in iPSC-derived 9 / 16 Pigmented Induced Pluripotent Stem Cell-Derived Skin Models Fig 3. iPSC-derived keratinocytes internalize melanosomes in a PAR-2-dependent manner. a) Normal epidermal keratinocytes and iPSC-derived keratinocytes internalize freshly isolated melanosomes within 24 hours via a mechanism involving PAR-2.Melanosomes, + Melanosomes, + Melanosomes & STI. Scale bar = 100m. b) Quantification of Fontana-Masson staining in a). = p<0.05, = p<0.01, = p<0.001. doi:10.1371/journal.pone.0136713.g003 Fig 4. iPSC-derived melanocytes and keratinocytes participate in melanin transfer. normal epidermal melanocytes and iPSC-derived keratinocytes in co-culture for 7 and 24 hours. iPSC-derived melanocytes and normal epidermal keratinocytes in co-culture for 7 and 24 hours. iPSC-derived melanocytes and iPSC-derived keratinocytes in co-culture for 7 and 24 hours. Inset. doi:10.1371/journal.pone.0136713.g004 10 / 16 Pigmented Induced Pluripotent Stem Cell-Derived Skin Models keratinocytes was concentrated in the perinuclear region of the cell, suggesting that the melanosomes had taken up a supranuclear cap arrangement. Next, iPSC-derived melanocytes were co-cultured with normal epidermal keratinocytes to investigate their ability to synthesize and transfer melanin in this context. After 7 hours in co-culture the keratin-1 positive normal epidermal keratinocytes were also positive for BQ 123 site gp-100 staining suggesting that the iPSC-derived melanocytes had successfully made and transferred melanin. Again, after 24 hours in co-culture the melanosomes had taken up the supranuclear cap arrangement in the normal epidermal keratinocytes. Finally, iPSC-derived melanocytes and iPSC-derived keratinocytes were co-cultured to investigate whether the desired abilities these cell types had displayed with their normal counterparts would be retained. After 7 and 24 hours in co-culture results closely resembled those in Fig 4a4d suggesting that our iPSC-derived cells would be capable of making a functional epidermal-melanin unit when placed in a 3D skin equivalent. Moreover, keratin-14 positive iPSC-derived keratinocytes were also able to participate in melanin transfer. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729663 Generation of 3D skin equivalents from iPSC-derived fibroblasts, keratinocytes and melanocytes As reported previously, we have successfully generated 3D skin equivalents from iPSC-derived fibroblasts and keratinocytes. In order PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19728767 to determine whether our iPSC-derived melanocytes could be incorporated into this model, we generated 3D skin equivalents from iPSCderived fibroblasts, keratinocytes and melanocytes and tissue architecture and cellular localization were determined by hematoxylin and eosin staining, Fontana-Masson staining and immunofluorescence. After 2 weeks at the air-liquid interface 3D skin equivalents consisting of iPSC-derived fibroblasts, keratinocytes and melanocytes exhibited normal tissue architecture as determined by H+E staining with a stratified and differentiated epidermis as Fig 5. 3D skin equivalents produced from iPSC-derived 
fibroblasts, keratinocytes and melanocytes have normal architecture and are functional. a) Hematoxylin and eosin, b) Ki67, c) gp-100, d) FontanaMasson, e) loricrin, f) keratin-1, g) keratin-14. Blue, dashed line. Inset c). Inset d). h & i) Control and forskolin-treated iPSCderived 3D skin equivalents, respectively, after 14 days at the air-liquid interface. j) Quantification of melanin increase in 3D skin equivalents in response to 40M forskolin. Scal
