Basement membrane formation during wound healing is dependent on epidermal transplants

The purpose of the study was to compare directly the effect of healing and the formation of the basement membrane during wound healing from two autologous primary keratinocyte cultures in the liquid environment in full-thickness wounds in pigs. Wounds were either transplanted with cultured epidermal autografts (n = 26) or autologous keratinocyte suspensions (n = 24) or treated with saline alone (n = 40) and covered with a chamber. All wounds transplanted with cultured epidermal autografts and keratinocyte cell suspensions had positive "take" after transplantation. Healing times were significantly shorter for wounds treated with either cultured epidermal autografts or keratinocyte suspensions (p = 0.0001) compared with saline-treated wounds but were not different from each other (p = 0.1835). There were no differences in cytokeratin and laminin expression; however, staining with monoclonal antibody against collagen type VII showed a lower signal for cultured epidermal autografts only on days 8 and 16 compared with keratinocyte suspensions. Electron microscope evaluation showed a higher incidence of anchoring fibrils and a more mature dermal-epidermal junction in wounds treated with keratinocyte cell suspensions at day 8. These findings may be due to the single, noncontact-inhibited cells and the early formation of an in vivo neodermis to the wet wound environment. These data suggest that wounds transplanted with autologous keratinocyte suspensions in a wet environment may be an alternative method in the treatment of wounds.     

Mouse epidermal cell cultures : II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin

In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10⁶ viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%.The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.     

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.     

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.     

Proliferation of human embryonic and fetal epidermal cells in organ culture

The morphology of human embryonic and fetal skin growth in organ culture at the air-medium interface was examined, and the labeling indices of the epidermal cells in such cultures were determined. The two-layered epidermis of embryonic specimens increased to five or six cell layers after 21 days in culture, and the periderm in such cultures changed from a flat cell type to one with many blebs. The organelles in the epidermal cells remained unchanged. Fetal epidermis, however, differentiated when grown in this organ culture system from three layers (basal, intermediate, and periderm) to an adult-type epidermis with basal, spinous, granular, and cornified cell layers. Keratohyalin granules, lamellar granules, and bundles of keratin filaments, organelles associated with epidermal cell differentiation, were observed in the suprabasal cells of such cultures. The periderm in these fetal cultures formed blebs early but was sloughed with the stratum corneum in older cultures. The rate of differentiation of the fetal epidermis in organ culture was related to the initial age of the specimen cultured, with the older specimens differentiating at a faster rate than the younger specimens. Labeling indices (LIs) of embryonic and fetal epidermis and periderm were determined. The LI for embryonic basal cells was 8.5% and for periderm was 8%. The fetal LIs were 7% for basal cells, 1% for intermediate cells, and 3% for periderm. The ability to maintain viable pieces of skin in organ culture affords a model for studying normal and abnormal human epidermal differentiation from fetal biopsies and for investigating proliferative diseases.     

Effects of ultraviolet radiation on human epidermal cell alloantigen presentation: initial depression of Langerhans cell-dependent function is followed by the appearance of T6- Dr+ cells that enhance epidermal alloantigen presentation

The effects of ultraviolet radiation (UV) on the immune parameters of human epidermis were studied. We determined the effects of both in vitro and in vivo UV on human epidermal cell surface markers and on epidermal immune function in the allogeneic epidermal cell-lymphocyte reaction (ELR). Epidermal cells obtained immediately after in vitro and in vivo UV exposure exhibited a dose-dependent decrease in alloantigen-presenting function in the ELR. This was not the result of a decrease in the number of T6+ Dr+ Langerhans cells but was due to their being less efficient at alloantigen presentation than equivalent numbers of Langerhans cells from unirradiated skin. The reduced stimulation in the ELR immediately after UV was not reversible by the addition of exogenous IL 1 or indomethacin and thus appeared to be due to a direct effect of UV on the alloantigen-presenting function of Langerhans cells. In contrast to this suppression of the epidermal immune function when epidermal cells were obtained immediately after UV, epidermal cells harvested 24 hr or later after in vivo UV exhibited a dose-dependent enhancement of allostimulatory capacity in the ELR that peaked 3 days after UV. The time course of the enhancement of allostimulation in the ELR after in vivo UV coincided with a decrease in the percentage of Langerhans cells and the appearance within the epidermis of T6- Dr+ cells, which are derived from the bone marrow, as evidenced by their expression of the bone marrow derivation markers HLe 1 and T200. Removal of Dr+ cells but not of T6+ cells from epidermal cell suspensions harvested 3 days after in vivo UV abrogated allostimulation in the ELR, demonstrating that the T6- Dr+ cells were responsible for the observed UV-induced enhancement of alloantigen presentation. Taken together, the results indicate that the timing and dosage of UV exposure are critical factors determining whether suppression or enhancement of epidermal immune function follows UV.     

