Comparison of Protocols for Neural Differentiation of Dental-pulp Stem Cell

Objectives: To compare three different xeno-free protocols for neural differentiation of dental pulp stem cell (DPSC)
Methods: DPSC were cultured in normal growth media (Control; DMEM+10% FBS) and three different neural induction media namely N1: DMEM alone for 5 days, N2: Pluripotent stem cells (PSC) neural induction media for 7 days and N3: Neural induction media with 40ng/ml bFGF and 20ng/ml EGF for 21 days. Proliferation of cells was evaluated at early, intermediate and late stages of differentiation using MTS assay. Expression of neural gene namely Nestin, Vimentin, neuron specific enolase (ENO-2), beta III tubulin (TUB-3), neurofilament-medium (NF-M) and neurofilament-heavy (NF-H) and stemness genes namely octamer-binding transcription factor 4 (OCT-4), Nanog Homeobox (NANOG), SRY (sex determining region Y)-box 2 (SOX-2) were evaluated by RT-PCR at same time points. Protein expression for nestin, TUB-3 and NF-H was evaluated at the end of differentiation using flow cytometry. All assays were performed in triplicates and statistical analysis was performed with either one way or two way ANOVA and t test (α=0.05).
Results: Differentiated cells proliferated less compared to control (p>0.005; N3 vs. control) at every stage of differentiation. The neural genes expression was higher in cells treated with N3 than cell treated with N1 and N2 at every stage of differentiation. The stemness genes expression reduced in all the treated groups compared to control at every stage of differentiation (p>0.005 for N3 vs. Control). N3 presented higher decrease in stemness genes expression compared to N1 and N2. The neural protein expression was higher in cell treated with N3 than cells treated with N1 and N2.
Conclusions: The results suggest that neural induction media with bFGF and EGF (N3) induced higher neural gene and protein expression of differentiated DPSC compared to other two differentiation media (N1 and N2).