Use of a Serum-Free Epidermal Culture Model to Show Deleterious Effects of Epidermal Growth Factor on Morphogenesis and Differentiation

Abstract

The presence of serum has limited the utility of many culture models for the study of cytokine effects because its complexity and variability can confound the interpretation of data. In the present study, a serum-free skin co-culture model was used to investigate the effect of exogenous epidermal growth factor (EGF) on epidermal proliferation and differentiation. Human keratinocytes cultured on collagen rafts at the air-liquid interface produced a well-differentiated epithelium that resembled normal epidermis. Keratin filaments, membrane-coating granules, and keratohyalin granules were all observed. Epidermal differentiation markers keratin K1/K10, involucrin, and transglutaminase were localized in most of the suprabasal layers, whereas profilaggrin/filaggrin was confined to the granular layers and stratum corneum. In the continual presence of 10-20 ng/mL EGF, the epidermis was less organized, thinner, and less proliferative. EGF also depressed several indicators of differentiation: The number of keratohyalin granules and membrane-coating granules was greatly decreased; antigen expression of profilaggrin/filaggrin appeared diminished by immunocytochemical staining; frequent nuclear retention was noted in the relatively thickened stratum corneum-like layers. As detected by immunohistochemical staining, the expression of EGF receptor in the epidermis was reduced by exogenous EGF. These data illustrate that EGF cannot be considered a simple mitogen. Our findings also underscore the importance of using sophisticated culture models to assess complex cytokine effects that may be dependent on the architecture of a differentiating epidermis.

Chemically Defined Protein-Free in vitro Culture of Mammalian Embryo Does Not Restrict Its Developmental Potential for Differentiation of Skin Appendages

“…The appropriateness of the serum-free medium for the investigation of factors that influence differentiation of epidermis and its appendages, also recognized by others [Chen et al, 1995;Fujie et al, 1996;Bates et al, 1997], prompted the present study. The degree of epidermal differentiation in serum-free and serum-supplemented media was investigated in a more precise manner than before by a classical histological analysis and for the first time at the ultrastructural level.…”

“…Various in vitro culture models have been used for the investigation of embryonic development in mammals [Cockroft, 1997;Karabulut et al, 1999] as well as for the development of some specific organs or tissues such as skin and its appendages [Chen et al, 1995;Inamatsu et al, 1998;Stark et al, 1999]. Organ culture of the embryonic part of the postimplantation rat embryo presents such an example in which the gastrulating embryo during 2 weeks in vitro acquires an irregular teratoma-like feature [Š kreb and Švajger, 1973;Š kreb and Crnek, 1980].…”

EDC cross-linking improves skin substitute strength and stability

“…For inoculation, the area of the collagen-GAG scaffold was calculated by measuring the width and length of the rectangular or square sponge. HF (passage 2) were inoculated onto rinsed control and cross linked scaffolds at a density of 0.5 Â 10 6 cells/cm 2 and cultured at 37 1C and 5% CO 2 in CSS medium [37,41]. After 1 day of culture, the area of the HF-collagen substrates was measured again and sponges were inoculated with HK (passage 2) at a density of 1 Â 10 6 cells/cm 2 .…”

Contributions of the epidermal growth factor receptor to keratinocyte motility

“…Interestingly, the number of EGF receptors is elevated and persistently expressed in abnormally differentiating outer layers of psoriatic epidermis and targeted overexpression of amphiregulin to basal keratinocytes in the epidermis results in a psoriatic phenotype (Cook et al, 1997). In organotypic models of keratinocyte differentiation, EGF suppresses epidermal morphogenesis, stratification, and overall differentiated phenotype (Chen et al, 1995;Ponec et al,1997). Expression of specific markers for keratinocyte terminal differentiation such as fillagrin, keratin 1 and keratinocyte transglutaminase is substantially reduced after exposure to EGF (Chen et al, 1995;Marchese et al, 1990;Monzon et al, 1996;Poumay and Pittelkow, 1995) and, conversely, inhibition EGF receptor tyrosine kinase activity induces expression of the differentiation markers, keratin 1 and keratin 10 (Peus et al, 1997).…”

“…In organotypic models of keratinocyte differentiation, EGF suppresses epidermal morphogenesis, stratification, and overall differentiated phenotype (Chen et al, 1995;Ponec et al,1997). Expression of specific markers for keratinocyte terminal differentiation such as fillagrin, keratin 1 and keratinocyte transglutaminase is substantially reduced after exposure to EGF (Chen et al, 1995;Marchese et al, 1990;Monzon et al, 1996;Poumay and Pittelkow, 1995) and, conversely, inhibition EGF receptor tyrosine kinase activity induces expression of the differentiation markers, keratin 1 and keratin 10 (Peus et al, 1997). The underlying basis for such widespread inhibition of differentiation dependent gene expression remains unclear, but appears to involve both transcriptional and post-transcriptional mechanisms (Drozdoff and Pledger, 1993;Monzon et al, 1996).…”