Induction and regulation of contact hypersensitivity by resident, bone marrow-derived, dendritic epidermal cells: Langerhans cells and Thy-1+ epidermal cells

Circumstantial evidence suggests strongly that epidermal Langerhans cells (LC) alone among epidermal cells (EC) are responsible for generating an immunogenic signal for contact hypersensitivity (CH) after epicutaneous application of hapten. However, data obtained from previous studies performed with intact skin or isolated EC do not address the immunogenic capacity of a second dendritic, bone marrow-derived population of cells that resides within the epidermis, Thy-1+ epidermal cells. To identify the cellular source(s) of the antigenic signals emerging from the epidermis, purified preparations of LC, Thy-1+ cells, and keratinocytes were prepared from CBA/J mouse skin. Each cell type was derivatized in vitro with TNBS and inoculated via various routes into syngeneic mice that were assayed for the induction of CH and specific unresponsiveness. IA+ LC, when derivatized with hapten and inoculated into mice, induced CH without evidence of down-regulation regardless of the route of immunization. Derivatized Thy-1+ EC did not deliver a positive signal for CH. Rather, Thy-1+ EC possessed the capacity to initiate down-regulation of the CH response when they were delivered i.v. We conclude that all cellular elements necessary for the induction and regulation of CH after epicutaneous application of hapten to skin reside within the epidermis. The resident, dendritic, bone marrow-derived populations within the epidermis have the capacity to determine the outcome of an epicutaneous antigenic encounter.     

Remyelination of spinal cord axons by olfactory ensheathing cells and Schwann cells derived from a transgenic rat expressing alkaline phosphatase marker gene

Transplantation of cell suspensions containing olfactory ensheathing cells (OECs) has been reported to remyelinate demyelinated axons in the spinal cord with a Schwann cell (SC)-like pattern of myelination. However, questions have been raised recently as to whether OECs can form SC-like myelin. To address this issue we prepared SCs and OECs from transgenic rats in which a marker gene, human placental alkaline phosphatase (hPAP), is linked to the ubiquitously active promoter of the R26 gene. SCs were prepared from the sciatic nerve and OECs from the outer nerve-fiber layer of the olfactory bulb. Positive S100 and p75 immunostaining indicated that >95% of cells in culture displayed either SC or OEC phenotypes. Suspensions of either SCs or OECs were transplanted into an X-irradiation/ethidium bromide demyelinating lesion in the spinal cord. We observed extensive SC-like remyelination following either SC or OEC transplantation 3 weeks after injection of the cells. Alkaline phosphatase (ALP) chromagen reaction product was associated clearly with the myelin-forming cells. Thus, cell suspensions that are enriched in either SCs or OECs result in peripheral-like myelin when transplanted in vivo.     

Immunofluorescent studies of epidermal protein during UV induced carcinogenesis.

Previous studies with agar diffusion technique demonstrated that antibodies produced in rabbits by injection of urea extractable proteins of rat cornfied cells cross react with proteins extracted from normal epidermis of hairless mice using the same technique. In the present study we investigated by indirect immunofluorescence microscopy the immunoreactivity of epidermal proteins in normal and ultraviolet light (UVB) induced hyperplasia and malignant transformation. Reactivity to the antibody was seen over the entire epidermis of nontreated skin and hypertrophied epidermis which occurred at 6-8 weeks after initiation of UVB irradiation. However, the reactivity diminished when malignant changes took place in the epidermal cells. Almost complete disappearance of the immunoresponse was observed in squamous cell carcinoma produced by further UVB radiation. These results suggest that the reactivity of this urea extractable protein serves as an additional immunologic marker for normal epidermal cells. Alterations in the immunoreactivity parallels UVB induced carcinogenesis.     

