Two‐photon microscopy of dermal innervation in a human re‐innervated model of skin

When skin is injured, innervation can be severely disrupted. The subsequent re‐innervation processes are poorly understood notably because of the inability to image the full meandering course of nerves with their ramifications and endings from histological slices. In this letter, we report on two‐photon excitation fluorescence (TPEF) microscopy of entire human skin explants re‐innervated by rodent sensory neurons labelled with the styryl dye FM1‐43. TPEF imaging of nerve fibres to a depth up to roughly 300 μm within the dermis was demonstrated, allowing three‐dimensional reconstruction of the neural tree structure. Endogenous second‐harmonic imaging of type I fibrillar collagen was performed in parallel to TPEF imaging using the same nonlinear microscope, revealing the path of the nerves through the dermis.     

Effects of the re-innervation of organotypic skin explants on the epidermis

The nervous system takes part in skin homeostasis and interacts with skin cells. In in vitro organotypic skin models, these interactions are lost owing to the absence of nerve endings. We have developed an in vitro organotypic skin model based on a re-innervated human skin explant using primary sensory neurons from the dorsal root ganglia of rats. After 10 days of co-culture between skin explant and neurons, a dense network of nerve fibres was observed. The epidermis and dermis presented nerve fibres associated with cellular body from sensory neurons introduced in the co-culture. Epidermal thickness, cell density and quality of re-innervated skin explant were all higher when skin explants were re-innervated by sensory neurons at 10 days of culture. Proliferation of epidermal cell was not modified, but the apoptosis was significantly diminished. Hence, this innovative model of co-cultured skin explants and neurons allows better epidermal integrity and could be useful for studies concerning interactions between the skin and its peripheral nervous system.     

Human umbilical cord blood cells form epidermis in the skin equivalent model

Please cite this paper as: Human umbilical cord blood cells form epidermis in the skin equivalent model. Experimental Dermatology 2010. Abstract: Recently, human embryonic stem cells have been differentiated in vitro into functional epidermal keratinocytes. Here, we demonstrated that these cells can be generated also from non‐embryonic, human umbilical cord blood (hUCB) cells that have the potential to differentiate into cells of non‐hematopoietic lineage. Human UCB mono‐nucleated cells were cultivated in monolayer and in three‐dimensional skin equivalent cultures and assayed for the presence of phenotype‐specific markers. Our results determined that after one month of culturing in serum containing medium, the hUCB cells produced morphologically homogeneous colonies of epithelial cells expressing keratinocyte‐specific markers. They also formed stratified epidermis in organ cultures that contained sporadic CD1a‐positive cells within the accurate strata. We concluded that hUCB cells have the capacity to differentiate into functional epidermal keratinocytes and may serve as a source of high‐quality keratinocytes for clinical applications.     

Effects of retinoic acid on adult human epidermis in whole skin organ culture

Summary Adult human skin was cultured in wholeskin organ culture under chemically defined conditions. Retinoic acid was added to the culture at final concentrations of 5×10^-7 and 5×10^-6 M. Both concentrations elicited cell death in the upper epidermal layers and prevented the terminal differentiation of the cells to mature corneocytes. The inhibition of terminal differentiation was not permanent, as the corneocytes produced later during the culture showed no signs of inhibition. The upper vital cells in epidermis cultured with retinoic acid were very flattened and contained reduced amounts of cytoskeleton components. Fine, granular material not present in normal epidermis was oberved in both the intercellular spaces and the intracytoplasmic vesicles of retinoid-treated epidermis. The present results indicate that the response of normal human skin to retinoic-acid treatment involves the same kind of modulation of the epidermal structure previously described in embryonic avian and diseased human skin.     

Mannosylerythritol lipids inhibit melanogenesis via suppressing ERK‐CREB‐MiTF‐tyrosinase signalling in normal human melanocytes and a three‐dimensional human skin equivalent

Hyperpigmentation is caused by excessive production of melanin in melanocytes. Mannosylerythritol lipids (MELs) are glycolipid biosurfactants that are abundantly produced by yeasts and used commercially in cosmetics. However, the potential depigmenting efficacy of MELs has not been evaluated. In this study, the depigmentary effect of MELs was tested in primary normal human melanocytes (NHMs), α‐melanocyte‐stimulating hormone (MSH)‐stimulated B16 cells (murine melanoma cells) and a human skin equivalent (MelanoDerm) using photography, Fontana‐Masson (F&M) staining and two‐photon microscopy. Mannosylerythritol lipids significantly decreased the melanin contents in NHMs and α‐MSH‐stimulated B16 cells. Consistent with these findings, MELs treatment had a clear whitening effect in a human skin equivalent, brightening the tissue colour and reducing the melanin content. The molecular mechanism underlying the anti‐melanogenic effect of MELs treatment was examined by real‐time PCR and Western blotting. Mechanistically, MELs clearly suppressed the gene expression levels of representative melanogenic enzymes, including tyrosinase, Tyrp‐1 and Tyrp‐2, by inhibiting the ERK/CREB/MiTF signalling pathway in NHMs. This work demonstrates for the first time that MELs exert whitening effects on human melanocytes and skin equivalent. Thus, we suggest that MELs could be developed as a potent anti‐melanogenic agent for effective whitening, beyond their use as a biosurfactant in cosmetics.     

