AimThe aim of this study was to differentiate human embryonic stem cells (hESCs) into odontoblastic lineage in an optimized culture milieu.
MethodologyIn Phase 1, hESCs were differentiated into mesenchymal stem cells (H9-MSCs). In Phase 2, H9-MSCs were then differentiated into odontoblast-like cells (H9-Odont) under the stimulation of FGF-8 and BMP-4. Alternatively, H9-MSCs were differentiated into osteogenic lineage (H9-Osteo). In Phase 3, H9-Odont were seeded on 17% EDTA-treated dentine substrates in the presence of FGF-8 and BMP-4 for further differentiation. All experiments were performed in triplicate (n=3). One-way anova was used to test hESC differentiation into different cell types. Post hoc Tukey's test was used to compare between groups. P
ResultsH9-Odont expressed the odontoblastic marker DSPP gene 125.470.1 (SD)-folds higher compared with H9-MSCs at mRNA level (real-time RT-PCR). Additionally, the flow cytometry results revealed 53.1 +/- 3.4 (SD) % of DSP (+) cells in H9-Odont. Alternatively, H9-Osteo expressed 5.9 +/- 2.2 (SD) % of DSP (+) cells. Moreover, the SEM results demonstrated that H9-Odont were found to undergo morphological changes from a fibroblast-like shape into more rounded shapes with cytoplasmic extensions into the dentinal tubules when seeded on 17% EDTA-treated dentine substrate in the presence of FGF-8 and BMP-4. However, H9-Osteo and H9-MSCs did not show similar morphological changes under similar culture milieu.
ConclusionThis study supports the potential of hESCs as a stable, consistent, unlimited and off-the-shelf' cell source to obtain odontoblastic cells for future clinical and research applications.