Microarray analysis of gene expression in cultured skin substitutes compared with native human skin

Cultured skin substitutes (CSS), prepared using keratinocytes, fibroblasts, and biopolymers, can facilitate closure of massive burn wounds by increasing the availability of autologous tissue for grafting. But because they contain only two cell types, skin substitutes cannot replace all of the functions of native human skin. To better understand the physiological and molecular differences between CSS and native skin, we undertook a comprehensive analysis of gene expression in native skin, cultured keratinocytes, cultured fibroblasts, and skin substitutes using Affymetrix gene chip microarrays. Hierarchical tree clustering identified six major clusters of coordinately regulated genes, using a list of 1030 genes that were the most differentially expressed between groups. These clusters correspond to biomarker pools representing expression signatures for native skin, fibroblasts, keratinocytes, and cultured skin. The expression analysis revealed that entire clusters of genes were either up- or downregulated upon combination of fibroblasts and keratinocytes in cultured skin grafts. Further, several categories of genes were overexpressed in CSS compared with native skin, including genes associated with hyperproliferative skin or activated keratinocytes. The observed pattern of expression indicates that CSS in vitro, which display a well-differentiated epidermal layer, exhibit a hyperproliferative phenotype similar to wounded native skin.     

Phenotypic evaluation of cultured human mast and basophilic cells and of normal human skin mast cells

In order to evaluate various markers for human mast cells, two human mast/basophilic cell lines (HMC-1/KU812), cultured mast cells from the peripheral blood monocytic fraction and peripheral blood monocytes were compared with mast cells in tissue sections from normal skin, using histochemistry, enzyme histochemistry and immunohistochemistry. All reagents stained normal skin mast cells, with toluidine blue, tryptase reactivity and antibodies against the FcRI and the stem cell factor receptor (c-kit) being most active. The cell lines and mast cells cultured from peripheral blood were negative for avidin, safranin and chymase, strongly positive for c-kit and variably reactive with all other reagents. All antibodies except AA1 against tryptase also stained one or several epidermal and dermal cell types or blood monocytes. Histochemical stains (toluidine blue, avidin) and reagents for the enzymes tryptase and chymase are thus specific markers for mast cells. The frequent reactivity of antibodies against mast cells with other cell types indicates interesting functional and ontogenetic relationships between these cells.     

Allotransplantation of cultured human skin

Summary Human skin allografts cultured for approximately 6 weeks on a solid medium were transplanted to non-related human recipients with skin defects of various etiology. Although based on this study no answer could be given to the question whether these grafts survived or were replaced by cells of the recipient, it was observed that in most cases the grafts were not rejected and remained in situ several months up to over a year.     

Aging and cultured human skin fibroblasts.

We have studied aging at the cellular level by examining skin fibroblast cultures derived from skin biopsy samples obtained from old and young members of a longitudinal study on human aging. Results from a number of studies already completed indicate that cell cultures from old human donors, unlike cultures from young donors, have impaired cell proliferation and reduced induction of sister chromatid exchanges. A number of other parameters, however, are not affected by the age of the human fibroblast donor. These include cell protein and RNA content and the rate of cellular macromolecular synthesis. Studying cell cultures derived from members of an ongoing longitudinal study permits examination of correlations between in vivo and in vitro measurements on the same people. In addition, it allows for future longitudinal studies on human cellular aging.     

Reorganization of hair follicles in human skin organ culture induced by cultured human follicle-derived cells

Studies of human hair follicle (HF) induction by follicle-derived cells have been limited due to a lack of suitable test systems. In this study, we established a skin organ culture system which supports HF formation by follicle-derived cells. Long-term skin organ cultures were set up from human retroauricular skin specimens and maintained in culture for up to 8 weeks. In vitro expanded human HF-derived cells from the dermal papilla (DP) and the outer root sheath (ORS) were injected together into the skin specimens and evaluated for their ability to induce reorganization of HFs. Macroscopic analysis of the cultured skin specimens demonstrated the growth of velus-like hair after 4 weeks in culture. Histologic evaluation of the cultured skin specimens after 8 weeks of culture revealed multiple miniaturized HFs with sebaceous glands. In addition, cell clusters of various differentiation stages could be demonstrated in serial sections of the cultured skin specimens. Labeling of HF-derived cells with the fluorescence dye CFDA-1 prior to injection suggested a de novo reorganization of HFs out of the injected cells. In conclusion, the study demonstrated HF formation by HF-derived cells in an in vitro skin organ culture model.     

