Comparative study of xeno-free induction protocols for neural differentiation of human dental pulp stem cells in vitro.
Arch Oral Biol. 2019 Sep 25;109:104572. doi: 10.1016/j.archoralbio.2019.104572. [Epub ahead of print]
Madanagopal TT1, Franco-Obregón A2, Rosa V3.
- Faculty of Dentistry, National University of Singapore, Singapore; Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, Canada.
- Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Institute for Health Innovation & Technology, iHealthtech, National University of Singapore, Singapore.
- Faculty of Dentistry, National University of Singapore, Singapore; National University Centre For Oral Health Singapore, National University Hospital System, Singapore. Electronic address: [email protected].
To compare three different xeno-free protocols for neural differentiation of human dental pulp stem cells (DPSC).
DPSC were treated with three different media to induce neural differentiation namely N1 (DMEM for 5 days), N2 (PSC neural induction media for 7 days) and N3 (neural media with B27 supplement, 40 ng/ml bFGF and 20 ng/ml EGF for 21 days). Cell proliferation (MTS assay), morphology, gene (qPCR for NESTIN, VIMENTIN, TUB-3, ENO2, NF-M and NF-H) and protein expression (flow cytometry) of neurogenic markers were assessed at different time points and compared to untreated cells (DMEM supplemented with 10% FBS). Statistical analysis was performed with global significance level of 5%.
N1 and N2 formulations increased the genetic expression of two out of six genes TUB-3, NF-M and TUB-3, NF-H, respectively, whereas N3 elevated the expression of all genes by the late stage. N3 also stimulated protein expression for NESTIN, TUB-3 and NF-H. Cells treated with both N2 and N3 presented neuron-like morphology, decreased proliferation and expression of stemness genes at protocol end point.
N3 was the most effective formulation in promoting a neurogenic shift in gene and protein expression. Cells provided with the N3 formulation exhibited neuron-like morphology, elaborating axonal-like projections concomitant with cell cycle withdrawal and reduced expression of stemness genes indicating greater commitment to a neurogenic lineage.
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