Effect of human skin explants on the neurite growth of the PC12 cell line

The skin is a densely innervated organ. After a traumatic injury, such as an amputation, burn or skin graft, nerve growth and the recovery of sensitivity take a long time and are often incomplete. The roles played by growth factors and the process of neuronal growth are crucial. We developed an in vitro model of human skin explants co‐cultured with a rat pheochromocytoma cell line differentiated in neuron in presence of nerve growth factor (NGF). This model allowed the study of the influence of skin explants on nerve cells and nerve fibre growth, probably through mediators produced by the explant, in a simplified manner. The neurite length of differentiated PC12 cells co‐cultured with skin explants increased after 6 days. These observations demonstrated the influence of trophic factors produced by skin explants on PC12 cells.     

Eosinophil localization to the basement membrane zone is autoantibody‐ and complement‐dependent in a human cryosection model of bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by antibodies (IgG and IgE) targeting cell‐substrate adhesion proteins. A variety of BP models suggest that autoantibody‐dependent neutrophil degranulation is essential for blister formation. However, lesional biopsies reveal a predominance of eosinophils and few neutrophils. Our goal was to evaluate the role of antibodies and complement in eosinophil localization, degranulation and split formation at the dermo‐epidermal junction (DEJ) utilizing a human skin cryosection model of BP paired with a human eosinophilic cell line, 15HL‐60. Expression of receptors for IgG (FcγRII), IgE (FcεRI) and complement (CR1 and CR3) was confirmed on 15HL‐60 cells using flow cytometry. 15HL‐60 expression of granule protein [eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO)] mRNA and their degranulation in vitro was confirmed using RT‐PCR and ELISA, respectively. For cryosection experiments, BP or control sera or IgG and IgE antibodies purified from BP sera were utilized in combination with 15HL‐60 cells ± fresh complement. Both BP serum and fresh complement were required for localization of 15‐HL60 cells to the DEJ. Interestingly, eosinophil localization to the DEJ was dependent on IgG, but not IgE, and complement. However, no subepidermal split was observed. Additionally, the 15HL‐60 cells did not degranulate under any experimental conditions and direct application of cell lysate to cryosections did not result in a split. Our observation that eosinophil localization to the DEJ is dependent on IgG mediated complement fixation provides additional insight into the sequence of events during the development of BP lesions.     

Epithelial deletion of podoplanin is dispensable for re‐epithelialization of skin wounds

The mucin‐like transmembrane protein podoplanin (PDPN) is prominently represented in tumor‐associated gene expression signatures of numerous types of cancer including squamous cell carcinoma, and gain‐of‐function and knockdown approaches in tissue culture strongly suggested an important role of PDPN in cell proliferation, migration and adhesion. PDPN is absent during epidermal homeostasis but is highly expressed in basal keratinocytes during cutaneous wound healing. Enhanced motility of immortalized keratinocytes upon ectopic PDPN overexpression argues for wound healing defects upon podoplanin deficiency in keratinocytes; however, in vivo data that unequivocally define the impact of PDPN by functional studies in a physiologically relevant system are still missing. Here, we have applied an in vivo loss‐of‐function approach by generating a novel transgenic mouse line with keratinocyte‐specific podoplanin deficiency. Performing cutaneous full‐thickness excisional wounds to examine re‐epithelialization capacity, unexpectedly, no defects were observed in wound healing properties of mutant mice. Similarly, PDPN‐deficient primary keratinocytes showed no impairment in migration, adhesion or proliferation. Thus, PDPN function is not rate‐limiting for re‐epithelialization but may be functionally compensated by an as yet unknown protein. Our data also call for in vivo functional studies on PDPN in settings of skin tumor development and progression to clarify PDPN's role in skin pathology.     

Effects of a ceramide‐containing water‐in‐oil ointment on skin barrier function and allergen penetration in an IL‐31 treated 3D model of the disrupted skin barrier

Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL‐31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL‐31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide‐containing water‐in‐oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide‐containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and β4‐integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide‐containing skin care ointment reduced IL‐31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.     

