Investigating human eyelash hair follicle growth <i>in situ</i> and <i>ex vivo</i> : a pilot study
Authors: C.I. Platt, J. Laybourne, P. Cannon, R. Paus
Journal: British Journal of Dermatology (2020)

Dear Editor, Eyelashes play a crucial role in protecting the eye from airborne particles and are intimately connected with human beauty.1 Clinically, trichomegaly, hypotrichosis and trichiasis are the result of aberrant eyelash growth, which can compromise eyelash function, result in secondary morbidity and, in severe cases, result in blindness. Paradoxically, given their importance in dermatology and ophthalmology, very little is known about the biology of human eyelashes.1 Previous studies have investigated the growth rate, growth duration and morphology of human anagen eyelash hair follicles (ELHFs) in vivo.2-4 However, these studies provide little information about the molecular controls of normal and pathological human eyelash growth. For this it is essential to examine ELHFs at the tissue level, and to develop ELHF organ culture assays that permit analysis ex vivo.5, 6 The current pilot study represents a first step in this direction. Eyelid biopsies were collected from three patients (mean age 78 ± 13 years; mean number of ELHFs examined per biopsy 14 ± 3) undergoing lower-lid correction at the Manchester Royal Eye Hospital. All patients provided informed consent and the biopsies were stored under UK Human Tissue Authority approval. All ELHFs within a biopsy were microdissected, snap frozen and cryosectioned at 7 µm. Cell proliferation and apoptosis were evaluated immunohistochemically (Ki-67/TUNEL activity) and pigmentation was assessed by Masson–Fontana histochemistry.5, 6 Lef-1 immunoreactivity was assessed indirectly using a rabbit anti-Lef-1 primary antibody (1: 100; Abcam, Cambridge, UK). Anagen ELHFs were microdissected from an additional three patients as above and organ cultured in supplemented William's E medium as described previously.6 ELHFs in the anagen, early-anagen, telogen and catagen stages were identified. Human anagen ELHFs were characterized by an asymmetric bulb, a fully differentiated outer and inner root sheath, Ki-67+ cells in the hair matrix and the basal layer of the outer root sheath, an oval dermal papilla (DP) and prominent melanin granules in the precortical hair matrix and hair shaft (Figure 1a, e).1, 4 Apoptotic (TUNEL+) cells were absent in anagen ELHFs in situ. Telogen ELHFs were identified by an unpigmented club hair, sparse Ki-67+ cells and a round, compacted DP positioned below an unpigmented epithelial strand (Figure 1b, f). ELHFs in early anagen showed a club hair and compacted DP, partially surrounded by a crescent-shaped secondary hair germ containing localized Ki-67+ cells (Figure 1c, g). Catagen ELHFs were characterized by a wide DP stalk with clear TUNEL+ cell nuclei in the HF matrix (Figure 1d, h). Nuclear localization of Lef-1 was observed in the proliferative and precortical regions of anagen ELHFs, suggesting active Wnt signalling in the growing HF (Figure 1i). The percentage of ELHFs at each stage of the cycle was determined following histological characterization: anagen (44 ± 9%), telogen (35 ± 5%), catagen (11 ± 8%) and early anagen (10 ± 2%). Ex vivo, all four microdissected, organ-cultured ELHFs increased in length over time, albeit with a high degree of variability (Figure 1j, k) and appeared to retain an anagen morphology at 72 h, as judged by gross light microscopy. However, from 120 h onwards ELHFs showed reduced pigmentation and development of a club hair-like structure (Figure 1j). Also, fewer Ki-67+ cells were seen in the proximal bulb, along with a more compact DP and thinner hair matrix, compared with freshly isolated anagen ELHFs (Figure 1l, m), suggesting catagen development. This shows for the first time that organ culture of human ELHFs is possible in principle, where the clinically crucial switch from anagen to catagen, and eyelash production as such, can be studied and pharmacologically manipulated ex vivo. The current study extends previous human ELHF work4 by examining not only anagen, but also telogen, catagen and early-anagen ELHFs and by showing that these stages are similar to those in human scalp HFs.7 The percentage of eyelashes in the anagen phase of the HF cycle reported here is greater than previously documented for lower-lid eyelashes;2 however, this may be due to differences in the methods used to identify ELHF cycle stages across different studies. Although some cultured human ELHFs can show eyelash production beyond 72 h under the current assay conditions, they tend to enter into catagen rapidly. Of interest is the appearance of a club hair-like structure in ELHFs at 120 h of culture, as this occurs during telogen scalp HF development in vivo7 and ex vivo.8 Although limited by the low number of organ-cultured anagen ELHFs that could be examined here, our pilot study demonstrates that ELHFs can be microdissected and organ cultured, thus substantially expanding the methodological repertoire of human eyelash research. Chris Platt: Conceptualization (lead); Data curation (lead); Formal analysis (lead); Funding acquisition (equal); Investigation (lead); Methodology (equal); Project administration (lead); Resources (equal); Software (lead); Supervision (lead); Validation (equal); Visualization (lead); Writing-original draft (lead); Writing-review & editing (equal). James Laybourne: Conceptualization (equal); Data curation (supporting); Formal analysis (equal); Funding acquisition (supporting); Investigation (equal); Methodology (supporting); Project administration (equal); Resources (lead); Software (supporting); Supervision (supporting); Validation (equal); Visualization (supporting); Writing-original draft (equal); Writing-review & editing (lead). Paul Cannon: Conceptualization (equal); Data curation (supporting); Formal analysis (supporting); Funding acquisition (supporting); Investigation (equal); Methodology (equal); Project administration (supporting); Resources (supporting); Software (supporting); Supervision (equal); Validation (equal); Visualization (supporting); Writing-original draft (equal); Writing-review & editing (equal). Rauf Paus: Conceptualization (supporting); Data curation (supporting); Formal analysis (lead); Funding acquisition (lead); Investigation (supporting); Methodology (equal); Project administration (supporting); Resources (lead); Software (supporting); Supervision (equal); Validation (equal); Visualization (supporting); Writing-original draft (supporting); Writing-review & editing (lead).