α6 Integrin (α6high)/Transferrin Receptor (CD71)low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells.

The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.

Elastogenic potential and antisagging properties of a novel Murraya koenigii extract.

BACKGROUND: The process by which functional elastic fibers are produced, namely elastogenesis, is complex and difficult to assess in vitro. Identifying efficient elasticity-boosting ingredients thus represents a challenge. AIMS: The elasticity-boosting properties of a novel extract of Murraya koenigii leafy stems were assessed in vitro in 3D culture models before being evaluated in human female volunteers. METHODS: Synthesis of elastic fiber related proteins was evaluated in a skin-equivalent model. Using multiphoton microscopy, the structural organization of elastin deposits was studied within a scaffold-free dermal microtissue. Biomechanical properties of the 3D microtissue were also measured by atomic force microscopy. In vivo, fringe-projection and image analysis were used to evaluate nasogenian fold severity in a panel of Caucasian female volunteers. The impact of gravity on visible signs of facial aging was assessed by clinical scoring carried out alternatively in the supine and sitting positions. RESULTS: We showed the Murraya koenigii extract increased protein expressions of elastin and fibrillin-1 in a 3D skin equivalent model. Using scaffold-free dermal microtissue, we confirmed that Murraya koenigii extract allowed a proper and ordered network of elastin deposits and consequently improved tissue elasticity. Clinical data showed that a twice-daily application for 98 days of the extract formulated at 1% allowed to visibly reduce nasogenian fold severity, jowl severity and to mitigate the impact of gravity on the facial signs of aging. CONCLUSION: The newly discovered extract of Murraya koenigii leafy stems represents an innovative antiaging ingredient suited for elasticity-boosting and antisagging claims.

Chronological aging impacts abundance, function and microRNA content of extracellular vesicles produced by human epidermal keratinocytes.

The disturbance of intercellular communication is one of the hallmarks of aging. The goal of this study is to clarify the impact of chronological aging on extracellular vesicles (EVs), a key mode of communication in mammalian tissues. We focused on epidermal keratinocytes, the main cells of the outer protective layer of the skin which is strongly impaired in the skin of elderly. EVs were purified from conditioned medium of primary keratinocytes isolated from infant or aged adult skin. A significant increase of the relative number of EVs released from aged keratinocytes was observed whereas their size distribution was not modified. By small RNA sequencing, we described a specific microRNA (miRNA) signature of aged EVs with an increase abundance of miR-30a, a key regulator of barrier function in human epidermis. EVs from aged keratinocytes were found to be able to reduce the proliferation of young keratinocytes, to impact their organogenesis properties in a reconstructed epidermis model and to slow down the early steps of skin wound healing in mice, three features observed in aged epidermis. This work reveals that intercellular communication mediated by EVs is modulated during aging process in keratinocytes and might be involved in the functional defects observed in aged skin.

Maintenance of Chronological Aging Features in Culture of Normal Human Dermal Fibroblasts from Old Donors.

Chronological aging is defined as a time-dependent decline of tissue homeostasis which severely impacts skin. Understanding the mechanisms of skin aging is an active research area limited by the lack of relevant in vitro models. Being a component of aging, replicative or stress-induced senescence is repeatedly used to mimic skin aging in vitro, thus presenting only a partial view of the complexity of aging. Herein, we aimed to clarify whether primary normal human dermal fibroblasts retained age-related characteristics when cultured in 2D monolayer, and could be used as a relevant model for aging research. We compared three groups of fibroblasts isolated from different aged donors. We observed strongly decreased population doubling capacities, a reduced clonogenic ability, an impairment in extracellular matrix production together with modifications of respiratory metabolism with an increase in age. These disruptions were particularly marked when comparing fibroblasts isolated from old individuals (over 70 years old) to those isolated from young individuals (18-37 years old), while cells from middle-aged donors exhibited an intermediate profile. These alterations of cell features can be related to the signs of dermis aging, thus showing that cultured primary cells indeed retain some characteristics of the original tissue from which they were extracted.

A new organotypic model containing dermal-type macrophages.

Human skin equivalents (SEs) are popular three-dimensional (D) cell culture systems in fundamental and applied dermatology. They have been made to contain dendritic cells, but so far no study on the incorporation of potentially anti-inflammatory dermal macrophages has been performed. Here, we show that monocyte-derived dermal-type macrophages can be introduced into a rigid scaffold with dermal fibroblasts. They maintain their cell surface markers CD163, DC-SIGN/CD209 and HLA-DR, which discriminate them from monocytes and dendritic cells. They retain the ability to produce the anti-inflammatory cytokine IL-10 in response to lipopolysaccharide (LPS) and to phagocytose latex beads. We thus demonstrate the feasibility of creating macrophage-fibroblast 3D cultures as a first step towards generating SEs with dermal macrophages.

