A primer for studying cell cycle dynamics of the human hair follicle

Abstract

Abstract:The cell cycle is of major importance to human hair follicle (HF) biology. Not only is continuously active cell cycling required to facilitate healthy hair growth in anagen VI HFs, but perturbations in the cell cycle are likely to be of significance in HF pathology (i.e. in scarring, non-scarring, chemotherapy-induced and androgenic alopecias). However, cell cycle dynamics of the human hair follicle (HF) are poorly understood in contrast to what is known in mouse. The current Methods Review aims at helping to close this gap by presenting a primer that introduces immunohistological/immunofluorescent techniques to study the cell cycle in the human HF. Moreover, this primer encourages the exploitation of the human HF as a powerful and clinically relevant tool to investigate mammalian cell cycle biology in situ. To achieve this, we describe methods to study markers of general 'proliferation' (nuclei count, Ki-67 expression), apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, cleaved caspase 3), mitosis (phospho-histone H3, 'pS780'), DNA synthesis (5-ethynyl-2 0 -deoxyuridine) and cell cycle regulation (cyclins) in the human HF. In addition, we provide specific examples of dual immunolabelling for instructive cell cycle analyses and for investigating the cell cycle behaviour of specific HF keratinocyte subpopulations, such as keratin 15+ stem/ progenitor cells.

In vitro skin three-dimensional models and their applications

“…Stratum basale, Stratum spinosum Undefined role in cell division, rRNA synthesis, maintenance of mitotic spindle [100][101][102] Cleaved caspase 3 Stratum granulosum Execution of apoptosis [102][103][104] epidermis shows two germinal layers (stratum basale and spinosum), a secretory layer (stratum granulosum), and a layer of dead cells aka corneocytes, and secreted lamellar bodies (stratum corneum). Thick skin on palms of hands and feet features an additional layer, stratum lucidum, between stratum granulosum and stratum corneum.…”

“…Nuclei were labeled with Draq5. Scale bars, 100 m. Ki67 is a well-known marker to differentiate between dividing and postmitotic cells [102]. This is found within the nucleus during G 1 , S and G 2 , while in mitosis it is bound to dividing chromosomes.…”

“…Caspase 3 is a major executioner caspase in all types of apoptosis. Its prodomain is cleaved upon apoptosis induction thereby activating its function that leads to the execution of apoptotic cell death [102,104,105].…”

Visualization of Global RNA Synthesis in a Human (mini-) Organ in Situ by Click Chemistry

“…Comparative analysis of EU incorporation within the matrix, pre-cortex, IRS and DP of the HF bulb [9,10] showed relative compartmental homogeneity in levels of RNA synthesis, as no significant differences in EU signal were seen between these cell populations (Figure 1CD). Supporting this, visualization of proliferation (Ki-67) [9,10] against EU incorporation revealed no distinguishable pattern of RNA synthesis that associated with either cell cycle activity or arrest in this compartment (Figure 1E).…”

“…Moreover, we document that active RNA synthesis can be modulated ex vivo and that this can be sensitively quantified in situ . The simple new technique reported here enriches future toxicological ex vivo studies of human tissues by adding global RNA and protein synthesis to the customary proliferation and apoptosis read-out parameters [9,15], using scalp HFs as a model human (mini-) organ.…”