Approach for the Derivation of Melanocytes from Induced Pluripotent Stem Cells  Tamihiro Kawakami, Tatsuro Okano, Sora Takeuchi, Kayoko Osumi, Yoshinao Soma, Munenari Itoh, Tomohisa Hirobe, Kowichi Jimbow  Journal of Investigative Dermatology  Volume 138, Issue 1, Pages 150-158 (January 2018) DOI: 10.1016/j.jid.2017.07.849 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Generation of human epidermal melanocytes derived from human iPS cells. (a) Human iPS cell-derived melanocytes appear as black dots and slightly black areas in iDMM on 60-mm gelatinous dishes coated with Matrigel. (b) High concentrations of mature human iPS cell-derived melanocytes were generated from the border of human iPS cell nests. Scale bar =100 μm. (c) Human iPS cell-derived melanocytes containing many melanin granules in the cytoplasm. Scale bar =100 μm (left), 20 μm (right). (d) Human iPS cell-derived melanocytes fixed on coverslips, stained with an antityrosinase antibody (green) and imaged by immunofluorescence microscopy. Scale bar =20 μm. (e) RT-PCR analysis of human iPS cell-derived melanocytes. Black bars show differences among human iPS cells and normal human adult epidermal HEMa-LP melanocytes under various conditions. RT-PCR analysis of human iPS cell-derived melanocytes revealed significantly decreased mRNA levels of the pluripotency gene NANOG compared with human iPS cells. In contrast, the mRNA expression levels of melanocyte-specific markers (MITF, tyrosinase, and TYRP1) were significantly higher in human iPS cell-derived melanocytes than in human iPS cells. NANOG, MITF, tyrosinase, and TYRP1 mRNA expression levels in human iPS cell-derived melanocytes were similar to HEMa-LP cells. (f) Human iPS cell-derived melanocytes reached confluence in 60-mm gelatinous dishes coated with Matrigel in 8 weeks. We found many colonies and individual cells on the dishes. (g) Two black colonies from confluent plates adhered and were growing in a new human iPS cell-derived melanocyte culture on Matrigel. Melanocytes in (g) were picked up from (f). The two colonies are shown as enlarged views in the insets. Scale bar =100 μm. (h) Pigmented cells retrieved from human iPS cell-derived melanocyte medium adhered and grew to slightly black areas with new human iPS cell-derived melanocytes on Matrigel (left). The presence of human iPS cell-derived melanocytes induced using pipetted colonies and retrieved medium was triggered to increase new melanocyte proliferation from human iPS cells on the gelatinous dishes coated with Matrigel (right). Pigmented cells in the black areas are shown as enlarged views in the insets. (h) Human iPS cell-derived melanocyte was cultured for 9 days after retrieving the precipitated melanocytes in the medium. Scale bar =100 μm. (i) Numerous human iPS cell-derived melanocytes were seen in each 60-mm-diameter gelatinous dish coated with Matrigel after 12 days from pipetting colonies and retrieving medium. HEMa-LP, human epidermal melanocytes adult-lightly pigmented; iDMM, iPS cell-derived melanocyte medium; iPS, induced pluripotent stem; MITF, microphthalmia-associated transcription factor; RT-PCR, reverse transcription PCR; TYRP1, tyrosinase-related protein 1. Journal of Investigative Dermatology 2018 138, 150-158DOI: (10.1016/j.jid.2017.07.849) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Generation of human epidermal melanocytes derived from human iPS cells. (a) Human iPS cell-derived melanocytes appear as black dots and slightly black areas in iDMM on 60-mm gelatinous dishes coated with Matrigel. (b) High concentrations of mature human iPS cell-derived melanocytes were generated from the border of human iPS cell nests. Scale bar =100 μm. (c) Human iPS cell-derived melanocytes containing many melanin granules in the cytoplasm. Scale bar =100 μm (left), 20 μm (right). (d) Human iPS cell-derived melanocytes fixed on coverslips, stained with an antityrosinase antibody (green) and imaged by immunofluorescence microscopy. Scale bar =20 μm. (e) RT-PCR analysis of human iPS cell-derived melanocytes. Black bars show differences among human iPS cells and normal human adult epidermal HEMa-LP melanocytes under various conditions. RT-PCR analysis of human iPS cell-derived melanocytes revealed significantly decreased mRNA levels of the pluripotency gene NANOG compared with human iPS cells. In contrast, the mRNA expression levels of melanocyte-specific markers (MITF, tyrosinase, and TYRP1) were significantly higher in human iPS cell-derived melanocytes than in human iPS cells. NANOG, MITF, tyrosinase, and TYRP1 mRNA expression levels in human iPS cell-derived melanocytes were similar to HEMa-LP cells. (f) Human iPS cell-derived melanocytes reached confluence in 60-mm gelatinous dishes coated with Matrigel in 8 weeks. We found many colonies and individual cells on the dishes. (g) Two black colonies from confluent plates adhered and were growing in a new human iPS cell-derived melanocyte culture on Matrigel. Melanocytes