BMP2, FGF2, and Dlx3 in Tissue-Specific Dental Stem Cell Differentiation

Stem cell differentiation into target dental tissues is characterized by complex tissue-specific changes in gene profiles facilitated by factors such as BMP2, FGF2, and Dlx3. Objectives: To characterize trends in tissue-specific cell differentiation toward dental follicle (DF), dental pulp (DP), and periodontal ligament (PDL) lineages as they relate to BMP2 and FGF2 growth factors and the Dlx3 transcription factor. Methods: Cells were treated with either BMP2 (80ng/ml), FGF2 (10ng/ml), or a combination of both for a period of 6 days in order to test the effect on cultured MSC, DF, DP, and PDL progenitor cells. To determine the effect of Dlx3 knockdown, MSC and PDL were transfected with Dlx3 siRNA. After 18 days culture, total RNA was extracted and expression of collagen I, Dlx3 and osteocalcin was evaluated by real time RT-PCR. To quantify mineralization, cell cultures were stained with Alizarin Red S (AR-S). Results: FGF2 and BMP2 significantly upregulated collagen I in MSC (4.5-fold for BMP2, 2-fold for FGF2, and 4.7-fold for BMP2 and FGF2 together) while downregulating collagen I in DP (1.37, 1.1, and 3.14-fold, respectively) and PDL (2.39, 1.64, and 3.48-fold, respectively). Microarray comparison indicated significantly higher Dlx3 expression in DF, DP and PDL compared to MSC. Dlx3 knockdown caused a significant reduction in mineralization parameters: AR-S staining was reduced by 27-56% depending on cell type and osteocalcin gene expression by 3.15-7.12-fold. Conclusion: These data indicate that BMP2 and FGF2 upregulated collagen I gene expression in MSC cells and display the opposite effect in DP and PDL cells, suggesting that the effect of growth factors on matrix gene expression depends on the progenitor cell differentiation status. Dlx3 knockdown significantly reduced mineralization indicators, suggesting a role for Dlx3 in the mineralization and differentiation of dental tissues. Support by NIDCR grant DE15425 to TGHD is gratefully acknowledged.