Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts

The immortal human keratinocyte line HaCaT is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differenti ation capacity. HaCaT cells form a nearly regular epidermal architecture wh en transplanted onto subcutaneous tissue of athymic mice. In order to analy ze further their differentiation capacity in vitro, HaCaT cells were studie d in organotypic cocultures on top of collagen gels containing human dermal fibroblasts, Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of fibroblasts in the co llagen gel, With further culture time of up to 3 wk a remarkably well struc tured and differentiated squamous epithelium developed, After 1 wk, keratin s 10 and 16, involucrin, and transglutaminase I were expressed in suprabasa l layers, whereas filaggrin, keratin 2e, and loricrin appeared after 2-3 wk , Within this time, a nearly complete basement membrane had formed includin g hemidesmosomes and anchoring fibrils, Epithelial cell proliferation becam e restricted to the basal layer after 2 and 3 wk, Using the TdT-mediated dU TP nick end labeling assay, fragmentation of DNA was detectable in nuclei o f the parakeratotic stratum corneum, Ultrastructurally, many features of ke ratinization accumulated after 2 and 3 wk, though an orthokeratotic keratin ization was not achieved, in contrast to HaCaT transplants, This differenti ation deficiency - as compared with normal keratinocytes - might be due to a lack of paracrine factors important for keratinocyte differentiation or t o a reduced sensitivity of these cells, Nevertheless, this high degree of d ifferentiation under organotypic conditions qualifies this cell Line as an appropriate model for elucidation of the molecular mechanisms regulating ke ratinocyte growth and differentiation and for use in pharmacotoxicology.