Research Article: Key Regulatory Role of Dermal Fibroblasts in Pigmentation as Demonstrated Using a Reconstructed Skin Model: Impact of Photo-Aging

Date Published: December 9, 2014

Publisher: Public Library of Science

Author(s): Christine Duval, Catherine Cohen, Corinne Chagnoleau, Virginie Flouret, Emilie Bourreau, Françoise Bernerd, Andrzej T. Slominski.


To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

Partial Text

In human skin, melanocytes that produce the melanin pigment accounting for the tegument color, lie at the dermal epidermal junction (DEJ), the boundary structure between the two major skin compartments, the epidermis and dermis. Melanocytes in the basal layer of epidermis produce various amounts and types of melanins (eu/pheomelanins) within specific organelles called melanosomes that are transferred to neighboring epidermal keratinocytes. During the keratinocyte differentiation program, melanosomes are more or less degraded depending on the skin type, leading to melanin dust in fair skin or unaltered melanosomes all the way up to the outermost epidermal layers in black skin. The large variety of constitutive colors of human skin arises from these complex and gradual processes.

To demonstrate that the 3D skin model is capable of detecting the role of fibroblasts in the pigmentation process, we studied the effects of drastically different fibroblast conditions in the dermal equivalents. For this, two experimental approaches were designed using the pigmented reconstructed skin model (PRS): one consisted in producing PRS with or without living fibroblasts, and the second in producing PRS with fibroblasts from either fetal or adult skin. The epidermis was reconstructed with the same strains of keratinocytes and melanocytes for all models. The impact of modifying the fibroblast component on the level of pigmentation and melanocyte biomarkers was investigated.

In order to study cutaneous pigmentation in a physiological context, we have developed a pigmented reconstructed skin which reproduces i) the 3-D architecture of the melanocyte environment, ii) the interactions between melanocytes and their active cellular partners, keratinocytes and fibroblasts, iii) the functionality of the pigmentary system [40]. One of the advantages of this model is the possibility to vary the source of the epidermal cells (melanocytes or keratinocytes) or the dermal cells independently. Thus, analysis of interactions between the different cells types is possible and the role of each on skin pigmentation can be individually evaluated. Particularly, this skin model constitutes a unique tool to study the global impact of fibroblasts on skin pigmentation. This can occur through multiple mechanisms and, in particular, through the secretion of soluble factors or the production of various extracellular matrix proteins that make up the epidermal-dermal junction. The PRS used here is also the only in vitro model that allows macroscopic observation of skin pigmentation changes. The purpose of this study was to highlight the influence of fibroblasts on pigmentation in the PRS model by modifying only the fibroblastic component within the dermal equivalent and to prove that the nature/origin or the cell history can have a significant impact on this phenomenon.