Clonal Expansion of Epithelial Stem Cells from Human ERM

Background: The Epithelial Cell Rests of Malassez (ERM) are the only existing dental-origin epithelial cells found in the periodontal ligament (PDL), around all erupted teeth, throughout life. Several studies have shown that the ERM is a good candidate stem cell niche for enamel producing epithelia. In vitro differentiation of epithelia from the human PDL has been achieved, but the stemness of human ERM (hERM) and their ability for clonal expansion remains unexplored. Human dental epithelial stem cells would find application in post-tramautic facial reconstruction, crown replacement or enamel regeneration in vitro.

Objectives: To develop an in vitro system for the clonal isolation and expansion of hERM and to test their potential stem cell properties.

Methods: hERM cells were clonally isolated using cell surface markers and flow cytometry and were expanded through inhibition of anoikis and exploitation of cell proliferation pathways. hERM clones were tested for self-renewal and karyotype stability, for the expression of stem-cell markers using immunofluorescence microscopy and PCR arrays and for their ability to express enamel specific proteins upon osteogenic induction.

Results: We report a detailed protocol for the sorting and propagating of single live hERM expressing integrin-alpha6 and the undifferentiated cell markers Notch-1 and CD34. We have specifically examined hERM clones for the expression of the epithelial stem cell super-marker LGR5 and the sharing of overlapping genetic programs with other stem and progenitor epithelial cells. Our studies of ERM in the PDL identify epithelial stem cell markers in a novel context.

Conclusion: The isolation and characterization of hERM stem cells should help elucidate the molecular pathways that govern enamel tissue development and represents an advance in application of tooth regeneration in humans without genetic interference.