Modulation of CD86 expression in skin dendritic cells does not always correlate with changes in DC motility, migration and allostimulatory functions.

CD86 expression is a well-known activation marker of dendritic cells (DC). In this study, we compared the level of CD86 expression in monocyte-derived skin DC with their motility, migratory abilities and allostimulatory capabilities. We show that motility and migration could be uncoupled from activation and that the immune response-modulating effects of certain compounds may correlate with down-regulation of CD86 expression rather than with effects on motility and migration.

Tspan8 Drives Melanoma Dermal Invasion by Promoting ProMMP-9 Activation and Basement Membrane Proteolysis in a Keratinocyte-Dependent Manner.

Melanoma is the most aggressive skin cancer with an extremely challenging therapy. The dermal-epidermal junction (DEJ) degradation and subsequent dermal invasion are the earliest steps of melanoma dissemination, but the mechanisms remain elusive. We previously identified Tspan8 as a key actor in melanoma invasiveness. Here, we investigated Tspan8 mechanisms of action during dermal invasion, using a validated skin-reconstruct-model that recapitulates melanoma dermal penetration through an authentic DEJ. We demonstrate that Tspan8 is sufficient to induce melanoma cells' translocation to the dermis. Mechanistically, Tspan8+ melanoma cells cooperate with surrounding keratinocytes within the epidermis to promote keratinocyte-originated proMMP-9 activation process, collagen IV degradation and dermal colonization. This concurs with elevated active MMP-3 and low TIMP-1 levels, known to promote MMP-9 activity. Finally, a specific Tspan8-antibody reduces proMMP-9 activation and dermal invasion. Overall, our results provide new insights into the role of keratinocytes in melanoma dermal colonization through a cooperative mechanism never reported before, and establish for the first time the pro-invasive role of a tetraspanin family member in a cell non-autonomous manner. This work also displays solid arguments for the use of Tspan8-blocking antibodies to impede early melanoma spreading and therefore metastasis.

Evolution of three dimensional skin equivalent models reconstructed in vitro by tissue engineering.

Since the culture of keratinocytes on feeder layers, research to produce skin equivalents has been motivated by the challenge of treating large burns and chronic wounds and by European regulations which both require proof of the innocuousness and the effectiveness of cosmetic products, and which forbid animal testing. The dynamism in fundamental research, dermocosmetology and the pharmaceutical industry has led to the evolution and complexification of reconstructed skin. The Collagen-GAG-Chitosan sponge, as well as the self-assembly model, allow dermal reconstruction in which the neosynthesized extracellular matrix contains all of the desired macromolecules. It is deposited forming an ultrastructurally organised architecture. The quality of the dermis obtained allows the development and regeneration of a pluristratified and differentiated epidermis firmly anchored by an organised dermal-epidermal junction. Evolution of reconstructed skin into models which are more and more similar to the physiological skin results in higher graft take rates in the treatment of burns and chronic wounds, and brings to research, to dermocosmetology and to the pharmaceutical industry, a wide range of products such as pigmented, endothelialized, immunocompetent, and now adipose reconstructed skins. The present review will mainly concentrate on the latest developments in skin engineering and will mostly concern the studies carried out by our groups.

Variations in the characteristics of keratocytes in culture in relation to their location in human cornea.

To reconstruct artificial stroma close to corneal stroma, it is necessary to use keratocytes with high proliferative potential that maintain the keratocyte phenotype as characterised by CD34. To select such cells, we tested the proliferative potential and characterised the keratocytes isolated from 4 different areas of the human cornea: superior perilimbal, inferior perilimbal, superior central and inferior central. Keratocytes isolated from these different areas had significantly different growth rates (p<0.05), as measured by population doublings: superior perilimbal (42.59+/-11.78) > inferior perilimbal (38.23+/-12.67) > superior central (35.69+/-8.07) > inferior central (25.35+/-7.63). Their clonogenic potential evolved in the same order. Moreover, CD34 labelling gave higher levels in the central areas in relation to the perilimbal areas. We found the best location for isolating keratocytes for stromal reconstruction. The superior perilimbal area had the greatest capacity for proliferation, as well as the best clonogenic potential and the average CD34 level (70%) remained high.

Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny.

The epidermis undergoes constant renewal during its lifetime. This is possible due to a special population of keratinocyte stem cells (KSCs) located at the basal layer. These cells are surrounded by their direct progeny, keratinocyte progenitors or transient amplifying cells (TAs), which arise from cell division. Skin is exposed every day to sun radiation; in particular, UVA radiation penetrates through the epidermis and induces damage to KSCs and TAs. Although keratinocytes in the basal layer are the most likely skin carcinomas and/or photoaging cells of origin, surprisingly few studies have addressed the specific responses of these cells to UV radiation. In this study, we showed for the first time that keratinocyte stem cells were more resistant to UVA irradiation than their direct progeny, transient amplifying cells. Using both the MTT assay and clonogenic assay, we found that KSCs were more photo-resistant compared to TAs after exposure to different doses of UVA (from 0 to 50 J/cm2). Moreover, KSCs had a greater ability to reconstruct human epidermis (RHE) after UVA exposure compared with TAs. Finally, investigations of DNA repair using the comet assay showed that DNA single-strand breaks and thymine dimers were repaired quicker and more efficiently in KSCs compared with TAs. In a previous work, we showed that the same stem cell population was more resistant to ionizing radiation, another carcinogenic agent. Collectively, our results combined with other observations demonstrate that keratinocyte stem cells, which are responsible for epidermal renewal throughout life, are equipped with an efficient arsenal against several genotoxic agents. Our future work will try to identify the factors or signaling pathways that are responsible for this differential photo-sensitivity and DNA repair capacity between KSCs and TAs.

Long-term Genoprotection Effect of Sechium edule Fruit Extract Against UVA Irradiation in Keratinocytes.

Photoprotection is essential to prevent the long-term deleterious effects of ultraviolet (UV), including skin cancer and photoaging. So far, there has been an increase in the use of natural bioactive phytochemicals for the development of more effective skin photoprotective agents. However, the molecular mechanisms underlying the photochemoprotection activity of such compounds remain largely unknown. The objective of this study was to investigate the effects of a Sechium edule fruit extract (SEE) in terms of photoprotection against UVA in primary human keratinocytes. We found that SEE protected keratinocytes against UVA-induced cytotoxicity, decreased the intracellular amounts of reactive oxygen species, and reduced oxidatively induced DNA lesions after UVA exposure. Furthermore, SEE decreased the induction of CPD lesions in UVA-irradiated keratinocytes and exhibited increased DNA repair of such photoproducts at 24 h postexposure. Finally, using DNA repair biochips, we demonstrated that SEE-treated keratinocytes had DNA enzymatic repair activities more efficient for abasic sites, CPD and thymine glycols. Therefore, the benefits of SEE against UVA could be explained by a combination of antioxidant activity, the reduction in DNA damage, and the enhancement of DNA repair capacities.

Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light.

BACKGROUND: Artificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. OBJECTIVE: The purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. METHODS: The spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. RESULTS: We developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450 nm, 525 nm and 625 nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99 J/cm2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the "mitochondria" and "integrin signaling" categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. CONCLUSION: Artificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede normal cellular functions and contribute to premature skin aging. The present results extend our knowledge of the effects of the low-energy wavelengths that are increasingly used to treat skin disorders.

Effects of solar ultraviolet radiation on engineered human skin equivalent containing both Langerhans cells and dermal dendritic cells.

Exposure of human skin to solar ultraviolet (UV) light induces local and systemic immune suppression. It is known that alterations of immune functions of Langerhans cells (LCs) and dermal dendritic cells (DDCs) mediate this phenomenon. The purpose of this study was to mimic in vitro the early UV-induced skin disruption to better understand the involvement of the skin micro-environment in triggering this immunosuppressive state. We therefore developed skin equivalents (SEs) integrating LCs and DDCs derived from monocytes (mo-LCs and mo-DDCs, respectively). First, we showed that Langerin(+) mo-LC and dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (SIGN)(+) mo-DDCs were immunolocalized in situ in epidermal and dermal compartments of SEs, respectively. The SE micro-environment without immune cells displayed full cytokine profile that may ensure and maintain differentiation, localization, and immaturity of LCs and DDCs in situ, as shown by secretion of granulocyte-macrophage colony-stimulating factor, transforming growth factor beta (beta)-1, interleukin (IL)-4, IL-13, and IL-15 involved in cell differentiation; presence of complete chemokine network as macrophage inflammatory protein 3 alpha (alpha); low secretion of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), IL-1 beta, IL-6, and IL-8; and surprising secretion of immunosuppresive cytokine IL-10. Second, we demonstrated that skin micro-environment homeostasis was greatly disrupted under solar UV irradiation of SEs. In fact, we showed a pro-inflammatory state characterized by high secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 and low secretion of IL-10. This breakdown of immune homeostasis was visualized at the same time as in situ migration of mo-LCs and mo-DDCs into the dermal equivalent of SEs. Moreover, this tissue migration of mo-LCs and mo-DDCs into SEs was in accordance with the chemokine (C-C motif) receptor 7 expression and the DC-lysosome-associated membrane glycoprotein acquisition only on mo-LCs. Our results highlighted major participation of the skin micro-environment in the triggering and modulating of UV-induced skin immune responses. In addition, it could be concluded that these SEs are reliable tools for modeling biological events inaccessible in humans.

