Isolation and charaterization of stem cell populations in the periodontium.

Abstract

Stem cells represent promising candidates for tissue engineering due to their capacity for self-renewal and their potential for differentiating into multiple cell lineages. The periodontal tissues are composed of various cell types, such as periodontal ligament fibroblasts, osteoblasts, cementoblasts, endothelial cells, the Epithelial Cell Rests of Malassez (ERM). Studies have previously identified periodontal ligament stem cells (PDLSC) within these tissues, which have the capacity to form periodontal ligament, cementum and bone. Another potential source of progenitor cells described in periodontal tissues are ERM, which are the only odontogenic epithelial cells in the adult periodontium. The present study identified that ERM contained a unique multipotential stem cell population with similar properties as described for PDLSC. Furthermore, the present proposal investigated the cell surface protein expression of PDLSC to identify unique markers for the isolation and purification of PDLSC. The present study demonstrated that ovine Epithelial Cell Rests of Malassez contain a subpopulation of stem cells that could undergo epithelial-mesenchymal transition into mesenchymal stem-like cells with multi lineage potential. Ex vivo-expanded ERM expressed both epithelial (cytokeratin-8, E-cadherin and Epithelial Membrane Protein-1) and bone marrow stromal/stem cell markers (CD44, CD29, Heat Shock Protein-90β). Integrin α₆/CD49f could be used for the enrichment of clonogenic cell clusters (colony-forming units-epithelial cells [CFU-Epi]) which was weakly expressed by PDLSC. Importantly, ERM demonstrated a capacity to differentiation into bone, fat, cartilage and neural cells in vitro, and form bone, cementum-like and Sharpey’s fibre-like structures when transplanted into immunocompromised mice. Additionally, gene expression studies showed that osteogenic induction of ERM triggered an epithelial-mesenchymal transition. The present study also examined the cell surface protein expression of human PDLSC using CyDye cell surface labelling and two-dimensional electrophoresis coupled with liquid chromatography--electrospray-ionization tandem mass spectrometry. In addition to the expression of well known mesenchymal stem cell associated cell surface antigens such as CD73 (ecto-5'-nucleotidase) and CD90 (Thy-1), PDLSC were also found to express two novel cell surface proteins, Annexin A2 and sphingosine kinase 1. Interestingly, previous studies have implicated CD73, CD90, Annexin A2 and sphingosine kinase 1 expression in the maintenance of various stem cell populations. Comparative analyses investigated the expression of CD73, CD90, Annexin A2 and sphingosine kinase-1 in human gingival fibroblasts, human keratinocytes, ovine PDLSC and ovine ERM cells. Importantly, this study found that human skin epithelial cells lacked any cell surface expression for CD73, CD90 and Annexin A2. In summary, ERM and PDLSC are both important stem cell sources that could play a pivotal role in periodontal homeostasis and regeneration following insult or disease. As periodontal regeneration is essentially a re-enactment of the periodontal tissue development process, it is plausible to suggest that the combination of ERM and PDLSC would hold greater potential for periodontal regeneration compared to established bone marrow-derived mesenchymal stem cells.