Multilayered Epidermis Research Articles

Transdifferentiation of Umbilical Cord-Derived Mesenchymal Stem Cells Into Epidermal-Like Cells by the Mimicking Skin Microenvironment.

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are multipotent, primitive, and have been widely used for skin tissue engineering. Their transdifferentiation is determined by the local microenvironment. In this study, we investigated the potential epidermal differentiation of UC-MSCs and the formation of epidermis substitutes in a 3-dimensional (3D) microenvironment, which was fabricated by UC-MSCs embedded into collagen-chitosan scaffolds (CCSs) combined with an air-liquid interface (ALI) culture system. Using fluorescence microscope, we observed that UC-MSCs were spindle-shaped and evenly distributed in the scaffold. Methyl thiazolyl blue tetrazolium bromide assay and Live/Dead assay indicated that the CCSs have good biocompatibility with UC-MSCs. Immunohistochemistry and western blotting assay showed that UC-MSCs on the surface of the CCSs were positive for the epidermal markers cytokeratin 19 and involucrin at 14 days. In addition, hematoxylin-eosin staining indicated that multilayered epidermis substitutes were established. The constructed epidermis substitutes were applied to treat full-thickness wounds in rats and proved to promote wound healing. In conclusion, manipulating the 3D microenvironment is a novel method for inducing the epidermal differentiation of MSCs to engineer epidermal substitutes, which provides an alternative strategy for skin tissue engineering.

表皮の多層構造を考慮した両親媒性物質会合体による化粧品製剤の浸透設計

化粧品製剤の重要な役割の一つに，有用素材を皮膚内へ運ぶことが挙げられる。皮膚には，外界からの異物の侵入を防いだり体内からの過剰な水分蒸散を防いだりする角層という強固なバリアが備わっているため，これまでの製剤技術開発者は，皮膚上に塗布した有用素材を角層より下の細胞に届けることに注力し，成果を上げてきた。したがって，開発された製剤技術の多くが角層への浸透性向上に対するアプローチであり，その下の表皮生細胞層での拡散浸透性はほとんど検討されてこなかった。われわれは角層とその下の表皮生細胞層で水分量が大きく異なることに着目し，両者に対する製剤の浸透性は異なるのではないかと仮説をたて，各々の層に分けて両親媒性物質会合体の浸透性を評価した。その結果，仮説どおり同一の製剤でも角層とその下の表皮生細胞層とでは浸透性が異なることが明らかとなり，角層と比較してこれまであまり着目されなかった表皮生細胞層に対する浸透性も，角層と同様に表皮全層の浸透性に大きく寄与していることが示唆された。すなわち，有用素材を目的の表皮細胞まで効率的に運ぶには，角層に加えて表皮生細胞層に対する浸透性も考慮することが重要であると考えた。

Development and characterization of an equine skin-equivalent model.

There is increasing interest in the biological and pathological study of equine skin owing to the high prevalence of cutaneous diseases in horses. However, knowledge of equine skin cell biology and cultures is limited by the low number of in vitro studies in the literature. The objective of the study was to develop and characterize an in vitro equine skin equivalent. Cultures of pure equine keratinocytes and dermal fibroblasts were obtained by enzymatic digestion of skin biopsies. Fibroblasts were embedded into type I collagen matrices to obtain dermal scaffolds, the surface of which was seeded with keratinocytes. The three-dimensional cultures were exposed to the air-liquid interface to enable epidermal stratification. After 14 days in air-exposed conditions, histological analysis showed that keratinocytes underwent differentiation into a multilayered epidermis. Immunohistochemical studies revealed the expression of epidermal cytokeratin in keratinocytes, whereas vimentin was expressed in dermal fibroblasts, as expected in equine skin. Immunostaining of Ki67 showed proliferative keratinocytes in the stratum basale. A continuous basement membrane at the dermo-epidermal junction was also detected immunohistochemically through the expression of its major components (type IV collagen and laminin 5). Ultrastructural analysis by electron microscopy showed desmosomes located among keratinocytes in all layers and hemidesmosomes among the basal keratinocytes and lamina densa. This study reports, for the first time, the development of an in vitro equine skin-equivalent model that resembles equine skin morphologically, immunohistochemically and ultrastructurally.

