Insulin-like Growth Factor-Binding Protein Contributes to the Proliferation of Less Proliferative Cells in Forming Skin Equivalents

In this study, the effects and the mediating factors of dermal cells on the epidermal regenerative ability were investigated. Human epidermal cells were separated into rapidly adhering (RA) cells and slowly adhering (SA) cells and used for culturing skin equivalents (SEs). For dermal part, normal human fibroblasts, dermal sheath cells (DSCs), and dermal papilla cells were used. SEs produced using SA cells and DSCs showed a thicker epidermis and higher expressions of alpha(6)- and beta 1-integrin than SEs using SA cells and normal fibroblasts showed. We hypothesized that DSCs may secrete specific cytokines that can influence the regenerative potential of epidermal cells, and compared cytokine secretion by DSCs and normal human fibroblasts. Using RayBio (R) human cytokine antibody array C (series 1000), 120 cytokines were tested. Results showed that DSCs produced a much greater amount of insulin-like growth factor-binding protein (IGFBP-2), angiogenin, and BMP-6 than normal human fibroblasts produced. On the basis of the cytokine antibody array, we next investigated whether IGFBP-2, angiogenin, or BMP-6 has effects on SEs reconstruction. The addition of IGFBP-2 induced a thicker and more mature epidermis and higher expressions of alpha(6)- and beta 1-integrin, whereas BMP-6 exhibited little effect. Thus, the SEs with IGFBP-2 showed almost the same morphology of the SEs using DSCs. Further, p63, a putative keratinocyte stem cell marker, was more frequently observed in the basal layer of SE with IGFBP-2. In conclusion, IGFBP-2 is a major factor from DSCs that affects epidermal regenerative capacity of skin and may play an important role for stemness maintenance in human epidermal keratinocytes.