Keratinocytes of the basal layer function are to maintain tissue homoeostasis and to fulfil skin repair in response to an external aggression. In wound‐healing, during re‐epithelialization phase, ...epithelial precursor cells gradually migrate from the edges of the wound. The epidermal reconstruction model called standard model allows the vertical skin regeneration process (proliferation/differentiation) to being investigated, and keratinocyte function in preserving skin homoeostasis to being assessed. Here, we developed and characterized a 3D migration model, which introduces a step of keratinocytes migration such as the one observed in the phase of re‐epithelialization in wound‐healing process. We validated the added value and the discriminative potential of this model by demonstrating pro‐epithelializing effects of compounds. This new model allows the role of keratinocytes in different biomechanical and environmental requests to being better understood, and brings a new tool for compound screening and the study of mechanisms involved in skin regeneration.
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
.
Objectives
The present study was designed to asses hHF, hEpi, and hHFDP precursors and stem cell properties in response to clock pathway alteration due to long-term deregulated circadian rhythm
in vivo
.
Materials & Methods
A clinical study protocol was designed to include two groups of women: diurnal workers (control) and shift workers (deregulated). After informed consent, two 3-mm fresh punch biopsies were taken from the occipital region of each donor (10 donors/group). Cell culture characterization, measurement of colony area, culture medium analysis, and RT-qPCR analysis were carried out.
Results
Long-term circadian rhythm deregulation affected clock pathway protein expression and correlated with alterations in hHF, hEpi, and hHFDP properties.
Conclusions
This study provides, for the first time in humans, evidence that
in vivo
deregulation of the clock pathway affects regenerative properties of human skin and hair precursor cells.
The decline of tissue regenerative potential of skin and hair is a hallmark of physiological ageing and may be associated with age‐related changes in tissue‐specific stem cells and/or their ...environment. Human hair follicles (hHF) contain keratinocytes having the property of stem cells such as clonogenic potential. Growth capacity of hHF keratinocytes shows that most of the colony‐forming cells are classified as holoclones, meroclones or paraclones when analysed in a clonal assay (Cell, Volume 76, page 1063). Despite the well‐known impact of ageing on human hair growth, little is known about changes in hHF keratinocyte clonogenic potential with age. This study aimed at assessing the clone‐forming efficiency (CFE) of hHF keratinocytes from three age groups of human donors. It demonstrates that ageing affects hHF keratinocyte CFE.
A number of clinical observations have indicated that the regenerative potential and overall function of the epidermis is modified with age. The epidermis becomes thinner, repairs itself less ...efficiently after wounding, and presents modified barrier function recovery. In addition, the dermal papillae fatten out with increasing age, suggesting a modification in the interaction between epidermal and dermal compartments. As the epidermal regenerative capacity is dependent upon stem and progenitor cell function, it is naturally of interest to identify and understand age-related changes in these particular keratinocyte populations. Previous studies have indicated that the number of stem cells does not decrease with age in mouse models but little solid evidence is currently available concerning human skin. The objective of this study was to evaluate the clonogenic potential of keratinocyte populations isolated from the epidermis of over 50 human donors ranging from 18 to 71 years old. The data indicate that the number of epidermal cells presenting high regenerative potential does not dramatically decline with age in human skin. The authors believe that changes in the microenvironment controlling epidermal basal cell activity are more likely to explain the differences in epidermal function observed with increasing age.
The regenerative capacity of human interfollicular epidermis is closely linked to the potential of immature keratinocytes present within its basal layer. The availability of selection methods and ...culture systems allowing precise assessment of basal keratinocyte characteristics is critical for increasing our knowledge of this cellular compartment. This report presents a multi-parametric comparative study of basal keratinocytes selected according to two different principles: 1) high adhesion capacity on a type-I collagen-coated substrate Adh⁺⁺⁺, 2) high cell-surface expression of α6-integrin Itg-α6 (high). Importantly, analysis performed at the single-cell level revealed similar primary clone-forming efficiency values of 45.5% ± 6.7% Itg-α6(high) and 43.7% ± 7.4% Adh⁺⁺⁺, which were markedly higher than those previously reported. In addition, both methods selected keratinocytes exhibiting an extensive long-term growth potential exceeding 100 cell doublings and the capacity for generating a pluristratified epidermis. Our study also included a global transcriptome comparison. Genome-wide profiling indicated a strong similarity between Adh⁺⁺⁺ and Itg-α6(high) keratinocytes, and revealed a common basal-associated transcriptional signature. In summary, cross-analysis of Adh⁺⁺⁺ and Itg-α6(high) keratinocyte characteristics showed that these criteria identified highly equivalent cellular populations, both characterized by unexpectedly high growth capacities. These results may have broad impacts in the tissue engineering and cell therapy fields.