A developmental study of murine epidermal Langerhans cells

Prospective skin prior to invasion by neural crest cells was dissected from 10.5-day mouse embryos and cultivated in chick embryo hosts. The graft tissue was prepared for the demonstration of both mouse and chick cells, pigment cells, and Langerhans cells. Chick cells were not found in the graft mouse epidermis; however, ATPase-positive and osmium iodide-positive cells were present. Electron microscopic examination revealed that, in younger grafts, only indeterminate cells could be found among the keratinocytes. In older grafts, both indeterminate cells and Langerhans cells with granules were seen. The evidence affirms that epidermal Langerhans cells are not related to pigment cells.Based on the developmental nature of Birbeck (Langerhans) granules from the cytomembrane, it is proposed that the granule no longer be considered as specific to and characteristic of epidermal Langerhans cells. Rather, Langerhans cells should be defined as ATPase-positive, desmosome-free cells within stratified squamous, potentially keratinizing, epithelia. Thus epidermal, ATPase-positive indeterminate cells and such cells with Birbeck granules both should be considered as components of the Langerhans cell series.Normal chick skin does not show ATPase-positive cells. However, when 10.5-day mouse embryo ectoderm was inserted under the ectoderm of chick embryos, the resulting chimeric epidermis possessed ATPase-positive cells. It is proposed that epidermal Langerhans cells are of ectodermal origin.     

Modulation of KB and A431 cell adhesion by epidermal growth inhibitory pentapeptide.

A pentapaptide, pyroGlu-Glu-Asp-Ser-GlyOH (EPP), isolated from mouse epidermis, inhibits mitoses and enhances differentiation in primary cultures and in transformed mouse epidermal cells in vitro (Elgjo et al. 1986 b). The present work demonstrates that EPP also modulates the adhesiveness of two human tumour cell lines (KB and A431) of epidermal origin to uncoated plastic and to plastic coated with fibrinogen or collagen type 1. The adhesion modulatory effect of EPP was observed over a broad range of concentrations (10(-12)-10(-6) M), and depended on the substrate the cells were growing on. Thus, when cells were seeded on plastic or collagen, the attachment to the substrate was suppressed at the highest concentrations of EPP, and stimulated at the lowest ones. The opposite concentration-response pattern was observed when fibrinogen was used as substrate.     

Role of HLA-DR bearing Langerhans and epidermal indeterminate cells in the in vitro generation of alloreactive cytotoxic T cells in man

Human epidermal cells (EC) act as stimulator cells in the mixed-skin cell lymphocyte culture reaction (MSLR). To analyze the role of human epidermal Langerhans cells (LC) and indeterminate cells (IC), which are the only cells expressing the DR-Ia-like antigens in normal epidermis, in the generation of alloreactive cytotoxic T lymphocytes (CTL) in cell-mediated cytolysis, 18-hr 51Cr-release assays against PBL targets (targets autologous to stimulator EC) were conducted after allogeneic human MSLR. MSLR and CTL assays were conducted with, as stimulator cells, suspensions of normal human EC as controls, and EC after: (1) preincubation with anti-HLA-DR or OKT6 (specific for LC in EC suspension) monoclonal antibodies; (2) panning, a monolayer technique used to deplete EC suspensions in OKT6 or DR-positive cells. The generation of alloreactive CTL was found to occur only after allogeneic MSLR and when targets and stimulator cells were from the same donor; it was reduced by EC incubation: cytotoxic activity 26.66 +/- 3.84 (controls); 8.8 +/- 3.6 and 7.7 +/- 3.7 (EC incubated with OKT6 or anti-DR, respectively); it was reduced or abolished when the EC used in MSLR were depleted in OKT6 or DR-positive cells by panning. These findings demonstrate that human LC and IC are necessary for an optimal in vitro sensitization in MSLR and the subsequent in vitro generation of alloreactive CTL in man.     

