Long-Term Culture of Human Gingival Keratinocyte Progenitor Cells by Down-regulation of 14-3-3σ

Abstract

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.

Hypoxia Induces an Undifferentiated Phenotype of Oral Keratinocytes in vitro

“…The ability of hypoxic cells to maintain an undifferentiated state was the other major hallmark in this study, confirmed by the expression pattern of the suprabasal layer specific, differentiated keratinocyte phenotypic marker, 14-3-3σ (Kirschner et al, 2006). Moreover, basal cell phenotypic markers such as α6 integrin and p75 NTR (Rezvani et al, 2011; Nakamura et al, 2007) were noted to be expressed in cells under 0.5% oxygen pressure.…”

Differentiation of Dermal Multipotent Cells Into Odontogenic Lineage Induced by Embryonic and Neonatal Tooth Germ Cell–Conditioned Medium

“…The colony-forming unit assay was performed as previously described [1,28]. Single-cell suspensions of dermal fi broblasts (1 × 10 3 cells or 1 × 10 4 cells) within DMEM containing 15% FBS were seeded into culture plates (Corning, Lowell, MA, USA) and then incubated at 37°C in 5% carbon dioxide.…”

Characterization of mesenchymal stem cells from human normal and hyperplastic gingiva

“…Histologically and functionally, gingiva belongs to masticatory mucosa, which is defined by its ability to resist the friction of food during chewing and is distinguishable from lining mucosa of cheeks and inner lips. However, despite several indications for identification of gingival epithelial stem/progenitor cells (Kirschner et al, 2006), little attention was directed towards demonstrating the MSC identity within human gingival propria. To date, multiple types of cranial neural crestderived mesenchymal stem cells (MSC) have been identified and proved not only to play essential role in local homeostasis but also to hold promise for regenerative therapy (Gronthos et al, 2000;Miura et al, 2003;Seo et al, 2004;Sonoyama et al, 2006;Fujii et al, 2008).…”