Insulin/IGF-1 Controls Epidermal Morphogenesis via Regulation of FoxO-Mediated p63 Inhibition

SummaryThe multilayered epidermis is established through a stratification program, which is accompanied by a shift from symmetric toward asymmetric divisions (ACD), a process under tight control of the transcription factor p63. However, the physiological signals regulating p63 activity in epidermal morphogenesis remain ill defined. Here, we reveal a role for insulin/IGF-1 signaling (IIS) in the regulation of p63 activity. Loss of epidermal IIS leads to a biased loss of ACD, resulting in impaired stratification. Upon loss of IIS, FoxO transcription factors are retained in the nucleus, where they bind and inhibit p63-regulated transcription. This is reversed by small interfering RNA-mediated knockdown of FoxOs. Accordingly, transgenic expression of a constitutive nuclear FoxO variant in the epidermis abrogates ACD and inhibits p63-regulated transcription and stratification. Collectively, the present study reveals a critical role for IIS-dependent control of p63 activity in coordination of ACD and stratification during epithelial morphogenesis.

Skin wound healing in different aged Xenopus laevis

Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8- and 15-month-old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re-epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti-inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti-tumor necrosis factor (TNF)-α, iNOS, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti-α-SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar-like pattern. The quantitative PCR analysis demonstrated an up-regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar-like tissue formation.

Tissue Engineered Skin Substitutes Created by Laser-Assisted Bioprinting Form Skin-Like Structures in the Dorsal Skin Fold Chamber in Mice

Tissue engineering plays an important role in the production of skin equivalents for the therapy of chronic and especially burn wounds. Actually, there exists no (cellularized) skin equivalent which might be able to satisfactorily mimic native skin. Here, we utilized a laser-assisted bioprinting (LaBP) technique to create a fully cellularized skin substitute. The unique feature of LaBP is the possibility to position different cell types in an exact three-dimensional (3D) spatial pattern. For the creation of the skin substitutes, we positioned fibroblasts and keratinocytes on top of a stabilizing matrix (Matriderm®). These skin constructs were subsequently tested in vivo, employing the dorsal skin fold chamber in nude mice. The transplants were placed into full-thickness skin wounds and were fully connected to the surrounding tissue when explanted after 11 days. The printed keratinocytes formed a multi-layered epidermis with beginning differentiation and stratum corneum. Proliferation of the keratinocytes was mainly detected in the suprabasal layers. In vitro controls, which were cultivated at the air-liquid-interface, also exhibited proliferative cells, but they were rather located in the whole epidermis. E-cadherin as a hint for adherens junctions and therefore tissue formation could be found in the epidermis in vivo as well as in vitro. In both conditions, the printed fibroblasts partly stayed on top of the underlying Matriderm® where they produced collagen, while part of them migrated into the Matriderm®. In the mice, some blood vessels could be found to grow from the wound bed and the wound edges in direction of the printed cells. In conclusion, we could show the successful 3D printing of a cell construct via LaBP and the subsequent tissue formation in vivo. These findings represent the prerequisite for the creation of a complex tissue like skin, consisting of different cell types in an intricate 3D pattern.

Human Eccrine Sweat Gland Cells Turn into Melanin-Uptaking Keratinocytes in Dermo-Epidermal Skin Substitutes

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

Cystatin M/E knockdown by lentiviral delivery of shRNA impairs epidermal morphogenesis of human skin equivalents

The protease inhibitor cystatin M/E (CST6) regulates a biochemical pathway involved in stratum corneum homeostasis, and its deficiency in mice causes ichthyosis and neonatal lethality. Cystatin M/E deficiency has not been described in humans so far, and we did not detect disease-causing mutations in the CST6 gene in a large number of patients with autosomal recessive congenital ichthyosis, who were negative for mutations in known ichthyosis-associated genes. To investigate the phenotype of CST6 deficiency in human epidermis, we used lentiviral delivery of short hairpin RNAs that target CST6 in a 3D reconstructed skin model. Surprisingly, CST6 deficiency did not cause an ichthyosis-like phenotype, but prevented the development of a multilayered epidermis. From this study, we conclude that CST6 deficiency may be incompatible with normal human foetal development.

