Long-term culture of human gingival keratinocyte progenitor cells by down-regulation of 14-3-3 sigma

Human gingival keratinocytes in culture stop proliferating after a limited number of passages. This limitation is associated with a gradual depletion of the stem cell compartment of the cell population. Human skin keratinocytes have a three- to five-fold higher proliferation capacity under similar culture conditions, and previous studies indicated that stable down-regulation of the 14-3-3 sigma protein in these cultures prevents stem cell differentiation and generates immortal cell lines without the effects of tumorigenic transformation, e.g., genotypic alterations. In this report, we demonstrate the creation of an immortalized human gingival keratinocyte stem cell line by stable down-regulation of the 14-3-3 sigma protein. Keratinocyte cultures were generated from human subjects ranging from 17 to 92 years of age and retrovirally transduced with a 14-3-3 sigma antisense RNA expression construct. In contrast to the control cultures, which propagated for only 2-5 passages and 25-35 cell doublings, the 14-3-3 sigma-transduced cultures propagated for 11 passages and 110 cell doublings so far. The percentage of stem cells measured by clonal analysis, which gradually decreased in the control cultures, increased to a steady level of over 90% in the 14-3-3 sigma down-regulated culture. This gingival keratinocyte stem cell line and others, which can be generated using the same procedure, have the potential to be useful for studies on stem cell differentiation, for developing gene therapy procedures that target the gingival epithelium, as well as a stable platform for testing oral hygiene products and as potential material for preprosthetic surgery.