Induction of connective tissue growth factor expression by sphingosylphosphorylcholine in cultured human skin fibroblasts

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In an effort to find downstream effectors of SPC, we performed microarray analysis and found that the expression of the gene for connective tissue growth factor (CTGF) was significantly affected in human skin fibroblasts cultured in vitro. Northern blot analysis showed that SPC markedly induced CTGF mRNA expression in a dose- and time-dependent manner. Consistent with this result, Western blot analysis also showed that SPC significantly induced the CTGF production. Pretreatment with cycloheximide did not prevent the CTGF induction by SPC, indicating that SPC stimulates CTGF mRNA expression without the increased synthesis of a regulatory protein. Inhibition by pretreatment with Y27632, but not by PD98059 (a mitogen-activated protein kinase 1/2 inhibitor) and LY294002 (a phosphatidylinositol 3-kinase inhibitor), indicated that rho-kinase pathway was involved in SPC-induced CTGF expression. Together, these results reveal the potential importance of CTGF induction as a downstream event in SPC-induced cellular responses.     

Lipase expression in human skin

We have used a monoclonal antibody against human pancreatic lipase to study the immunohistochemical expression of lipase in formalin-fixed, paraffin-embedded biopsy specimens from normal and a variety of tumoral and inflammatory skin diseases. In normal skin, lipase is detected in the sebaceous glands and in the external root sheath of the hair follicle. Antibody to lipase might be a valuable reagent to help confirm sebaceous and follicular differentiation in adnexal tumors. Immunoreactivity to lipase is also detected within mononuclear phagocytes in situations where extracellular release of lipids occurs, such as in xanthomas or in inflammation of the fat with significant lysis of adipocytes. The antibody to lipase identifies a major protein in pilosebaceous homogenates of 42.7 kDa following immunoblotting.     

Intracellular collagen fibrils in cultured human skin.

During cultivation, the collagen fibrils of skin explants are broken down. The cells of the explants participate in this resorption. The ultrastructure of the intracellular degradation of collagen fibrils of cultured human skin has been examined. Intracellular collagen fibrils occur in fibroblasts, macrophages, smooth muscle cells and unidentifiable cells. Normal collagen fibrils are engulfed and appear within membrane-bounded tubes of the cytoplasm. Primary lysosomes fuse with the tubes. Degraded intracellular collagen fibrils are frequently present in secondary lysosomes and show decreasing diameters, filamentous splitting, loss of axial periodicity, variable stainability and cross-banded filamentous aggregates. The changes in the intracellular collagen fibrils are identical with those seen in the extracellular space. The present study demonstrates that various cell types in dermis are involved in collagen fibril degradation and that the lysosomes play an important part in the intracellular resorption.     

Low doses of UVB or UVA induce chromosomal aberrations in cultured human skin cells

Chromosomal defects are frequently present in malignant and premalignant skin disorders; however, it is not known whether ultraviolet radiation from sunlight plays a role in their induction. To obtain information on the ability of ultraviolet A and ultraviolet B to induce chromosomal aberrations, cultured melanocytes and fibroblasts were exposed to physiologic doses of ultraviolet A or ultraviolet B and, for comparison, to gamma rays. As a measure of chromosomal aberrations, the formation of micronuclei was determined. To obtain sufficient statistical data on induced micronuclei and cell kinetics, a flow cytometry method has been modified and applied. The flow cytometry method analysis is based on staining the DNA with ethidium bromide and the cell membranes with 1,6-diphenyl-1,3,5,-hexatriene. We observed dose-dependent micronuclei formation after gamma or ultraviolet B irradiation in both cell types and also for ultraviolet A in fibroblasts. The yield of micronuclei induced in fibroblasts by ultraviolet A was only a factor 15 smaller than that induced by ultraviolet B (313 nm). The results indicate that 10 kJ per m2 (equivalent to 1 minimal erythema dose) of ultraviolet B and 150 kJ per m2 of ultraviolet A (0.2 minimal erythema dose) can induce 1% of micronuclei in fibroblasts, equivalent to the induction due to 0.6 Gy of gamma radiation. In conclusion, physiologic doses of sunlight can induce chromosomal aberrations at a level comparable with that observed after exposure to approximately 1 Gy of ionizing radiation. Therefore, sunlight can be considered a potential inducer of chromosomal aberrations in skin cells, which may contribute to skin carcinogenesis.     

