osome diameter is 0.5m. Therefore, iPSC-derived keratinocytes were co-cultured with red fluorescent microspheres to investigate the capability of these cells to internalize melanosome-sized particles and to determine how the efficiency of this process compared to that in normal epidermal keratinocytes. At all time points investigated, the uptake of the microspheres in normal and iPSC-derived keratinocytes was not significantly different from one another. Moreover, as the length of incubation with the microspheres increased from 2 to 6 hours, the microspheres began to self-organize into a typical supranuclear cap configuration, representing a characteristic internal localization pattern that allows melanin to protect DNA within the nucleus from the damaging effects of ultraviolet radiation . DMXB-A price Internalization of freshly isolated melanosomes by iPSC-derived keratinocytes To further investigate the capability of our iPSC-derived keratinocytes to participate in the melanin transfer process, we incubated these cells with freshly isolated melanosomes. 8 / 16 Pigmented Induced Pluripotent Stem Cell-Derived Skin Models Fig 2. iPSC-derived keratinocytes can internalize and intracellularly transport microspheres 0.5m in diameter. Normal epidermal keratinocytes and iPSC-derived keratinocytes co-cultured with red fluorescent microspheres 0.5m in diameter for different time points. Quantitative analysis of microsphere internalization. Parallel fluorescence and phase contrast microscopy showing method used to determine that only internalized beads were counted in the quantitative analysis. Blue, Red. doi:10.1371/journal.pone.0136713.g002 Following a 24 hour incubation with these organelles, we confirmed by Fontana-Masson staining that they had been 
internalized by the iPSC-derived keratinocytes. Moreover, it was also clear that the melanosomes had been correctly transported within the cells, resulting in their hallmark supranuclear localization. Protease-activated receptor-2 is known to be involved in melanosome phagocytosis in normal epidermal keratinocytes. Therefore, in order to provide mechanistic insight into how the melanosomes had been internalized by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729663 the iPSC-derived keratinocytes, we repeated the 24 hour melanosome incubation in the presence of soybean trypsin inhibitor, a known PAR-2 inhibitor. STI reduced melanosome uptake by around 50% in both the normal and iPSC-derived keratinocytes confirming the involvement of PAR-2 in this process. Surprisingly, iPSC-derived keratinocytes appeared to internalize melanosomes more efficiently than normal keratinocytes, as determined by more extensive Fontana-Masson staining. Melanin transfer between iPSC-derived melanocytes and iPSC-derived keratinocytes We next investigated the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729642 capability of our iPSC-derived cell types to synthesize and transfer melanin, characteristic of the epidermal-melanin unit in normal human skin. Normal epidermal melanocytes were co-cultured with iPSC-derived keratinocytes and gp-100 expression was investigated to determine the ability of the latter cell type to take up melanin in a 2D context. gp-100 has previously been shown to be a useful marker in studying melanosome transfer in melanocyte/keratinocyte co-cultures. After 7 hours in co-culture, the keratin-1 positive iPSC-derived keratinocytes were also positive for gp100 staining. At this time point, gp-100 staining was somewhat diffuse and localized throughout the cytoplasm. However, by 24 hours in co-culture gp-100 staining
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