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. 2014 Jul;22(4):328-33.
doi: 10.4062/biomolther.2014.065.

Adipose-derived stem cells improve efficacy of melanocyte transplantation in animal skin

Won-Suk Lim  1 , Chang-Hyun Kim  1 , Ji-Young Kim  1 , Byung-Rok Do  2 , Eo Jin Kim  3 , Ai-Young Lee  1
Affiliations

Adipose-derived stem cells improve efficacy of melanocyte transplantation in animal skin

Won-Suk Lim et al. Biomol Ther (Seoul). 2014 Jul.

Abstract

Vitiligo is a pigmentary disorder induced by a loss of melanocytes. In addition to replacement of pure melanocytes, cocultures of melanocytes with keratinocytes have been used to improve the repigmentation outcome in vitiligo treatment. We previously identified by in vitro studies, that adipose-derived stem cells (ADSCs) could be a potential substitute for keratinocytes in cocultures with melanocytes. In this study, the efficacy of pigmentation including durability of grafted melanocytes and short-term safety was examined in the nude mouse and Sprague-Dawley rat after grafting of primary cultured human melanocytes, with or without different ratios of primary cultured human ADSCs. Simultaneous grafting of melanocytes and ADSCs, which were separately cultured and mixed on grafting at the ratios of 1:1, 1:2, or 1:3, showed better efficacy than that of pure melanocytes. Grafting of melanocytes cocultured with ADSCs resulted in a similar outcome as the grafting of cell mixtures. Skin pigmentation by melanocytes : ADSCs at the ratios of 1:1 and 1:2 was better than at 1:3. No significant difference was observed between the 1-week and 2-week durations in coculturing. Time-course microscopic examination showed that the grafted melanocytes remained a little longer than 6-week post-grafting. No inflammatory cell infiltration was observed in the grafted skin and no melanocytes were detectable in other organs. Collectively, grafting of melanocytes and ADSCs was equally safe and more effective than grafting of melanocytes alone. Despite the absence of significant differences in efficacy between the group of 1:1 and that of 1:2 ratio, 1:2 ratio for 1-week coculturing may be better for clinical use from the cost-benefit viewpoint.

Keywords: Adipose-derived stem cells; Animal; Efficacy; Grafting; Melanocytes; Short-term safety.

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Figures

Fig. 1.
Fig. 1.
Simultaneous grafting of melanocytes, with or without ADSCs in nude mice. (A) Light microscopy of skin specimens stained with H&E (×400). Skin was grafted with indicated cells, such as melanocytes alone (a, 3.3×104 cells), melanocytes to ADSCs ratio as 3:1 (b), 1:1 (c) and 1:3 (d) for 2 weeks. Scale bar=200 μm (B) Relative number of melanocytes, which was determined by calculating the ratio of melanocyte number cultured in each condition to that cultured melanocyte alone for 1 week, for 4 weeks after grafting of melanocytes, with or without ADSCs as indicated ratios. The number of melanin-containing melanocytes was counted in 5 random microscopic fields (high power, ×400) for statistical analysis. The data represent the mean ± SD of 5 nude mice. *p<0.05 vs. melanocyte monoculture (C) H&E staining of skin specimens 1 week (left) and 2 weeks (right) after grafting of melanocytes alone to compare their residing location. ×400, Scale bar=200 μm.
Fig. 2.
Fig. 2.
(A) Number of melanocytes at 1 week (Aa), 2 weeks (Ab), and 3 weeks (Ac) after grafting of melanocytes-ADSCs cocultures vs. melanocyte monocultures in nude mice. The data represented the mean ± SD of 5 nude mice. *p<0.05 vs. melanocyte monoculture (B) Melanocyte number in nude mice skin at 2-week post-grafting of melanocytes alone (a, 1.0×105 cells), fixed ratio of melanocytes to ADSCs (b, 1:2), and ADSCs alone (c). The cells were cultured in vitro for 1 or 2 weeks. The data represent the mean ± SD of 5 nude mice. *p<0.05 vs. melanocyte monoculture (C) Light microscopy of different internal organs, such as lymph nodes (a), lung (b), liver (c), spleen (d), kidney (e), heart (f), and ovary (g) 12 weeks after melanocyte grafting.
Fig. 3.
Fig. 3.
Number of melanocytes at 1 week and 2 weeks after grafting of melanocytes-ADSCs cocultures at a fixed ratio (1:2) vs. melanocyte monocultures in SD rats. The cells were cultured in vitro for 2 weeks. The data represent the mean ± SD of 5 SD rats. *p<0.05 vs. melanocyte monoculture.
Fig. 4.
Fig. 4.
Durability of grafted cells in nude mice. The data represent the mean ± SD of 5 nude mice. *p<0.05 vs. melanocyte monoculture.
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