Morphological differentiation and changes in polypeptide synthesis pattern during regeneration of human epidermal tissue developed in vitro

• Published in: Differentiation (London) Vol. 47; no. 1; pp. 37 - 48
• Main Authors: Jensen, Peter K.A., Fey, Stephen J., Larsen, Peter Mose, Nørgård, Jan O.R., Bolund, Lars
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier B.V 01.05.1991; Blackwell Publishing Ltd
• Subjects: Cell Differentiation; Cells, Cultured; Electrophoresis, Gel, Two-Dimensional; Epidermis - cytology; Epidermis - metabolism; Epidermis - physiology; Humans; In Vitro Techniques; Isoelectric Focusing; Keratinocytes - metabolism; Keratinocytes - physiology; Keratinocytes - ultrastructure; Keratins - metabolism; Microscopy, Electron; Peptides - metabolism; Regeneration - physiology
• ISSN: 0301-4681; 1432-0436
• DOI: 10.1111/j.1432-0436.1991.tb00219.x

By incubating multilayered primary cultures of human keratinocytes in low-calcium medium the suprabasal cell layers can be stripped off leaving a basal cell monolayer. When this monolayer is re-fed normal calcium medium a reproducible series of cell kinetic, morphological, and biochemical changes takes place resulting in the reestablishment of a multilayered tissue. Analysis of cell-cycle-specific proteins indicated that, during regeneration, a large cohort of cells became synchronized undergoing DNA replication after 3 days. Examination of culture morphology at the ultrastructural level confirmed the capacity of the basal cell monolayer to gradually reestablish a multilayered, differentiated epithelium. The ultrastructural appearance at 7 days post-stripping was similar to that of unstripped cultures and was indicative of a tissue in steady state. Quantitation of cornified envelope formation at different times during regeneration showed that an increasing proportion of the cells were able to undergo terminal differentiation. In general, the pattern of keratin synthesis in the original epidermal explant labelled in vitro was similar to the pattern observed in human epidermis in vivo; however, in contrast to epidermis in vivo the explant also synthesized the hyperproliferative keratins 6 and 16. The in vitro differentiated keratinocytes showed underexpression of several proteins identified as differentiation markers, whereas several basal cell markers were overex-pressed compared to the original explant. In addition, the in vitro differentiated keratinocytes synthesized some new proteins, notably keratins 7, 15 and 19. The basal layer remaining after stripping mainly expressed basal cell markers; however, during recovery, some of the differentiation-specific markers (e.g. keratin 10 and 15) were again expressed together with keratin no. 19, which is also expressed during wound healing in vivo. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model to investigate certain aspects of the regulation of epidermal tissue homeostasis.