Mitotic activity of keratinocytes in regeneration and tissue homestasis

Abstract

Despite decades of research, the exact regulation of epidermal development and homeostasis remains elusive. Proliferation in the epidermis is controversially discussed and knowledge is mostly derived from studies of mouse skin. However, it is well established that mouse and human skin differ regarding anatomy and likely also proliferative regulation. To address this question, mitoses were systematically assessed in a long-term human fibroblast-derived matrix-based skin equivalent (fdmSE). Keratinocytes in our fdmSE divided in 4 different ways: horizontal, oblique, or perpendicular to the basement membrane (BM) or suprabasally. The largest proportion of divisions occurred in horizontal orientation (< 80 %) at all time points. The second most common division type was oblique division (< 50 %). Perpendicular divisions were found at a low frequency (< 20 %) at intermediate time points only. They were absent at early and late time points. Thus, it appears that in the human interfollicular epidermis (IFE) all types of divisions are active. Importantly, we also observed suprabasal mitoses present at all analysed time points in the SE. Suprabasal division in epidermis has so far been restricted to embryogenesis, wound healing and diseased skin. We could confirm that it is also part of the normal human epidermis in situ thus suggesting that this spatial mitotic organisation is part of tissue homeostasis in human epidermis. These cells are in an early stage of differentiation as suggested by their expression of keratin 10 with a connection to the BM still detectable in some cases. Furthermore, we aimed at investigating asymmetric cell division in the IFE. To maintain the delicate interplay between self-renewal and differentiation, progenitor cells have to divide asymmetrically. Differential daughter cell fate can be established in two ways: oriented division which displaces one daughter cell from the stem cell niche, or asymmetric distribution of cell fate determinants to the daughter cells. Several components of oriented division have been proposed in invertebrate and vertebrate systems including the PAR-complex, or the adaptor proteins NuMA, Inscuteable and LGN. However, antibodies available for those proteins did not allow detecting these markers here. Instead, we identified the Notch inhibitor Numb as a possible marker for asymmetric keratinocyte division. Numb was segregated asymmetrically during some divisions of the human keratinocytes in 2D cultures. To determine its function, we established a protocol to stably knock down Numb in the human keratinocytes using CRISPR/Cas9. Notably, Numb deletion did not affect proliferation in short term culture (14 days), suggesting that it is not essential for mitosis per se. Instead, Numb may be important for the regulation of cell fate in epidermal regeneration, a question that needs to be addressed in future studies.