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

Abstract

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.

Keywords: Adult stem cells; Cell differentiation; Mesenchymal stem cells; Periodontal ligament; Periodontal ligament stem cells; Pluripotency; Progenitor cells; Regeneration; Stem cells; Tissue engineering.

Fig. 1

Cell proliferation of human periodontal ligament derived MSCs (Top panel) at Day 3 and Day 18 after seeding. Spindle like morphology was observed during the cell proliferation. The cells started to from colonies and 80% confluence was observed around Day 18. Bottom panel represents PDLSC’s (A, C, E, G – all unstained) and BMMSC’s (B, D, F, H – all unstained) under the induction of Osteogenic (A and B) adipogenic (C and D) and chondrogenic (E and F) medium on Day 16 and Day24. G and H depicts cells cultured under regular culture medium that served as negative control.

Fig. 2

Immunophenotypic analysis of PDLSC’s (A) and BMMSC’s (B) by flow cytometry. Cultured cells were incubated with specific antibodies against the cell surface antigens CD73, CD105, CD90, CD166, HLA-DR, CD34 and 45. Both PDLSC’s and BMMSC’s showed similar expression profile.

Fig. 3

Picture labelled 1, 2 and 3 represents osteogenic, adipogenic and chondrogenic differentiation of periodontal ligament mesenchymal stem cells respectively. Periodontal cells (A, B and C) stained with Alizarin Red solution (panel1), Oil Red O (panel2) and Alcian blue (panel3) after 28 days under ×10 and ×20 magnification. Pictures D, E and F in each panel (1,2 and3) depicts bone marrow derived cells stained with the same stain solution under ×10 and ×20 magnification. Differentiated osteoblasts and calcium deposits from both the cell types are stained red. Similarly, in panel 2 the differentiated cells demonstrate lipid droplets inside them that are stained bright red by Oil Red O solution while the undifferentiated cells are negative for the stain. Similarly, in panel 3 Cells demonstrate chondrogenic glycosaminoglycans that are stained blue by the Alcian blue solution. While the undifferentiated cells are negative for the stain. Pictures labelled C and F in panel 1,2 and 3 depict cells cultured under regular culture medium that served as negative control. (A, D, C and F - ×10 magnification and B, E-×20 magnification). Panel 4 depict the mRNA expression of pluripotency markers from the PDLSC’s. Both SOX 2 and GADPH were highly expressed and Oct-4 and NANOG showed relatively a lower level of expression. Graph depicting the band intensity is also shown.