Human embryonic stem cell differentiation into odontoblastic lineage: an in vitro study

Abstract

Aim: The aim of this study was to differentiate human embryonic stem cells (hESCs) into odontoblastic lineage in an optimized culture milieu.

Methodology: In 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 < 0.05 was considered statistically significant.

Results: H9-Odont expressed the odontoblastic marker DSPP gene 125.47 ± 0.1 (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.

Conclusion: This 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.

Keywords: dentine; growth factors; human embryonic stem cells; mesenchymal stem cells; odontoblastic differentiation.