Mixed Langerhans cell and interstitial/dermal dendritic cell subsets emanating from monocytes in Th2-mediated inflammatory conditions respond differently to proinflammatory stimuli.

The skin harbors two dendritic cell (DC) subsets, Langerhans cells (LC) and interstitial/dermal DC (IDDC), which traffic to lymph nodes after inflammation and ultraviolet stress. To demonstrate that monocytes may act as DC precursors for skin DC in postinflammatory recolonization, we generated LC and IDDC from monocytes by using cytokines related to the T helper cell type 2 environment [granulocyte macrophage-colony stimulating factor/transforming growth factor-beta/interleukin-13/tumor necrosis factor alpha (GM-CSF/TGF-beta/IL-13/TNF-alpha)]. In this study, skin DC [LC as Langerin/CD207(+) cells and IDDC as DC-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN)/CD209(+) cells] displayed desynchronized programs along their differentiation, activation/maturation processes in response to stimuli characteristics of a proinflammatory context. First, we demonstrate that monocytes are able to diverge simultaneously along two distinct pathways toward Langerin(+)-LC-type DC and DC-SIGN(+)-IDDC. Second, as TGF-beta is known to antagonize the TNF-alpha-induced maturation process of DC, we showed that IDDC did not mature and acquired a low CC chemokine receptor 7 (CCR7) receptor expression even when stimulated with prolonged incubation with TNF-alpha. It is striking that the LC subset is able to express a high level of CCR7 expression and the maturation marker DC-lysosome-associated membrane protein (DC-LAMP). Third, mixed LC and IDDC subsets secrete IL-10 and IL-12 when stimulated by CD40 ligand and lipopolysaccharide (LPS) but not after prolonged incubation with TNF-alpha. In contrast, LPS was a better activator of IL-10 secretion than the CD40 ligand for GM-CSF/IL-4-generated DC and for GM-CSF/TGF-beta/IL-13-generated LC and IDDC populations. To summarize, the phenotypic/migratory maturation status of LC may be more easily enhanced by stimuli mimicking a proinflammatory situation, and IDDC are more resistant. Moreover, our culture system provided a means of studying cross-talk between two skin DC outside of their respective skin compartment.

Development of an optimised culture medium for keratocytes in monolayer.

UNLABELLED: Our objective was to formulate a medium for monolayer culture optimising both keratocyte growth and preservation of the keratocyte phenotype. METHODS: An experimental matrix selected 14 media to test, using 7 components. Selection criteria were growth rates over 5 passages and expression of the CD34 marker. RESULTS: Acetylcholine, insulin and vitamin C had no effect on growth and differentiation. The DMEM + Ham F12 1 : 1 based medium was selected for its initial effect on growth. At concentrations of 5 ng/ml, b-FGF improved the percentage of CD34+ cells without reducing growth rates. New-born calf serum (NCS) had a greater effect on growth than foetal calf serum (FCS). We showed three major interactions: between b-FGF and IGF-1, FCS and IGF-1 and NCS and b-FGF. CONCLUSION: We selected the following medium, which provides optimal growth and preservation of the CD34+ phenotype: DMEM/HAM-F12 + 10% NCS + 5 ng/ml b-FGF + antibiotics.

Evolutive skin reconstructions: from the dermal collagen-glycosaminoglycan-chitosane substrate to an immunocompetent reconstructed skin.

