UVB exposure of a humanized skin model reveals unexpected dynamic of keratinocyte proliferation and Wnt inhibitor balancing.

We developed human dermo-epidermal skin substitutes that are presently applied in phase I and II clinical trials. Here, we used these very same skin equivalents containing melanocytes, named MelSkin, as an experimental skin model. We investigated the effects of ultraviolet B (UVB) irradiation on the skin grafts transplanted on immune-compromised rats. The irradiation induces a strong wound healing response going along with massive proliferation of basal keratinocytes, basically quiescent under nonirradiated, homeostatic conditions. As a consequence of UVB irradiation, the initially clearly defined basal keratinocyte (mono)layer expands into about 3 layers of keratinocytes, all of which still express the basal keratinocyte marker keratin 15. In contrast, epidermal melanocytes remain quiescent under these circumstances. Moreover, the Wnt inhibitors Dickkopf 3 and Wif1 are downregulated upon UVB irradiation in basal keratinocytes, whereas melanocytes continue to express Wnt inhibitors. These findings suggest that there is (a) a suprabasal population, proliferating in the homeostatic state, hence maintaining the integrity of the epidermis, and (b) a basal, usually quiescent keratinocyte population that is induced to massively proliferate upon irradiation. Importantly, the finding that MelSkin responds in a physiological fashion to UVB is of paramount importance in light of the planned clinical application.

Human Adipose Mesenchymal Cells Inhibit Melanocyte Differentiation and the Pigmentation of Human Skin via Increased Expression of TGF-ß1.

There is accumulating evidence that interactions between epidermal melanocytes and stromal cells play an important role in the regulation of skin pigmentation. In this study we established a pigmented dermo-epidermal skin model, melDESS, of human origin to investigate the effects of distinct stromal cells on melanogenesis. melDESS is a complex, clinically relevant skin equivalent composed of an epidermis containing both melanocytes and keratinocytes. Its dermal compartment consists either of adipose tissue-derived stromal cells, dermal fibroblasts (Fbs), or a mixture of both cell types. These skin substitutes were transplanted for 5 weeks on the backs of immuno-incompetent rats and analyzed. Gene expression and Western blot analyses showed a significantly higher expression of transforming growth factor-ß1 by adipose tissue-derived stromal cells compared with dermal Fbs. In addition, we showed that melanocytes responded to the increased levels of transforming growth factor-ß1 by down-regulating the expression of key melanogenic enzymes such as tyrosinase. This caused decreased melanin synthesis and, consequently, greatly reduced pigmentation of melDESS. The conclusions are of utmost clinical relevance, namely that adipose tissue-derived stromal cells derived from the hypodermis fail to appropriately interact with epidermal melanocytes, thus preventing the sustainable restoration of the patient's native skin color in bioengineered skin grafts.

Long-term expression pattern of melanocyte markers in light- and dark-pigmented dermo-epidermal cultured human skin substitutes.

PURPOSE: Transplantation of pigmented tissue-engineered human autologous skin substitutes represents a promising procedure to cover skin defects. We have already demonstrated that we can restore the patient's native light or dark skin color by adding melanocytes to our dermo-epidermal skin analogs. In this long-term study, we investigated if melanocytes in our skin substitutes continue to express markers as BCL2, SOX9, and MITF, known to be involved in survival, differentiation, and function of melanocytes. METHODS: Human epidermal melanocytes and keratinocytes, as well as dermal fibroblasts from light- and dark-pigmented skin biopsies were isolated and cultured. Bovine collagen hydrogels containing fibroblasts were prepared, and melanocytes and keratinocytes were seeded in a 1:5 ratio onto the gels. Pigmented dermo-epidermal skin substitutes were transplanted onto full-thickness wounds of immuno-incompetent rats and analyzed for the expression of melanocyte markers after 15 weeks. RESULTS: Employing immunofluorescence staining techniques, we observed that our light and dark dermo-epidermal skin substitutes expressed the same typical melanocyte markers including BCL2, SOX9, and MITF 15 weeks after transplantation as normal human light and dark skin. CONCLUSIONS: These data suggest that, even in the long run, our light and dark dermo-epidermal tissue-engineered skin substitutes contain melanocytes that display a characteristic expression pattern as seen in normal pigmented human skin. These findings have crucial clinical implications as such grafts transplanted onto patients should warrant physiological numbers, distribution, and function of melanocytes.