in (g) were picked up from (f). The two colonies are shown as enlarged views in the insets. Scale bar =100 μm. (h) Pigmented cells retrieved from human iPS cell-derived melanocyte medium adhered and grew to slightly black areas with new human iPS cell-derived melanocytes on Matrigel (left). The presence of human iPS cell-derived melanocytes induced using pipetted colonies and retrieved medium was triggered to increase new melanocyte proliferation from human iPS cells on the gelatinous dishes coated with Matrigel (right). Pigmented cells in the black areas are shown as enlarged views in the insets. (h) Human iPS cell-derived melanocyte was cultured for 9 days after retrieving the precipitated melanocytes in the medium. Scale bar =100 μm. (i) Numerous human iPS cell-derived melanocytes were seen in each 60-mm-diameter gelatinous dish coated with Matrigel after 12 days from pipetting colonies and retrieving medium. HEMa-LP, human epidermal melanocytes adult-lightly pigmented; iDMM, iPS cell-derived melanocyte medium; iPS, induced pluripotent stem; MITF, microphthalmia-associated transcription factor; RT-PCR, reverse transcription PCR; TYRP1, tyrosinase-related protein 1. Journal of Investigative Dermatology 2018 138, 150-158DOI: (10.1016/j.jid.2017.07.849) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Pigmentation changes in the nude mouse skin after injection of human melanocytes derived from iPS cells. (a) Human iPS cell-derived melanocytes pelleted by centrifugation on the bottom of the tube (left) and in the medium (right) appear black. (b) Blue to black pigmentation was found in the dorsal skin of each nude mouse that was consistent with the inoculation site on day 3. 1 × 106 cells/ml inoculation (left) and 3 × 106 cells/ml inoculation (right). (c) Postmortem examination disclosed black masses in the dorsal skin of the nude mouse consistent with the inoculation site. (d) Fontana-Masson staining revealing melanocyte aggregates in the pigmented lesion sections. Original magnification ×100 (left) and ×400 (right). Scale bar = 50 μm. (e) Melanocyte aggregates in the dorsal skin of the inoculated nude mouse were positive for anti-Melan-A immunostaining. Inoculated human iPS cell-derived melanocytes positive for staining with Melan-A antibodies were seen throughout the skin including the dermis and subcutaneous fat tissue (left) and at the dermal-epidermal junction and epidermis (right). Original magnification ×400 (left) and ×1000 (right). Scale bar = 50 μm. (f) Fontana-Masson staining revealed no melanocytes at the inoculation site (left). Immunohistochemical staining with anti-Melan-A antibodies revealed dermal melanocytes at the inoculation site (right). Original magnification ×200 (left and right). Scale bar = 50 μm. (g) Immunohistochemical staining with anti-Melan-A antibodies showed that the transplanted melanocytes appeared to migrate from the upper dermis to the epidermis. Original magnification ×1,000. Scale bar = 50 μm. iPS, induced pluripotent stem. Journal of Investigative Dermatology 2018 138, 150-158DOI: (10.1016/j.jid.2017.07.849) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Pigmentation changes in the nude mouse skin after injection of human melanocytes derived from iPS cells. (a) Human iPS cell-derived melanocytes pelleted by centrifugation on the bottom of the tube (left) and in the medium (right) appear black. (b) Blue to black pigmentation was found in the dorsal skin of each nude mouse that was consistent with the inoculation site on day 3. 1 × 106 cells/ml inoculation (left) and 3 × 106 cells/ml inoculation (right). (c) Postmortem examination disclosed black masses in the dorsal skin of the nude mouse consistent with the inoculation site. (d) Fontana-Masson staining revealing melanocyte aggregates in the pigmented lesion sections. Original magnification ×100 (left) and ×400 (right). Scale bar = 50 μm. (e) Melanocyte aggregates in the dorsal skin of the inoculated nude mouse were positive for anti-Melan-A immunostaining. Inoculated human iPS cell-derived melanocytes positive for staining with Melan-A antibodies were seen throughout the skin including the dermis and subcutaneous fat tissue (left) and at the dermal-epidermal junction and epidermis (right). Original magnification ×400 (left) and ×1000 (right). Scale bar = 50 μm. (f) Fontana-Masson staining revealed no melanocytes at the inoculation site (left). Immunohistochemical staining with anti-Melan-A antibodies revealed dermal melanocytes at the inoculation site (right). Original magnification ×200 (left and right). Scale bar = 50 μm. (g) Immunohistochemical staining with anti-Melan-A antibodies showed that the transplanted melanocytes appeared to migrate from the upper dermis to the epidermis. Original magnification ×1,000. Scale bar = 50 μm. iPS, induced pluripotent stem. Journal of Investigative Dermatology 2018 138, 150-158DOI: (10.1016/j.jid.2017.07.849) Copyright © 2017 The Authors Terms and Conditions