Epidermal chalones and squamous cell carcinomas. The growth inhibitory effects of aqueous epidermal extracts (G1 and G2 chalones) on the epidermis and on a transplantable keratinizing carcinoman in nude mice.

Balb/c/nu nude mice that had been transplanted with a moderately differentiated squamous cell carcinoma were injected i.p. with different doses of epidermal chalone, and control animals were injected with saline. The labelling indices (H3TdR) and the mitotic rate (stathmokinetic method with vinblastine sulphate) were determined. In the untreated animals, both the labelling index and the mitotic rate of the tumor were considerably higher than in the epidermis, and the rate of cell birth was almost twice that of the epidermis. Higher doses of chalone were needed to reduce the labelling index for the tumour than for the epidermis, and there was generally a less pronounced dose/response relationship in the tumours than in the epidermis. The same was true of the mitotic rate but here the results were not as obvious as for the labelling index. A possible explanation of the results may be that the tumour cells are less sensitive than epidermal cells to the injected chalones, or that reduced vascularization of the transplanted tumour may lead to reduced access of chalone, or that tumour necrosis may pay a role. However, it is evident that the tumour cells react less than the epidermis to both the G1 and the G2 chalone, and thus the findings of this study do not provide any evidence against the theory that epidermoid transplanted tumours are less sensitive to epidermal chalones than normal tissue of the same histogenetic origin.     

Multiple roles of Notch signaling in the regulation of epidermal development

Recent studies have shown that Notch signaling plays an important role in epidermal development, but the underlying molecular mechanisms remain unclear. Here, by integrating loss- and gain-of-function studies of Notch receptors and Hes1, we describe molecular information about the role of Notch signaling in epidermal development. We show that Notch signaling determines spinous cell fate and induces terminal differentiation by a mechanism independent of Hes1, but Hes1 is required for maintenance of the immature state of spinous cells. Notch signaling induces Ascl2 expression to promote terminal differentiation, while simultaneously repressing Ascl2 through Hes1 to inhibit premature terminal differentiation. Despite the critical role of Hes1 in epidermal development, Hes1 null epidermis transplanted to adult mice showed no obvious defects, suggesting that this role of Hes1 may be restricted to developmental stages. Overall, we conclude that Notch signaling orchestrates the balance between differentiation and immature programs in suprabasal cells during epidermal development.     

Stathmokinetic analysis of human epidermal cells in vitro

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling, have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7-15 days). The first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. The second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. The third subpopulation consists of non-cycling cells with G0/G1 DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. The results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.     

Autolysis and heterolysis of the epidermal cells in anuran tadpole tail regression

Autolysis and heterolysis of the degenerating epidermis of the tail fin of Rana japonica tadpoles during spontaneous metamorphosis were observed by transmission and scanning electron microscopy. In the early climactic stages of metamorphosis (st. 19–20), the outermost epidermal cells developed vacuoles that were acid phosphatase positive and showed apparent breakdown of the cell membrane. The cells shrunk, perhaps due to the rupture of the cell membrane, and sloughed off without typical cornification. As tail resorption proceeded, autolysis of the epidermal cells spread towards the inner layers, in which some epidermal cells lost desmosomal junctions. They also displayed atrophic figures with condensed cytoplasm, breakdown of the cell membrane, and pycnotic nuclei. Lymphocytes, neutrophils and macrophages were already present in the basal layers of the premetamorphic epidermis (st. 10). Based on ultrastructural observation, blood cells could be distinguished from autolysing epidermal cells. Only a few blood cells were found in the early climactic stages of metamorphosis (st. 19–20), but the number of the blood cells, especially macrophages, greatly increased during the final stages of metamorphosis (st. 23–24). During the final stages, many macrophages were observed to phagocytose the autolysing epidermal cells by projecting slender pseudopodia into the inner epidermis. Macrophages also were observed to pass through the degraded basal lamella. These results suggest that not only autophagy but also heterophagy of the epidermal cells by the macrophages is a major process in the regression of the tail fin epidermis.     