Adipose-Derived Stromal Cells Accelerate Wound Healing in an Organotypic Raft Culture Model

Adipose tissue is a known reservoir of multipotent mesenchymal stem cells, which can be manipulated in culture to produce cells with different phenotypes. The goal of this study was to determine whether the addition of these multipotential cells to organotypic, human skin equivalent cultures would accelerate wound healing after laser injury. For our initial studies, we were able to obtain 3-dimensional raft cultures from adult skin explanted directly onto the dermal equivalent containing human fibroblasts with or without adipose-derived stromal cells (ADSCs). Two days after laser injury, the raft cultures of skin explants that contained ADSCs had a completely healed multilayered epidermis, whereas the control raft culture without the adipose-derived cells still had areas of injury. With this encouraging outcome, these experiments were then repeated in a raft culture system initiated from dissociated primary adult human keratinocytes on the humanized dermal equivalent. Again, the cultures containing ADSCs healed faster than the control cultures. In conclusion, these data provide support to our hypothesis that ADSCs are an excellent and readily available source of factors necessary for accelerated wound healing and tissue regeneration.

In vitro development and characterization of canine epidermis on a porcine acellular dermal matrix

The aim of this study was to develop and to characterize a canine skin epidermal model able to form a proper epidermis on a porcine acellular dermal matrix (PADM). In addition, the role of fibroblasts in skin barrier formation was studied by incorporating or omitting canine dermal fibroblasts in the PADM. Canine epidermal composites were developed by seeding keratinocytes onto the surface of PADM that were previously seeded or non-seeded with dermal fibroblasts. After 14 days of culture under air-exposed conditions and in a special growth medium, skin composites were histologically processed and immunohistochemically characterized to determine the expression of cytokeratins and of vimentin and the presence of basement membrane. In all composites, keratinocytes underwent differentiation to a multilayer epidermis with 5-7 viable cell layers. The stratum basalis, stratum spinosum, stratum granulosum and stratum corneum were identified. The expression of cytokeratins was similar to that described in healthy canine epidermis. Laminin and collagen IV immunostaining revealed a homogeneous layer in the epidermal-dermal junction only when the matrix had been seeded by canine dermal fibroblasts. The model may become a simple, useful and cost-effective tool to investigate the biology and pathology of canine epidermis and could partially replace animal testing in several areas of dermatological research.

Keratinocyte Stem Cells and the Targets for Nonmelanoma Skin Cancer†

The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.

NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a “stimulatory” environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Zebrafish: A Model System to Study Heritable Skin Diseases

Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in ∼500 different genes. A number of model systems have been developed to advance our understanding of the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5-6 days post-fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphological and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provide a useful model system to study the molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases.

Poky/chuk/ikk1 is required for differentiation of the zebrafish embryonic epidermis

An epidermis surrounds all vertebrates, forming a water barrier between the external environment and the internal space of the organism. In the zebrafish, the embryonic epidermis consists of an outer enveloping layer (EVL) and an inner basal layer that have distinct embryonic origins. Differentiation of the EVL requires the maternal effect gene poky/ikk1 in EVL cells prior to establishment of the basal layer. This requirement is transient and maternal Ikk1 is sufficient to allow establishment of the EVL and formation of normal skin in adults. Similar to the requirement for Ikk1 in mouse epidermis, EVL cells in poky mutants fail to exit the cell cycle or express specific markers of differentiation. In spite of the similarity in phenotype, the molecular requirement for Ikk1 is different between mouse and zebrafish. Unlike the mouse, EVL differentiation requires functioning Poky/Ikk1 kinase activity but does not require the HLH domain. Previous work suggested that the EVL was a transient embryonic structure, and that maturation of the epidermis required replacement of the EVL with cells from the basal layer. We show here that the EVL is not lost during embryogenesis but persists to larval stages. Our results show that while the requirement for poky/ikk1 is conserved, the differences in molecular activity indicate that diversification of an epithelial differentiation program has allowed at least two developmental modes of establishing a multilayered epidermis in vertebrates.

