Terminal Epidermal Differentiation of Human Keratinocytes Grown in Chemically Defined Medium on Inert Filter Substrates at the Air-Liquid Interface

A fully differentiated epithelium having the features of epidermis was obtained in vitro by culturing second-passage normal human keratinocytes (NHK) in the chemically defined medium MCDB 153 on inert filter substrates at the air-liquid interface for 14 d. Vertical sections stained for histology and indirect immunofluorescence studies show a correct stratification and expression of differentiation markers. The presence of desmosomes, keratohyalin granules, and lamellar granules, and the formation of a more than ten-layers stratum corneum was evidenced by electron microscopy. Moreover, lipids typical for differentiated epidermis were present in these cultures, including ceramides, which are thought to be responsible for the relative impermeability of the stratum corneum. Under our culture conditions, i.e., in defined medium and at the air-liquid interface, the use of de-epidermized dermis as a substrate did not stimulate keratinocyte differentiation more than acetate cellulose or poly-carbonate filter membrane substrates.The obtaining of a well-differentiated epidermis grown in vitro on inert filters in a chemically defined medium should be useful as a standard system for studying epidermal differentiation, re-epidermization, cytotoxicity, epidermal permeation, and transepidermal drug delivery

La differenciation des keratinocytes humains normaux et transformes en milieu defini. (Application aux biomateriaux)

SIGLEINIST T 73726 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

A Characteristic Subset of Psoriasis-Associated Genes Is Induced by Oncostatin-M in Reconstituted Epidermis

The pathological manifestations of psoriasis are orchestrated by many secreted proteins, but only a handful, tumor necrosis factor-alpha, IFN-γ and IL-1, have been studied in great detail. Oncostatin-M (OsM) has also been found in psoriatic skin and we hypothesized that it makes a unique and characteristic contribution to the psoriatic processes. To define in-depth the molecular effects of OsM in epidermis, we used high-density DNA microarrays for transcriptional profiling of OsM-treated human skin equivalents. We identified 374 unambiguously OsM-regulated genes, out of 22,000 probed. OsM suppressed the expression of the “classical” epidermal differentiation markers, but strongly and specifically induced the S100A proteins. Cytoskeletal and complement proteins, proteases, and their inhibitors were also induced by OsM. Interestingly, a large set of genes was induced by OsM at early time points but suppressed later; these genes are known regulatory targets of IFN and thus provide a nexus between the OsM and IFN pathways. OsM induces IL-4 and suppresses the T-helper 1-type and IL-1-responsive signals, potentially attenuating the psoriatic pathology. The data suggest that OsM plays a unique role in psoriasis, different from all other, more thoroughly studied cytokines

Transcriptional Profiling of Epidermal Keratinocytes: Comparison of Genes Expressed in Skin, Cultured Keratinocytes, and Reconstituted Epidermis, Using Large DNA Microarrays

Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large “transcriptome.” To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis