Illustrations
Figure 1
A ) Experimental design. Two 3-mm fresh punch biopsies were collected from each subject at 9 a.m. Microdissection of the hair follicle was performed to collect hHF (Biopsy 1) and of the epidermis and the dermal papilla to collect hEpi and hHFDP (Biopsy 2) (diagram adapted from http://www.dermatology.ucsf.edu/skincancer/general/detection/biopsy.aspx). (B) Clock pathway protein expression in epidermal and hair precursor cells: (a-d) transversal sections of human skin labelled with antibodies against PER1 (green; a, c) or BMAL1 (green; b, d) and α6 integrin (red; a, b) or Ki67 (red; c, d), with nuclear Dapi dye (blue); (g-i) hEpi cultured in CFE conditions, labelled with antibodies against PER1 (g) or BMAL1 (h-i); (j-k) hHFDP in vitro labelled with antibodies against BMAL1 and nuclear Dapi dye (blue). (a-d) ×20 objective; (g) ×40 objective, 1.5x num; (h-i) ×40 objective.
Figure 1
Figure 2
Clock pathway deregulation and hEpi properties. A ) Images of hEpi CFE after 13 days of culture (representative of one donor per group). B -D ) Quantification of hEpi CFE from control and deregulated groups showing clone morphology (B ), number of clones (C ), and colony area measurement (D ) (10 donors per group; error bars represent SEM values; *p <0.05 and **p <0.01 based on the Student t-test, relative to the control group; ns: not significant). E ) hEpi clones in conditions used to measure CFE, labelled with antibodies against PER1 or BMAL1, with nuclear Dapi dye (blue) (×40 objective). F , G ) hEpi culture medium concentration (pg/mL) of oxytocin (F ) and cortisol (G ) after 13 days of culture for the control (n = 9) and deregulated (n = 8) groups (error bars represent SEM values; non-significance [ns] is based on the Student t-test, relative to the control group).
Figure 2
Figure 3
Clock pathway deregulation and hHF properties. A ) Images of hHF CFE after 13 days of culture (representative of one donor per group). B -D ) Quantification of hHF CFE from control and deregulated groups showing number of clones (B ), colony area measurement (C ), and clone morphology (D ) (10 donors per group, error bars represent SEM values; *p <0.05 and ** p <0.01 based on the Student t-test, relative to the control group; ns: not significant. E ) hHF clones in conditions used to measure CFE, labelled with antibodies against PER1 or BMAL1 (x40 objective). F , G ) hHF culture medium concentration (pg/mL) of oxytocin (F) and cortisol (G) after 13 days of culture for the control (n = 9) and deregulated (n = 10) groups (error bars represent SEM values; *p ≤0.05 based on the Student t-test, relative to the control group; ns: not significant).
Figure 3
Figure 4
A -C ) Clock pathway deregulation and hHFDP characteristics. hHFDP culture medium concentration (pg/mL) of orexin A (A ) or cortisol (B ) after 17 days of culture for the control (n = 10) and deregulated (n = 9) groups (*p <0.05 based on the Student t-test, relative to the control group). C ) Relative mRNA expression of Sox2 (n = 6/7), versican (n = 6/7), and nexin 1 (n = 5/7) in hHFDP from control and deregulated donors (n = control/deregulated) (**p <0.01 relative to the control group. D , E ) The circadian clock deregulation in vitro model showing representative images of hEpi CFE after 13 days of culture ± PF670462 (PF) at 2 μM (clock pathway inhibitor) and ± melatonin at 50 μM (clock pathway ligand) (D ), and quantification of hEpi CFE according to clone morphology criteria in the control group ± PF670462 (PF) at 2 μM and ± melatonin at 50 μM (E ) (n = 3; each condition was carried out in triplicate per experiment; error bars represent SEM values; *p <0.05 based on the Student t-test, relative to the control group; ns: not significant).
Figure 4
Auteurs
L’Oréal Research and Innovation, Aulnay-sous-Bois, France
Background
In mammals, desynchronized circadian rhythm leads to various biological symptoms. In skin and hair, human epidermal stem cell function in vitro is regulated by circadian oscillations, and thus contributes to tissue aging when deregulated. In mice, circadian arrhythmia of hair follicle stem cells contributes to age-related hair follicle cycling defects. Despite the well-described impact of circadian oscillations through a feedback loop involving the clock pathway on hair and skin stem cell function in vitro, little is known about the change in characteristics or regenerative properties of hHF (human hair follicle keratinocytes), hEpi (human interfollicular epidermal keratinocytes), and hHFDP (hair follicle dermal papilla stem cells) after long-term alteration of circadian rhythm in vivo .