Characterization of neurons from adult human skin-derived precursors in serum-free medium : a PCR array and immunocytological analysis

Abstract: Adult stem cells could be small sources of neurons or other cellular types for regenerative medicine and tissue engineering. Recently, pluripotent stem cells have been extracted from skin tissue, which opened a new accessible source for research. To routinely obtain a high yield of functional neurons from adult human skin stem cells with defined serum‐free medium, stem cells from abdominal skin were cultured in serum‐free medium. To differentiate them, we used a defined medium containing growth factors. Differentiated cells were identified using the following methods: (i) Oil‐red‐O staining for adipocytes, immunocytochemistry with antibodies recognising (ii) neurofilaments and PGP9.5 for neural differentiation, (iii) glial fibrillary acidic protein (GFAP) for glial differentiation, (iv) Ki‐67 for proliferative cells, (v) FM1‐43 staining to analyse vesicle trafficking in neuronal cells and (vi) a PCR array was used. Stem cells were floating in spheres and were maintained in culture for 4 months or more. They expressed nestin and Oct 4 and were proliferative. We induced specific differentiation into adipocytes, glial and neuronal cells. The yields of differentiated neurons were high and reproducible. They were maintained for long time (1 month) in the culture medium. Furthermore, these neurons incorporated FM1‐43 dye, which indicates a potent acquisition of synaptic features in neurons. Stem cells from adult human skin could be valuable and reproducible tools/source to obtain high numbers of functional specific cellular types, such as neurons, for tissue engineering. In this work, the possibility to obtain a high yield of differentiated neurons, with the ability of endocytosis and vesicle cell trafficking, was shown.     

HA modulation of epidermal morphogenesis in an organotypic keratinocyte‐fibroblast co‐culture model

Please cite this paper as: HA modulation of epidermal morphogenesis in an organotypic keratinocyte‐fibroblast co‐culture model. Experimental Dermatology 2010; 19 : e336–e339. Abstract: Hyaluronan (HA) is a major extracellular matrix component of the skin. Amongst its biological functions is the maintenance of epidermal homeostasis. The mechanisms of action, however, remain unclear. To explore the interaction of HA with the epidermis, we have looked at the effects of exogenous application of HA in an organotypic culture model containing a dermal substrate with and without fibroblast incorporation. The results demonstrate that exogenous HA enhances epidermal proliferation resulting in a thicker viable epidermis with an increase in the number and intensity of Ki67‐positive basal cells; HA also improves the basement membrane assembly as evidenced by an increased expression of laminin‐332 and collagen type IV but not the expression of Nidogen‐1 at the epidermal–dermal junctional zone; furthermore, the development of epidermal lipid barrier structure was enhanced. These findings provide evidence to support the therapeutic use of exogenous HA for treating skin disorders with aberrant epidermal homeostasis.     

Comparison of surface free energy between reconstructed human epidermis and in situ human skin

Background/aims: The objectives of this study were to characterise the Episkin^® model of reconstructed epidermis histologically and energetic, and to compare the results with the data obtained by Mavon (9, 10) on different areas of skin on the human body and on different skin types. Methods: Histologically, the two skin systems studied were stained using Goldner's trichome method. Surface free energy was determined by the measurement of contact angles, using the posed drop method. Results: Histologically, the two skin systems demonstrated the same cellular organisation in stratified layers. With regard to surface free energy, Episkin, skin from the forehead, normal and hyper‐seborrheic skins are hydrophilic surfaces, while skin from the forearm or of an oligo‐seborrheic type tends to be hydrophobic. Episkin and skin from the forehead exhibit similar surface free energy profiles. It is impossible to state whether Episkin has characteristics closer to those of normal or hyper‐seborrheic skin, but they differ significantly from those of oligo‐seborrheic skin. Conclusion: The results obtained during this study justify the use of Episkin in a range of tests where the use of in situ human skin raises problems.     

A diffuse reflectance spectroscopic study of UV‐induced erythematous reaction across well‐defined borders in human skin

Introduction: The colour of tissue is often of clinical use in the diagnosis of tissue homeostasis and physiological responses to various stimuli. Determining tissue colour changes and borders, however, often poses an intricate problem and visual examination, constituting clinical praxis, does not allow them to be objectively characterized or quantified. Demands for increased inter‐ and intra‐observer reproducibility have been incentives for the introduction of objective methods and techniques for tissue colour (e.g. erythema) evaluation. The aim of the present paper was to study the border zone of a UVB‐provoked erythematous response of human skin in terms of blood volume and oxygenation measured by means of diffuse reflectance spectroscopy using a commercial probe. Material and methods: A provocation model, based on partial masking of irradiated skin areas, defines two erythema edges at every skin site responding to the UV irradiation. In every subject, five test sites were exposed with a constant UV light irradiance (14 mW/cm²), but with different exposure times (0, 3, 6, 9 and 12 s). An analysis of the spectral data measured across the two edges was performed for every scan line. The oxygenized and deoxygenized haemoglobin contents were estimated in every measurement point, using a modified Beer–Lambert model. Results: The fit of the experimental data to the model derived by the modified Beer–Lambert law was excellent (R²>0.95). Analysing data for the chromophore content showed that the erythematous response in the provoked areas is dominated by the increase in oxyhaemoglobin. The widths for the left and right border zone were estimated to be 1.81±0.93 and 1.90±0.88 mm, respectively (mean±SD). The unprovoked area between the two edges was estimated to be 0.77±0.68 mm. Conclusion: While the chosen data analysis performed satisfactorily, the ability of the probe design to differentiate the spatial aspects of a reaction with abrupt borders was found to be suboptimal resulting in a probable overestimation of the erythematous edge slope. Probe modification or imaging techniques are possible solutions.