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Unlabelled: Fibroblasts isolated from the papillary and reticular dermis are different from each other in vitro. If papillary fibroblasts are subjected to prolonged serial passaging they will differentiate into reticular fibroblasts. Reticular fibroblasts have been shown to resemble myofibroblasts in several ways. TGF-β1 is the most important factor involved in myofibroblast differentiation., Aims: we investigated if TGF-β1 can induce differentiation of papillary fibroblasts into reticular fibroblasts, in monolayer cultures and in human skin equivalents., Method: Monolayer cultures of and human skin equivalents generated with papillary fibroblasts were stimulated with TGF-β1. The expression of markers specific for reticular and papillary fibroblasts was measured by qPCR and immunohistochemical analysis in monolayer cultures. In human skin equivalents, the morphology and the expression of several markers was analysed and compared to untreated papillary and reticular human skin equivalents., Results: Monolayer cultures of papillary fibroblasts started to express a reticular marker profile after stimulation with TGF-β1. Human skin equivalents generated with papillary fibroblast and stimulated with TGF-β1 were similar to papillary control equivalents and did not obtain reticular characteristics. Expression of reticular markers was only found in the lower layers of TGF-β1-stimulated papillary skin equivalents., Conclusions: TGF-β1 can induce differentiation to reticular fibroblasts in monolayer cultures of papillary fibroblasts. In skin equivalents no such effects were found. The major difference between these experiments is the presence of extracellular matrix in skin equivalents. Therefore, we hypothesize that the matrix secreted by papillary fibroblasts protects them from TGF-β1 induced differentiation.