Novel method for proliferation of oral keratinocyte stem cells

Abstract

Stem cell-based tissue engineering offers clear advantages over conventional normal cell approaches. Owing to their specific characteristics, oral keratinocyte stem cells represent an attractive solution for therapeutic applications. However, when cultured in vitro, these cells lose their unique properties, acquiring a limited capacity for self-renewal, and differentiate rapidly into normal functional keratinocytes. The main aim of the present study was to develop an in-vitro method for the expansion of oral keratinocyte stem cells using a biomaterial approach. Oral keratinocyte stem cells were isolated based on the identification of two surface markers - integrin α6β4 and CD71 - using a magnetic method. The cells were cultured on specific substrates formed from blends of polymers: poly(lactide-co-glycolide) (PLGA); poly(lactide-co-glycolide)+polyurethane (PLGA+PU); and poly(lactide-co-glycolide)+extracellular matrix (PLGA+ECM). The polymers were deposited using a laser-based technique - matrix-assisted pulsed laser evaporation. The cells were analyzed for cell size, cell proliferation, colony-forming efficiency, cell adhesion markers (such as E-cadherin and beta 1 integrin), keratinocyte stem cells and differentiation markers. The methods included ELISAs, immunofluorescence and atomic force microscopy imaging. After 14d in culture, cells seeded on PLGA+PU stained positive for p63, cd44H, cytokeratin 19 and integrin α6β4 and negative for involucrin, cytokeratin 14 and cytokeratin 10. The levels of adhesion molecules were significantly increased in cells grown on PLGA+PU: at 14d the E-cadherin levels were 5.4±0.2ng/mL (for cells grown on PLGA+PU) vs. 4.1±0.4ng/mL (for cells grown on control medium) (n=5, p<0.05 Bonferroni). Oral keratinocyte stem cells grown on PLGA+PU had the highest colony-forming efficiency and proliferation rate, together with the smallest cell size, compared with cells grown on control medium or other polymeric substrates. The present study demonstrates that by culturing oral keratinocyte stem cells on PLGA blended with PU it is possible to preserve their phenotype in vitro and to guide their short-term expansion and proliferation. Certain stem-cell characteristics are preserved and their short-term expansion may be enhanced.