Identification of functional markers in a self-assembled skin substitute in vitro

Some functional parameters were identified and assessed in a tissue-engineered self-assembled skin substitute. This skin substitute was produced using fibroblasts and keratinocytes isolated from adult human skin. Keratinocytes were seeded on a dermal layer, composed of two fibroblast sheets cultured for 35 d. The epidermal cells formed a stratified and cornified epidermis and expressed differentiation markers, notably involucrin and transglutaminase. Interestingly and for the first time, the receptor for vitamin D3 was detected in all of the epidermal cell layers of the skin substitute, as it is reported for normal human skin. This observation suggests that keratinocytes retain key receptors during their differentiation in the skin model. A network of collagen fibers was observed by electron microscopy in the dermal layer of the model. In the dermis, collagen fibers remodeling and assembly is dependent on enzymes, notably prolyl-4-hydroxylase. For the first time in a skin construct, the expression of prolyl-4-hydroxylase was detected in dermal fibroblasts by in situ hybridization. The secretion of collagenases by the cells seeded in our skin substitute was confirmed by zymography. We conclude that the self-assembly approach allows the maintenance of several functional activities of human skin cells in a skin model in vitro.     

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal kerationocytes in immunodeficient mice

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic “surface” epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Human epidermis reconstructed in vitro: A model to study keratinocyte differentiation and its modulation by retinoic acid

Summary It was possible to reconstruct epidermis in vitro by seeding dissociated keratinocytes on de-epidermized dermis and growing such recombined cultures for 1 wk, exposed to air, at the surface of the culture medium. These conditions were chosen to mimic the transdermal feeding and the exposure to the atmosphere that occur in vivo. Contrary to classical cultures performed on plastic dishes covered with culture medium, which show rudimentary differentiation and organization, the architecture of the stratified epithelium obtained in reconstructed cultures and the distribution of differentiation markers such as suprabasal keratins, involucrin, and membrane-bound transglutaminase were similar to those of the epidermis of skin biopsies; moreover, biochemical studies showed that the synthesis of the various keratins and the production of cornified envelopes was similar to what is found with skin specimens. The reconstructed epidermis model was found to be very useful to study in vitro the effect of retinoic acid on keratinocyte differentiation and epidermal morphogenesis.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation

Using chimeric human epidermal reconstructs, we previously demonstrated that epidermal pigmentation is dependent upon the phototype of melanocytes. We report here several lines of experimental evidence for dermal modulation of human epidermal pigmentation. First, phototype II-III epidermal reconstructs grafted on the back of immunotolerant Swiss nu/nu mice developed a patchy pigmentation dependent on the presence of colonizing human or mouse fibroblasts. Similarly, human white Caucasoid split-thickness skin xenografted on the same mouse strain became black within 3 months and histochemistry revealed a phototype VI pattern of melanin distribution. In vitro, human fibroblasts colonizing human dead de-epidermized dermis (DDD) induced a decrease in epidermal pigmentation whereas mouse (Swiss nu/nu) fibroblasts increased epidermal pigmentation. Conditioned medium from mice (Swiss nu/nu) fibroblasts also increased pigmentation whereas conditioned medium from human fibroblasts had no significant effect. Lastly, epidermal reconstructs made with normal or vitiligo keratinocytes and/or normal or vitiligo melanocytes from the same donor grown on DDD originating from several donors of the same clinical phototype did not pigment similarly and no specific dermal influence was noted for vitiligo. Thus, fibroblast secretion and acellular dermal connective tissue itself significantly influence melanocyte proliferation and melanin distribution/degradation. Our study suggests that murine fibroblasts are more potent than human fibroblasts in secreting soluble factors which can act directly on pigmentation, such as SCF, or activate keratinocytes to produce basement membrane proteins or melanogenic factors.     