Densities,distribution and phenotypic expression of T cells in human fetal skin

T cells are present in normal adult human skin, but their occurrence in fetal skin is unknown. T cell and Langerhans cell (LC) populations were studied using single or double immunohistochemical staining on cryostat-section. Skin samples taken from different body regions of 17 fetuses ranging from 18 to 30 weeks estimated gestational-age (w-EGA), were examined. In all specimens but one, we did not find any epidermal T cell. In contrast, dermal CD3⁺ T cells occurred at all w-EGA. The density of these cells increased with increasing age. Double staining showed that CD3⁺ T cells were predominantly CD4⁺/CD45RA⁺. On the other hand, LC, as assessed by CD1a expression, was evenly distributed within the interfollicular epidermis and papillary dermis at all gestational ages. Analysis of T cell and LC density in different body regions did not show significant topographic differences. We suggest that lack of epidermal T cells, although the LC network was fully represented, might reflect the scarce opportunity of fetal LC to contact foreign antigens in utero.     

Oxidative defense in cultured human skin fibroblasts and keratinocytes from sun-exposed and non-exposed skin

Skin fibroblasts and keratinocytes cultivated from chronically light-exposed skin sites have higher levels of the protective protein ferritin than cells derived from unexposed areas of the body, suggesting an adaptive response of cells exposed to chronic external insults. In the same line, ferritin levels were always found to be 2-to 7-fold higher in epidermal keratinocytes than in the underlying dermal fibroblasts of the same person thus providing the keratinocyte with continuous protection by the higher cellular ferritin content. The activation of ferritin by oxidative stress including UVA radiation could represent an important cellular defense mechanism that operates in human skin. Following low fluences of UVA radiation (2–4×10⁵ J/m²), ferritin levels increased by 20–30% in normal adult skin fibroblasts and showed a subsequent decrease at higher UVA fluences. In contrast, skin keratinocytes were not perturbed by UVA radiation exposure except for very high fluences (1.25× 10⁶ J/m²) where slight decreases in cellular ferritin were noted in 7 of the 12 cell lines. Fibroblasts derived from light-exposed skin sites that possessed higher levels of cellular ferritin were highly protected against UVA-induced membrane damage as measured by lactate dehydrogenase release compared with fibroblasts from nonexposed body sites with lower levels of ferritin. It is clear from our results that ferritin plays an important role in protection at the cellular level in human skin cells, but the role of this putative protective protein in vivo remains to be defined.     

Effects of glucocorticosteroids on cultured human skin fibroblasts

Summary An immediate depression of the rate of cell proliferation occurred upon addition of glucocorticosteroids to cultures of human skin fibroblasts in the early growth stages. A reduced sensitivity or even insensitivity of the fibroblasts to growth inhibition was found upon the addition of the steroids at later stages of cell growth, when the cell density has increased.The inhibition in the early growth stages is transient and is most pronounced if the cultured medium is not renewed. This transient inhibition is not due to the development of steroid-resistant cell lines, and resembles the effect called tachyphylaxis, as is also observed in vasoconstriction tests.     