The effect of the epidermal G2 chalone on the mitotic duration in nude mouse epidermis and in a transplanted squamous cell carcinoma.

Balb/c/nu nude mice transplanted with a moderately differentiated squamous cell carcinoma were injected intraperitoneally with different doses of aqueous skin extracts containing the epidermal G2 chalone. The mitotic counts and the mitotic rates were determined in histological sections using a stathmokinetic method with vinblastine sulphate. The mitotic duration was calculated from the mitotic rates and counts. Skin extracts containing epidermal G2 chalone increased the mitotic duration in the epidermis, and a similar trend was seen in the tumour. The higher the dose of chalone, the longer the mitotic duration tended to be. A straight line of best fit used to indicate the dose/response relationship was steeper for the epidermis than for the tumour. The study thus shows that the epidermal G2 chalone not only prevents epidermal cells from entering mitosis, it also prolongs the mitotic duration. Further, the results do not contradict the theory that tumour cells may be less sensitive to chalone than normal cells.     

In vivo ultraviolet-exposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressor-inducer T cells

In vivo UV exposure of human epidermis abrogates the function of CD1+DR+ Langerhans cells and induces the appearance of CD1-DR+ Ag-presenting macrophages. Epidermal cells from UV-exposed skin, in contrast to epidermal cells from normal skin, potently activate autologous CD4+ T cells, and, in particular, the CD45RA+ (2H4+) (suppressor-inducer) subset. We therefore determined whether UV-exposure in humans leads to a T cell response in which suppression dominates. Autologous blood T cells were incubated with epidermal cell suspensions from in vivo UV-irradiated skin. After activation, repurified T cells were transferred in graded numbers to autologous mononuclear cells (MNC) stimulated with PWM and the resultant IgG production analyzed by ELISA. Relative to T cells activated by unirradiated control epidermal cells, T cells activated by UV-exposed epidermal cells demonstrated enhanced capacity to suppress IgG production (n = 6; p less than or equal to 0.03). Within the T cell population, CD8+ cells stimulated by UV-exposed epidermal cells could be directly activated to suppress PWM-stimulated MNC Ig production if IL-2 was provided in the reaction mixture. The suppressive activity was also transferable with purified CD4+ T cells stimulated by UV-exposed epidermal cells (n = 10; p less than or equal to 0.01), and was radiosensitive. Suppression was decreased when PWM-stimulated MNC were depleted of CD8+ T cells before mixing with CD4+ T cells activated by UV-exposed epidermal cells, suggesting indirect induction of CD8+ Ts cells contained within the responding MNC populations. Indeed, physical depletion of CD45RA+ cells resulted in total abrogation of the suppressor function contained in the CD4+ T cells. Activation of suppressor function was critically dependent on DR+ APC contained in UV-exposed epidermis. The data suggest that UV-exposure modulates cutaneous APC activity in humans, as in mice, such that the dominant immune response is tilted toward suppression. These mechanisms in normal individuals may function to dampen responses to UV-induced endogenous Ag that are pathogenic in autoimmune disorders. However, these mechanisms might also facilitate the growth of UV-induced skin cancers.     

Some new observations and interpretations on the vital staining of leaf epidermal tissue by neutral red

Summary The lower leaf epidermis from 5 plant species was stained with neutral red at 2 pH's (7.1 and 5.6) in the light and dark when the stomata were open or closed. At pH 5.6 no globule (= droplet) formation was observed in the guard cells whether the stomata were open or closed and cell walls possessed a high affinity for the stain. At pH 7.1 globules appeared in guard cells of open stomata, but not closed stomata, within 15 minutes. Anaerobic conditions prevented this globule formation. InZea mays, globules also appeared in subsidiary cells when the stomata were closed and in certain epidermal cells. Where globule formation did not occur increased diffuse staining of certain epidermal cells was considered to be the indication of cell integrity. In old leaf material very large numbers of dark blue globules appeared in epidermal cells ofCommelina diffusa, C. communis andSenecio odoris and this was associated with cell senescence.The staining characteristics were discussed in terms of cellular K⁺, Cl^–, tannin and flavonoid content and vacuolar pH.