Stem cell properties of primary human periodontal ligament cells

Objectives: The aim of this study was to isolate mensenchymal multipotent progenitor cells from periodontal ligament (PDLMSCs) harvested from permanent and primary human teeth, and comparatively assess their stem cell properties, including proliferation rate and viability, multipotentiality, and the expression of stem-cell markers. Methods: PDL tissue was obtained from 12 teeth, 6 primary and 6 permanent, extracted from different patients and used to isolate CD105+ CD34- CD45- cells by magnetic activated cell sorting. To identify and quantitatively compare putative stem-cell markers and properties, the following assays were carried out: 1) mRNA levels of CD105, CD166 and Oct-4 genes by PCRq, 2) immunostaining for STRO-1, 3) osteogenic induction evaluated by von Kossa staining and expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), and type-I collagen (Col-I), 4) adipogenic induction assessed by oil red O staining and PPARgamma2 and LPL gene expression, and 5) proliferation rate and viability by trypan blue. Results: Data analysis demonstrated PDLMSCs obtained from both, permanent and primary teeth, expressed mesenchymal and embryonic stem-cell markers, including STRO-1, CD105, CD166 and Oct-4. An increased proportion of CD105+ (p<0.05) and CD45- (p<0.001), and increased mRNA levels of CD166 were found for PDLMSCs from permanent teeth (p<0.05). PDLMSCs treated with adipogenic supplementation showed Oil red O-positive staining and increased LPL/PPARgamma2 mRNA levels were found for PDLMSCs from primary teeth (p<0.05). In osteogenic differentiation, BSP, ALP and Col-I mRNA levels were significantly increased (p<0.05), with BSP mRNA levels higher in PDLMSCs from permanent teeth (p<0.05). Finally, proliferation rate was found to be higher in PDLMSCs from primary teeth as compared to permanent ones (p<0.01). Conclusion: Our findings demonstrate the presence of mesenchymal stem cells in the PDL region of both, primary and permanent teeth, and further indicate phenotypic dissimilarities that may significantly impact on their clinical applications.