanog; Cell proliferation; Multipotent differentiation; Pluripotency-associated transcription factors