Effects of glucocorticosteroids on cultured human skin fibroblasts

Summary As previously found, the glucocorticosteroid clobetasol-17-propionate inhibits cell proliferation during the early growth stage of normal baby foreskin fibroblasts and collagen synthesis in confluent cultures of these cells. The degree of inhibition of cell proliferation decreases with increasing cell density and, moreover, is transient.The anabolic steroids nandrolone and nandrolone-phenyl-propionate have similar effects on these cells. Likewise the magnitude of the inhibition is dose-dependent.When present together the two types of drug do not act in an additive manner. Even at low concentrations the anabolic steroids abolish the inhibitory effect of the glucocorticosteroid on cell proliferation. Furthermore, in this case only the inhibitory effect of the glucocorticosteroid on collagen synthesis is found and there is no further increase in this effect due to the presence of the anabolic steroids.Our results imply that the use of low concentrations of anabolic steroids combined with glucocorticosteroids in topical application to the skin may abolish some of the undesirable side effects of the glucocorticosteroids.     

Regulation of adrenomedullin secretion in cultured human skin and oral keratinocytes

Adrenomedullin, a potent vasoactive peptide, is actively secreted from primary cultures of human oral and skin keratinocytes, but nothing is known of the regulation of its release. This study describes the effects of a range of substances on adrenomedullin production from cultures of oral and skin keratinocytes. We have established that keratinocytes do not store adrenomedullin but secrete it constitutively. Cytokines interleukin-1alpha and -1beta, tumor necrosis factor-alpha and -beta, and the bacterial product, lipopolysaccharide, significantly stimulate adrenomedullin secretion from oral but not skin keratinocytes. Both transforming growth factor-beta1 and interferon-gamma are potent suppressors of adrenomedullin secretion from both cell types, as are forskolin, di-butyryl cyclic adenosine monophosphate, and adrenocorticotropin. The peptides thrombin and endothelin-1 increase adrenomedullin production, particularly from skin keratinocytes. These findings indicate that there are differences in the regulation of adrenomedullin production between oral and skin keratinocytes and that oral keratinocytes are particularly responsive to the action of inflammatory cytokines. This raises the possibility that adrenomedullin may serve a different functions in oral mucosa and skin.     

Plasminogen activator in cultured human epidermal cells

Primary cultures of human epidermal cells produce plasminogen activator (PA) as demonstrated by the ability of conditioned medium or cell lysates to hydrolyze fibrin in the presence of plasminogen, and to cleave [125I]plasminogen to characteristic fragments. The major molecular species of PA in human epidermal cells was inhibited by diisopropylfluorophosphate and comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the high molecular weight band of human urokinase (Mr 55,000). Production of PA by human epidermal cells was inhibited by cycloheximide, stimulated by colchicine, and not affected by cytochalasin B or the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Both cholera toxin and epidermal growth factor stimulated PA activity in human epidermal cells, and PA activity was maximal at concentrations that best support in vitro growth of human epidermal cells. Examination of individual cells indicated that at least 15% of cells within a culture produced detectable amounts of PA.     

Regulated expression of tumor-specific and MHC class I antigens in an ultraviolet radiation-induced murine skin cancer

Previous studies have shown that the ultraviolet (UV) radiation-induced tumor UV-1591 expresses a novel class I antigen that functions as a tumor-specific transplantation antigen (TSTA). The purpose of this study was to determine whether expression of novel class I TSTA and expression of endogenous class I antigens in UV-1591 are regulated independently or coordinately. Expression of both TSTA and H-2Kk antigens increased simultaneously after treatment of UV-1591 cells with either 5-azadeoxycytidine (5-azaC) or interferon-gamma (IFN-gamma). Furthermore, the kinetics and pattern of increased expression by 5-azaC and IFN-gamma were identical for both TSTA and H-2Kk antigens. The increased expression of TSTA and class I antigens induced by 5-azaC treatment was constitutive and did not require the continued presence of the drug. In contrast, the increased expression after IFN-gamma treatment was transient and required the continued presence of IFN-gamma. Neither 5-azaC nor IFN-gamma caused induction of class II antigens in UV-1591 tumor cells. These results indicate that expression of TSTA and class I antigens in UV-1591 tumor is coordinately regulated, whereas expression of class I and class II antigens is independently regulated.