Development of a bilayered living skin construct for clinical applications

An in vitro construct of human skin (living skin equivalent, LSE) has been engineered using serially passaged human epidermal keratinocytes and human dermal fibroblasts with a matrix of type I collagen. Cells are obtained from neonatal foreskin. LSE is cast, cultured, and shipped in a single culture insert. The size and shape of theinsert determines thesize and shape of the LSE. The dermal matrix consists of dermal fibroblasts within a condensed collagen lattice. The overlying epidermis is developed at the air-liquid interface to generate a protective cornified layer. Serum was not necessaryfor development of the epidermis. LSE for graft (Graftskin) has handling characteristics similar to split-thickness skin allowing it to be meshed, stapled, and sutured. LSE was cryopreserved using 65% glycerol an rapid freezing. Viability and in vivo performance on athymic mice were similar to fresh LSE. Cells derived from human eccrine gland were able to invade and form tubules rudimentary appendages may be possible.     

Crucial role of fibroblasts in regulating epidermal morphogenesis

Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether fibroblasts can substitute growth factors needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different fibroblast numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of fibroblasts the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of fibroblasts keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of fibroblasts showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3 weeks culture at the air-liquid interface, the proliferation index decreased irrespective of the number of fibroblasts present within the dermal matrix to levels observed also in native epidermis. Keratin 10 was localized in all viable suprabasal cell layers irrespective of the absence or presence of fibroblasts. Keratin 6 was downregulated with increasing numbers of fibroblasts, and keratins 16 and 17 were absent in fibroblast-populated matrices. The expression of involucrin or transglutaminase 1 showed a similar pattern as for the keratins. Irrespective of the number of fibroblasts incorporated into DED, the expression of alpha(3), alpha(6), beta(1), and beta(4) integrin subunits was upregulated. In fibroblast-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte growth factor. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with fibroblasts.     

Glucocorticoid receptor enhances involucrin expression of keratinocyte in a ligand-independent manner

In this study, we investigated the role of glucocorticoid receptor (GR) in epidermal keratinocytes. In adult normal human skin, GR was highly expressed in the upper layers of the epidermis. Consistent with normal skin, GR expression was increased after calcium treatment of HaCaT keratinocytes cultured in vitro, suggesting that GR is involved in keratinocyte differentiation process. Overexpression of GR using an adenovirus showed that expression of involucrin, an early differentiation marker of keratinocytes, was markedly increased due to GR overexpression. However, treatment with dexamethasone, a GR agonist, did not increase involucrin expression. Overexpression of GR led to phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK) in the absence of glucocorticoid, suggesting that the GR effect on involucrin expression is related to activation of intracellular signaling cascades. This idea was supported by the fact that GR-mediated involucrin induction was abolished after treatment with JNK and ERK inhibitors. In addition, GR mutants lacking the ligand-binding domain increased involucrin expression concomitantly with increase of ERK phosphorylation. Together, these results suggest that GR modulates involucrin expression of keratinocytes by regulating the intracellular signaling network in a ligand-independent manner.     

Reconstituted skin from murine embryonic stem cells

Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.     

Reconstitution of human epidermis in vitro is accompanied by transient activation of basal keratinocyte spreading

Frozen human cadaver skin obtained from the skin bank was thawed and incubated in serum-free medium for 1–2 days, after which the original epidermis could be removed mechanically. Transmission electron microscopic observations showed that the dermal matrix remaining behind contained intact bundles of collagen fibrils but no live cells and that a continuous lamina densa persisted in the basement membrane region. Indirect immunofluorescence analyses demonstrated linear staining of the basement membrane region by antibodies against laminin and type IV collagen and discontinuous staining with antibodies against fibronectin. Scanning electron microscopic observations revealed a normal topographical arrangement of dermal matrix papilla and interspersed crypts on the surface of the matrix. Epidermal cells placed on the dermal matrix attached in 1–2 h and spread by 24 h. After 1 week of culture the epidermis was reconstituted, at which time approximately 30% of the epidermal cells were basal keratinocytes and the remainder were more differentiated keratinocytes. A high degree of differentiation of the reconstituted epidermis was shown by the formation of hemidesmosomes along the basement membrane, the formation of desmosomes characterized by intercellular dense lines, and the presence of a cell layer containing keratohyalin granules. At various times during epidermal reconstitution, cells were harvested and tested in short-term assays for adhesion to fibronectin substrata. During the first several days there was a transient activation of basal keratinocyte spreading analogous to the modulation of keratinocyte spreading that we have observed during epidermal reconstitution in vivo.     