The keratinocyte in epidermal renewal and defence

Traditionally, keratinocytes have been considered inert constituents of the multilayered epidermis. Today's understanding has fundamentally changed. The keratinocyte is now recognized as an active player in epidermal renewal with key functions in the skin's immune defence. Under homeostatic conditions, keratinocyte progenitor cells are believed to divide symmetrically or asymmetrically, that is they continue to proliferate or go on to terminally differentiate and build up the overlaying epidermis. The fine-tuned process of epidermal renewal relies on an extraordinary network of signalling cascades which are governed by keratinocyte-receptor interactions with the environment through paracrine and autocrine circuits. Opposing this coordinated homeostatic process are signals of wounding and inflammation. They alter the fate of the keratinocyte and its response to the environment through changes in adhesion molecules and surface receptors, in addition to triggering an immediate inflammatory keratinocyte response in terms of secretion of cytokines, chemokines and antimicrobial peptides. If uncontrolled, the fundamental changes imposed by wounding and inflammation upon the homeostatic programme can lead to severe skin lesions including chronic inflammatory disorders. This review will describe the current knowledge of the regulatory signalling network which allows the keratinocyte to actively impact both epidermal homeostasis and the inflammatory response.

Imaging the electric field associated with mouse and human skin wounds

We have developed a noninvasive instrument called the bioelectric field imager (BFI) for mapping the electric field between the epidermis and the stratum corneum near wounds in both mouse and human skin. Rather than touching the skin, the BFI vibrates a small metal probe with a displacement of 180 mum in air above the skin to detect the surface potential of the epidermis through capacitative coupling. Here we describe our first application of the BFI measuring the electric field between the stratum corneum and epidermis at the margin of skin wounds in mice. We measured an electric field of 177+/-14 (61) mV/mm immediately upon wounding and the field lines pointed away from the wound in all directions around it. Because the wound current flows immediately upon wounding, this is the first signal indicating skin damage. This electric field is generated at the outer surface of the epidermis by the outward flow of the current of injury. An equal and opposite current must flow within the multilayered epidermis to generate an intraepidermal field with the negative pole at the wound site. Because the current flowing within the multilayered epidermis is spread over a larger area, the current density and subsequent E field generated in that region is expected to be smaller than that measured by the BFI beneath the stratum corneum. The field beneath the stratum corneum typically remained in the 150-200 mV/mm range for 3 days and then began to decline over the next few days, falling to zero once wound healing was complete. The mean wound field strength decreased by 64+/-7% following the application of the sodium channel blocker, amiloride, to the skin near the wound and increased by 82+/-21% following the application of the Cl- channel activator, prostaglandin E2.

Cellules souches embryonnaires et peau: du modèle cellulaire au potentiel thérapeutique

Embryonic stem (ES) cells are pluripotent cells able to differentiate into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported their efficient ability to recapitulate ectodermal and epidermal fates and form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. Thus, ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, characteristic of embryonic skin formation. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1+ neural precursors to undergo specific apoptosis while inducing epidermal differentiation. We further demonstrated that p63 stimulates ectodermal cell proliferation and is necessary for epidermal commitment. We provided further evidence that this unique cellular model provides a powerful tool to identify the molecular mechanisms controlling normal skin development and to investigate human ectodermal dysplasia congenital pathologies linked to p63 (in p63-ectodermal dysplasia human congenital pathologies). Epidermal stem cell activity has been used for years to repair skin injuries, but ex vivo keratinocyte amplification has limitations and grafted skin homeostasis is not totally satisfactory. Human ES cells raise hopes that the understanding of developmental steps leading to the generation of epidermal stem cells will once be translated into therapeutic benefit. We recently demonstrated that human embryonic stem cells can give rise to a stable somatic ectodermal cell population. Its finite population doubling, normal cell cycle kinetics and the absence of teratoma formation strongly suggest that, although derived from human embryonic stem cells, these ectodermal cells represent a clinically safe somatic cell population. They could thus be particularly useful as a source for committed, homogeneous, non-tumorigenic cell populations to be employed in clinical trials for epithelial stem cell loss.