The development of human skin models that have the same properties as genuine human skin is of particular significance. Very promising skin models are the three-dimensional artificial skin constructs, which, similar to genuine skin, consist of an epidermis of differentiated keratinocytes and a dermis. A skin equivalent based on a collagen-glycosaminoglycan-chitosan dermal substrate has been developed to meet the growing demand in tissue engineered skin equivalents. We used this model to investigate whether CD34-generated Langerhans/dendritic cell precursors could be integrated into this skin equivalent model and pursue their differentiation without addition of cytokine and growth factor. To address the issue of dendritic cell (DC) differentiation, an endothelialized skin equivalent coculture model was used to study the behaviour of haematopoietic progenitor cells (HPC) in epidermal and dermal environments. CD34(+) HPC were cultured for 6 days with GM-CSF, TGFbeta1 and TNFalpha and seeded in the endothelialized skin equivalent at different time points to favour dermal or epidermal integration. This integration (after keratinocyte seeding, only and in absence of exogenous GM-CSF, TNFalpha, TGFbeta1) gave rise both cutaneous DC, i.e. epidermal Langerhans cells (CD1a(+), HLA-DR(+)) and dermal DC (DC-SIGN(+), HLA-DR(+)) while endothelial cells are sufficiently activated to acquire HLA-DR expression. For the first time, the presence of a living dermal equivalent could provide a more complex environment integrating vascular components to study the differentiation of interstitial DC in a dermis equivalent. Such sophisticated skin equivalent may clarify some intriguing aspects of the numerous regulatory mechanisms controlling skin homeostasis.

In vitro glycation of an endothelialized and innervated tissue-engineered skin to screen anti-AGE molecules.

Glycation is one of the major processes responsible for skin aging through induction of the detrimental formation of advanced glycation end-products (AGEs). We developed an innovative tissue-engineered skin combining both a capillary-like and a nerve networks and designed a protocol to induce continuous AGEs formation by a treatment with glyoxal. We determined the optimal concentration of glyoxal to induce AGEs formation identified by carboxymethyl-lysin expression while keeping their toxic effects low. We showed that our tissue-engineered skin cultured for 44 days and treated with 200 µm glyoxal for 31 days displayed high carboxymethyl-lysine expression, which induced a progressively increased alteration of its capillary and nerve networks between 28 and 44 days. Moreover, it produced an epidermal differentiation defect evidenced by the lack of loricrin and filaggrin expression in the epidermis. These effects were almost completely prevented by addition of aminoguanidine 1.5 mm, an anti-glycation compound, and only slightly decreased by alagebrium 500 µm, an AGE-breaker molecule. This tissue-engineered skin model is the first one to combine a capillary and nerve network and to enable a continuous glycation over a long-term culture period. It is a unique tool to investigate the effects of glycation on skin and to screen new molecules that could prevent AGEs formation.

When integrated in a subepithelial mucosal layer equivalent, dendritic cells keep their immature stage and their ability to replicate type R5 HIV type 1 strains in the absence of T cell subsets.

Many potential targets of human immunodeficiency virus type 1 (HIV-1) reside in the human reproductive tract, including dendritic cells (DC). The ability of these cells to replicate HIV-1 is dependent on many factors such as their differentiation/maturation stage. Nevertheless, precise mechanisms underlying the early steps of transmucosal infection are still unknown. Our purpose was to investigate DC/HIV-1 interactions in a subepithelial mucosal layer equivalent (SEMLE) reconstructed in vitro. We used mixed interstitial DC (IntDC)/Langerhans cell (LC)-like cell subpopulations generated in vitro from CD34(+) progenitors. These cells were either integrated in SEMLE or maintained in suspension. Experimental infections were performed with a type X4 strain (HIV-1(LAI)) and a type R5 strain (HIV-1(Ba-L)). Proviral DNA was detected by in situ polymerase chain reaction (PCR) and viral replication was quantified by measuring p24 core protein release in the culture media. Our results showed that SEMLE enable DC to retain immature stage and reproduce the tropic selection that occurs in vivo. Indeed, IntDC/LC were infected by both types of HIV-1 strains, regardless of the infection schedule, whereas only type R5 virus replicated in DC in the absence of T cell subsets. Furthermore, the ability of DC to replicate HIV-1(BaL) was lost after 14 days of culture unless the cells had previously been integrated in SEMLE. These results suggest that this 3D model maintains the ability of DC to replicate type R5 virus by delaying their maturation. In conclusion, this in vitro model mimics human submucosa and can be considered as relevant for studying the preliminary steps of transmucosal HIV-1 infection.