Expression and regulation of PAX-9 and MSX-1 in embryonic and post-natal dental stem cells.

Introduction: Msx1and Pax9, are genes expressed in mesenchymal cells during the odontogenesis that are required for tooth formation. Our investigation aimed to study the expression of these genes in two sources of dental mesenchymal cells. Stem Cells from Human Exfoliated Deciduous teeth (SHED) and Human Embryonic Dental Mesenchymal Cells (HTM) were used. Regulation of gene expression in these cells by FGF, Wnt, BMP4 and Shh signalling pathways together with the effects of tooth matrix proteins (EMD) was investigated. Materials and Methods: Cells were grown in 6 well plates in 3 ml MSCGM medium (Lonza) containing Glutamax, 10% FBS and gentamicin Sulfate. Cells were grown to confluence and when 80% confluent, they were treated for 24 hours with either 20 ng per ml BIO (Gsk3 inhibitor), 1.5µl per ml of porcine dental matrix proteins (EMD), 0.4µl per ml of Fgf8 and 4µl per ml of DMSO (control). RNA was extracted and purified according the RNeasy-kit-protocol from Qiagen. RNA quantitation was done using a Bio Photometer (Eppendorf) in 2:100 dilutions of the samples with nuclease-free water. Quantitative analysis was performed using Rotor Gene Q-serier softhware. 10µl of mixture was made with 5µl of Sensimix- syber green from Quantace, 0.2µl F- primer , 0.2µl R-primer and 4.6µl of cDNA. Results: Complex patterns of changes in the levels of gene expression were observed that were both cell and treatment specific. In particular BMP4 treatment resulted in large upregulation of Msx1 and Pax9 gene expression in both cell types. Differences in gene expression observed between HTM and SHED cells identify possible reasons why SHED cells respond differently to HTM in tissue recombinations directed towards producing biological replacement teeth.