Transcriptional Control of the Differentiation Program of Interfollicular Epidermal Keratinocytes

Mammalian epidermis is a stratified squamous epithelium that serves as a protective barrier against external harmful elements. The development of the epidermis is a highly regulated process that begins by the commitment of a single layer of multipotent ectodermal cells to a keratinocyte cell fate. This is followed by stratification and a subsequent elaborate program of differentiation leading to the generation of a multilayered epidermis and patterned cutaneous appendages such as the hair follicles and sebaceous glands. The stratified epidermis occupying the space between skin appendages is referred to as the interfollicular epidermis (IFE) and is the focus of this review. Within the IFE, keratinocytes in the innermost basal layer are mitotically active. Upon specific cues, these cells undergo cell cycle arrest and execute a terminal differentiation program as they progress through spinous, granular, and cornified layers. This program operates continually throughout the life of an organism; dead cells sloughed off from the skin surface are replenished by basal cells moving outward in a highly synchronized fashion. Not surprisingly, at the heart of the control process is a dedicated group of transcription factors that ensure the integrity of the keratinocyte differentiation program by regulating gene expression in a temporally and spatially coordinated manner. Here we review the transcription factors that play important roles in the development and maintenance of IFE as evidenced by biochemical and genetic studies.

Embryonic stem cells as a cellular model for neuroectodermal commitment and skin formation

Embryonic stem (ES) cells can be differentiated into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells. These differentiated ES cells were able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1 + neural precursors to undergo specific apoptosis while inducing epidermal differentiation through ΔNp63 gene activation. We further demonstrated that ΔNp63 enhances ES-derived ectodermal cell proliferation and is necessary for epidermal commitment. This unique cellular model further provides a powerful tool for identifying the molecular mechanisms controlling normal skin development and for investigating p63-ectodermal dysplasia human congenital pathologies. To cite this article: D. Aberdam et al., C. R. Biologies 330 (2007).

Development and characterization of a canine skin equivalent

The development of a complex cellular model, which incorporates the basic cell components of the dog skin, would be a useful tool to investigate the biology and pathology of canine skin and also to replace animal testing partially. The aim of the present study was to develop and characterize a canine skin equivalent. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies from healthy dogs. Fibroblasts were embedded into a bio-matrix from collagen type I matrix protein; this built the scaffold where the keratinocytes were seeded, at air exposed conditions. At 3, 7, 15 and 21 days of culture in special growth media, skin equivalents were analysed by histological, immunohistochemical and electron microscopical techniques. At 15 days, keratinocytes underwent differentiation to a multilayer epidermis with stratum basal, stratum spinosum, stratum granulosum and stratum corneum. Expression of epidermal cytokeratins in keratinocytes was detected by immunhistochemistry, and followed the same pattern than in the normal canine epidermis. Fibroblasts from the skin equivalent expressed vimentin as dermal fibroblasts do. A basement membrane (BM) was observed underneath the epidermis; ultrastructurally, it was similar to the normal canine BM and collagen IV and laminin 5 were detected immunohistochemically as major components of this structure. Skin equivalents developed from canine cutaneous cells presented a similar morphological structure than healthy canine skin. Moreover, the immunohistochemical analysis revealed the expression of the major markers of the epidermis (keratins), dermis (vimentin) and BM (collagen type IV, laminin 5).