Capability of cultivated human hair-follicle cells to integrate with skin structure in vivo

In the present work, we labeled human epidermal keratinocytes and dermal papilla cells in order to study their behavior after intradermal transplantation. The cells were transduced by lentiviral vectors that bore a marker gene that encodes green fluorescent protein (copGFP) or red fluorescent protein (DsRed). A portion of the transgene expressing cells was evaluated by flow cytometry. The proposed genetic constructions have allowed one to achieve high efficiency (>95%) of the transduction of hair follicle cells. The in vitro transduced cells were injected under epidermis of human skin fragments, after which these fragments were transplanted under the skin of immunodeficient mice. The injected epidermal keratinocytes were found mainly in hair follicles and partially in the zone of interfollicular epidermis, while dermal papilla cells were found in the papilla of the derma. The results of the present study have shown that the chosen genetic constructions obtained based on human immunodeficiency lentivirus are capable of the effective and stable transduction of human skin cells. The injected cells survived and were found in the corresponding skin structures.     

Fabrication, quality assurance, and assessment of cultured skin substitutes for treatment of skin wounds

Advances in treatment of skin wounds depend on demonstration of reduced morbidity or mortality either during or after hospitalization. Tissue engineering of skin grafts from cultured cells and biopolymers permits greater amounts of grafts from less donor tissue than conventional procedures. Autologous keratinocytes and fibroblasts isolated from epidermis and dermis of skin may be combined with collagen-based substrates to generate cultured skin substitutes (CSS) with epidermal and dermal components. By regulation of culture conditions, CSS form epidermal barrier and basement membrane, and release angiogenic factors that stimulate vascularization. Prototypes of CSS may be tested for safety and efficacy by grafting to athymic mice which do not reject human tissues. Clinical application of CSS requires establishment of quality assurance assessments, such as, epidermal barrier by measurement of surface hydration, and anatomy by standard histology. Medical benefits of tissue engineered skin for treatment of burns are evaluated quantitatively by the ratio of healed skin to donor skin, and qualitatively by the Vancouver Scar Scale. These benefits may also be extended to other medical conditions including chronic wounds and reconstructive surgery.     

Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.     

Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites

In culture, keratinocytes generally express aberrant growth and differentiation programs, which are largely normalized in cell transplants. In order to study the underlying regulatory phenomena and to distinguish between intrinsic properties and external factors, different in vitro and in vivo models have been applied using human keratinocytes from foreskin and trunk skin. When transplanted onto nude mice, keratinocytes reformed a regular epithelium with expression of the differentiation markers, keratins K1 and K10, involucrin and filaggrin. Tissue homeostasis improved in later transplants, as made apparent by coexpression and regular distribution of K1 and K10. Since this was achieved in transplants, whether in contact with mesenchyme or separated by collagen matrix, renormalization was obviously mediated by diffusible factors. In vitro, the host-mesenchymal influence could largely be mimicked by recombining organotypic cultures (keratinocytes on lifted collagen gels) with de-epidermized dermis, but tissue homeostasis was apparently not achieved. Comparing keratinocytes from trunk skin and foreskin, differences observed in situ persisted in isolated cells and reconstituted tissues. The hyperproliferative character of foreskin epidermis, with its less-pronounced stratum granulosum, was maintained in recombinant cultures and transplants along with the expression of keratin K13 (typical for foreskin in situ) irrespective of the type of mesenchyme. Thus, we could demonstrate with these model systems that: (a) the regulation of keratinocyte growth and differentiation is mesenchyme-dependent; (b) it is mediated by diffusible factors; but that (c) differences between epidermis of different body sites are also controlled by intrinsic programs.     

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic surface epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.