HaCaT Keratinocytes: A Differentiation-Competent Platform for Episomal Replication of HPV Type 11

Few differentiation-competent models exist to study early intra-nuclear processes of human papillomavirus (HPV) in keratinocytes. Early HPV DNA replication is usually studied by transfecting transformed or tumor-derived cell lines (C33A, HEK293/HEK293T, CIN612). While these lines support episome replication, their transformed state and oncogene expression can confound interpretation, and they do not undergo the normal keratinocyte differentiation required for the HPV life cycle. We therefore evaluated HaCaT, a spontaneously immortalized, non-transformed keratinocyte line with reversible differentiation, as a model for HPV episomal replication. We optimized culture conditions—particularly extracellular calcium—to toggle HaCaT cells between basal-like proliferation and differentiation, and refined transfection parameters to deliver plasmid vectors required for HPV11 episomal replication. HaCaT cells display differentiation-associated morphological changes and keratin marker expression comparable to primary keratinocytes. In transient luciferase-based origin replicon assays, HPV11 plasmid replicons showed origin-dependent replication in both undifferentiated and differentiated HaCaT cells. Because Ca²⁺-driven differentiation rewires keratinocyte nuclear organization, this Ca²⁺-controlled HaCaT system enables evaluation of early viral nuclear processes, including episomal replication and differentiation-associated increases in replication activity, in a nuclear architecture–dependent epithelial context without exogenous viral oncogenes or cellular transformation.