Abstract
Despite decades of skin research, regulation of proliferation and homeostasis in human epidermis is still insufficiently understood. To address the role of mitoses in tissue regulation, we utilized human long-term skin equivalents and systematically assessed mitoses during early epidermal development and long-term epidermal regeneration. We now demonstrate four different orientations: (1) horizontal, i.e., parallel to the basement membrane (BM) and suggestive of symmetric divisions; (2) oblique with an angle of 45°–70°; or (3) perpendicular, suggestive of asymmetric division. In addition, we demonstrate a fourth substantial fraction of suprabasal mitoses, many of which are committed to differentiation (Keratin K10-positive). As verified also for normal human skin, this spatial mitotic organization is part of the regulatory program of human epidermal tissue homeostasis. As a potential marker for asymmetric division, we investigated for Numb and found that it was evenly spread in almost all undifferentiated keratinocytes, but indeed asymmetrically distributed in some mitoses and particularly frequent under differentiation-repressing low-calcium conditions. Numb deletion (stable knockdown by CRISPR/Cas9), however, did not affect proliferation, neither in a three-day follow up study by life cell imaging nor during a 14-day culture period, suggesting that Numb is not essential for the general control of keratinocyte division.
Highlights
Human epidermis is a stratified epithelium that follows a tightly regulated proliferation and differentiation program with a delicate interplay of stem cell self-renewal and differentiation in order to maintain tissue homeostasis
While the stem cell populations and their hierarchical order are well defined in murine epidermis and in the murine hair follicle [1,2,3,4,5], less is known about human epidermis
After an initial phase of wound-type regeneration, i.e., hyperproliferation with the consequence of hyperplasia, the cultures enter into a state of tissue homeostasis with reduced proliferation and an apparent reduction of cell layers
Summary
Human epidermis is a stratified epithelium that follows a tightly regulated proliferation and differentiation program with a delicate interplay of stem cell self-renewal and differentiation in order to maintain tissue homeostasis. As an acknowledged paradigm in homeostatic skin, basal epidermal cells divide either “symmetrically”, i.e., horizontally/in parallel to the basement membrane (BM), in order to replenish the basal precursor cell compartment or “asymmetrically” with the mitotic spindle in perpendicular orientation to the BM in order to produce progeny, initiating the differentiation program [7]. A recent study suggested that Numb is involved in maintaining the “the undifferentiated proliferating stem cell pool in the epithelial basal layer” [19] making Numb an excellent candidate for defining asymmetric division and prompting us to study its role in the mitotic regulation of human skin keratinocytes. These data exclude an essential role of Numb in human skin keratinocytes for traversing mitosis but rather support its function in the control of mitotic orientation
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