121 Characterization of different immortalized keratinocyte cell lines as models for epidermal differentiation studies

Abstract

In vitro reconstructed skin models have been widely used to study epidermal physiology. However, these models rely on primary cells that are limited by availability and donor variations. To overcome these issues we validated immortalized cell lines that were generated by the expression of SV40 large T antigen and hTERT (NHEK-SV/TERT) or HPV E6/E7 (NHEK-E6/ E7). Both cell lines show indefinite growth up to over 50 passages and displayed increased growth rates compared to primary cells. Primary keratinocytes present a mix of different epidermal cell populations such as keratinocyte stem and cells already committed to differentiation. Immortalized cells did not show distinct populations, arguing that immortalized cells are a more uniform mainly proliferative cell population that needs an exogenous stimulus to commit to differentiation. While NHEK-E6/E7 show a differentiation behavior comparable to primary cells, the highly proliferative NHEK-SV/TERT needed withdrawal of any proliferative stimulus to commit to a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and formed a tight and functional barrier. Both cell lines responded to inflammatory stimuli leading to an epidermal morphology mimicking Th1 or Th2 type dermatosis. In addition these cell lines are suitable for genome editing using CRISPR/Cas9 technology as different target genes could be successfully knocked out with NHEK-SV/TERT showing a higher transfection efficiency simplifying the isolation of edited single cell clones. Thus, the tested cell lines are suitable substitutes for primary cells and have the potential to serve as suitable models to investigate molecular pathomechanisms and test novel therapeutics during pre-clinical evaluation.