The use of epidermal stem cells and their progeny for tissue engineering and cell therapy represents a source of hope and major interest in view of applications such as replacing the loss of ...functionality in failing tissues or obtaining physiologic skin equivalents for skin grafting. The use of such cells necessitates the isolation and purification of rare populations of keratinocytes and then increasing their numbers by mass culture. This is not currently possible since part of the specific phenotype of these cells is lost once the cells are placed in culture. Furthermore, few techniques are available to unequivocally detect the presence of skin stem cells and/or their progeny in culture and thus quantify them. Two different sources of stem cells are currently being studied for skin research and clinical applications: skin progenitors either obtained from embryonic stem cells (ESC) or from selection from adult skin tissue. It has been shown that "keratinocyte-like" cells can be derived from ESC; however, the culturing processes must still be optimized to allow for the mass culture of homogeneous populations at a controlled stage of differentiation. The functional characterization of such populations must also be more thoroughly achieved. In order to use stem cells from adult tissues, improvements must be made in order to obtain a satisfactory degree of purification and characterization of this rare population. Distinguishing stem cells from progenitor cells at the molecular level also remains a challenge. Furthermore, stem cell research inevitably requires cultivating these cells outside their physiological environment or niche. It will thus be necessary to better understand the impact of this specific environmental niche on the preservation of the cellular phenotypes of interest.
•Giant-cell arteritis-related aortic dissection occurs earlier in patients with aortitis than in those without.•70% of patients presented Stanford type A aortic dissection.•Half of patients with type ...a dissection had a previous thoracic aorta dilation.•Aortic surgery is the single predictive factor for survival in patients with GCA-related aortic dissection.
To describe characteristics and outcomes of patients with giant cell arteritis (GCA)-related aortic dissection.
We retrospectively included, through a nationwide GCA network, all patients who had an aortic dissection either revealing GCA or occurring during follow-up.
A total of 46 patients were included in this study. Aortic dissection was inaugural and led to GCA diagnosis in 21 patients, whereas it occurred during follow-up in the 25 others, at a median of 53 1–265 months after GCA diagnosis.
Large-vessel vasculitis (LVV) was diagnosed through imaging before or at the time of aortic dissection in 31 (67%) patients. In patients who developed an aortic dissection during follow-up, the aortic event occurred 22 1–143 months post GCA diagnosis in the patients with previous aortitis, whereas it occurred after 72 19–265 months in patients without previously diagnosed aortitis (p = 0.005).
Aortic surgery was performed in 27 (59%) patients and 23 of them survived. A total of 15 (32%) patients died following the aortic dissection, including 11 who were not operated on. In a multivariate analysis, aortic surgery was the single predictor of survival (HR: 4.3; 95% CI: 1.47— 15.7; p = 0.007).
Patients with prior LVV are more prone to develop early aortic dissection and require close monitoring of aortic morphology. One third of patients died from the aortic dissection. Surgery remains the best predictive factor for survival.
DCMU N-(3,4-dichlorophenyl)-N-dimethylurea or diuron is a widely used herbicide, which can cause adverse effects on human, especially on immune cells, due to their intrinsic properties and wide ...distribution. These cells are important for fighting not only against virus or bacteria but also against neoplastic cell development. We developed an approach that combines functional studies and miRNA and RNA sequencing data to evaluate the effects of DCMU on the human immune response against cancer, particularly the one carried out by CD8+ T cells. We found that DCMU modulates the expression of miRNA in a dose-dependent manner, leading to a specific pattern of gene expression and consequently to a diminished cytokine and granzyme B secretions. Using mimics or anti-miRs, we identified several miRNA, such as hsa-miR-3135b and hsa-miR-21-5p, that regulate these secretions. All these changes reduce the CD8+ T cells’ cytotoxic activity directed against cancer cells,
in vitro
and
in vivo
in a zebrafish model. To conclude, our study suggests that DCMU reduces T-cell abilities, participating thus to the establishment of an environment conducive to cancer development.
Deciphering the molecular architecture of coat coloration for a better understanding of the biological mechanisms underlying pigmentation still remains a challenge. We took advantage of a rabbit ...French experimental population in which both a pattern and a gradient of coloration from white to brown segregated within the himalayan phenotype. The whole experimental design was genotyped using the high density Affymetrix® AxiomOrcun™ SNP Array and phenotyped into 6 different groups ordered from the lighter to the darker. Genome-wide association analyses pinpointed an oligogenic determinism, under recessive and additive inheritance, involving genes already known in melanogenesis (ASIP, KIT, MC1R, TYR), and likely processed pseudogenes linked to ribosomal function, RPS20 and RPS14. We also identified (i) gene-gene interactions through ASIP:MC1R affecting light cream/beige phenotypes while KIT:RPS responsible of dark chocolate/brown colors and (ii) a genome-wide epistatic network involving several others coloration genes such as POT1 or HPS5. Finally, we determined the recessive inheritance of the English spotting phenotype likely involving a copy number variation affecting at least the end of the coding sequence of the KIT gene. Our analyses of coloration as a continuous trait allowed us to go beyond much of the established knowledge through the detection of additional genes and gene-gene interactions that may contribute to the molecular architecture of the coloration phenotype.
•Deciphering the molecular architecture of coat coloration still remains a challenge.•Gene-gene interactions contribute to the coloration of body extremities.•RPS processed pseudogenes are likely involved in the coloration of body extremities.•A CNV affecting the KIT gene is likely involved in the English spotting phenotype.
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