Unilever Research, Trumbull, CT
Targeted expression of p16INK4a as a Model of Aging in an Organotypic Model of Skin
Keratinocytes are the most abundant cell type in the epidermis where they undergo a complex series of steps to form the stratum corneum, the body's primary barrier to the external environment. Because the complex steps of differentiation are inhibited in an aqueous environment, monolayer cell culture is a poor model of many aspects of skin physiology. We have therefore developed a three dimensional organotypic model of skin. The model was developed by seeding human dermal fibroblasts into a collagen gel then seeding normal human keratinocytes on top of the gel. After several days in culture keratinocytes begin to stratify and differentiate at which point the tissue is lifted to the air interface to promote further maturation. When keratinocytes from young donors (20-30 years old) were used, they almost invariably formed a uniform tissue with a well polarized stratum basale, several suprabasal layers and expressed differentiation-related proteins such as filaggrin, loricrin and caspase14. In contrast, when keratinocytes from older donors (50-60) were used, the tissues were highly variable, often exhibiting an atrophic phenotype with few suprabasal layers, a poorly organized basal layer and reduced expression of differentiation-related proteins. In this respect the organotypic model mimicked the effect of chronologic aging on skin. We then used lentiviral vectors to overexpress and silence expression of selected genes in order to study their function. P16INK4a (P16) codes for an inhibitor of cyclin-dependent kinase. As such it functions as a cell cycle checkpoint protein and its repression is required to maintain the proliferative capacity of the cell. We targeted expression of p16 or p16 miR to the basal layer using a keratin 14 promoter. When p16 was overexpressed in young donor cells the morphology changed dramatically and more closely resembled that of an older donor; in contrast, when p16 was repressed in older donor cells, the tissue formed was reminiscent of a younger donor. These results demonstrate that the donor phenotype is maintained in a skin equivalent and provide a model to study the molecular changes associated with that phenotype.