Abstract
Regeneration of human epidermis after injury is a key function of progenitor cells. Understanding of decision-making mechanisms for self-renewal and differentiation relies on the development of experimental approaches, including surrogate cell culture-based epidermal regeneration models for assessment of progenitor cell activation, autonomous growth and spontaneous differentiation at the clonal level. Here we propose a 3D epidermal cell culture system which satisfies aforementioned criteria. Growth from single epidermal cells in thick basal membrane-derived gel results in the formation of multilayered spheroids with an inward differentiation. Unlike classical human epidermal culture systems in which differentiation is induced experimentally by stress stimuli, differentiation in 3D is spontaneous, implying in vivo-like context. As such, 3D culture conditions allowed us to evaluate the behavior of primary human keratinocytes and commercially available keratinocyte-derived cell line N/TERT1 (in which telomerase is overexpressed), as well as lentiviral-infected N/TERT1-derivatives, produced in the lab. It is generally assumed that immortalization by telomerase does not interfere with differentiation and hence N/TERT1 is considered a reliable tool for epidermal studies. However, N/TERT1-derived spheroids displayed marked changes in their shape and in the content of Involucrin+ cells, suggesting that telomerase delayed differentiation. This was accompanied by an increased Ki67 index and expansion of K14+/p63+ cell subset. Furthermore, we observed modest disorganization of epidermal tight and adherence junctions, while epithelial polarity and stratification were preserved. Strikingly, secondary infection of N/TERT1 cells with “empty” lentivirus resulted in disruption of epithelial polarity, loss of ability for stratification and absence of Involucrin+ cells. Overall, we suggest a novel experimental approach for assessment of epidermal cell fate regulation and polarity. This approach might be also feasible for the elucidation of mechanisms driving epidermal cell transformation and oncogenesis.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have