acal silver solution for 1 hour at 37C. After further washing the cells twice with dH2O, they were examined by light microscopy using a Zeiss Axiovert 25 microscope. For staining of 3D skin equivalents the Fontana-Masson staining kit was used exactly as described in the manufacturer’s instructions. Quantification of Fontana-Masson staining was calculated using the analyze particles function of ImageJ. We counted 27 cells for each condition, randomly taken from 3 microscopic fields in 3 different experiments, and values are expressed as the mean value SEM. Statistical analysis was performed using the Student’s t test and significance level has been identified as p<0.05. Co-Culture Experiments For co-culture studies using normal or iPSC-derived melanocytes with normal or iPSC-derived keratinocytes; keratinocytes were first seeded onto gelatin-coated eight-well chamber slides at a cell density of 3.6 x 104 cells/well in CnT-07 media. 24 hours later 4 x 103 melanocytes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19731986 were then added to each well in CnT-07 media. The co-cultures were left for a further 7 or 24 hours before being examined by immunofluorescence. Generation of 3D Skin Equivalents For melanocyte localization experiments, 3D skin equivalents were generated by adapting a previously described protocol. Briefly, a type I collagen matrix was deposited onto polyethylene terephthalate membranes, and allowed to polymerize. After incubation of the polymerized matrix for 7 days, 1 x 106 iPSC-derived keratinocytes and 0.1 x 106 iPSC-derived melanocytes were seeded onto the matrix, and incubated for a further 7 days. The composite culture was raised to the 
air-liquid interface and fed from below to induce epidermal differentiation. 3D skin equivalents were harvested 14 days later and either snap frozen in LN2 or embedded in wax. For melanin quantification studies involving 40 M forskolin where we were aiming to determine the functionality of our iPSC-derived melanocytes in a 3D environment, the Nigericin (sodium salt) chemical information protocol was modified such that forskolin was added to the cell culture media at the same time as the epidermal cells were added to the skin construct and that the ratio of iPSC-derived keratinocytes to iPSCderived melanocytes was 1:1. Although this ratio was unlikely to result in a physiological number of melanocytes at the basal layer of the epidermis, these conditions have previously been shown to produce optimal results when studying the effects of drugs on melanogenesis in conventional 3D skin equivalents. 6 / 16 Pigmented Induced Pluripotent Stem Cell-Derived Skin Models Hematoxylin and Eosin Staining Following dewaxing, 7m sections were stained with Mayer’s Hematoxylin at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19730234 RT for 3 minutes. Blueing was achieved by rinsing in tap water while differentiation was achieved by rinsing in 1% acid ethanol. Counterstaining was achieved by rinsing with eosin for 30 seconds while dehydration was achieved by sequential washing with 95% ethanol, 100% ethanol and Histo-Clear. Slides were coverslipped with DPX and examined by light microscopy using a Zeiss Axioplan 2 microscope. Melanin Assay Quantification of melanin in iPSC-derived 3D skin equivalents was performed as described previously. Briefly, frozen 3D skin constructs were thawed at room temperature and melanin was extracted using Solvable. A 1 mg/ml melanin standard stock solution was prepared by dissolving synthetic melanin in Solvable and a series of melanin standards were prepared from the stock. Solvable was used as the 0.0 mg/ml standard. The