Establishing a technique for isolation and characterization of human periodontal ligament derived mesenchymal stem cells.

Unlabelled: Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs from extracted teeth is reasonably simple, being less invasive and presenting fewer ethical concerns than does the harvesting of MSC's from other sites. The objectives of this study were to isolate and characterize the PDL stem cells (PDLSC) from healthy adults' extracted teeth and then to characterize them by comparing them with bone-marrow derived MSCs (BMMSC).

Methods: The PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls.

Results: The cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs.

Conclusions: The periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC's derived from bone-marrow.