Adult skin specimens obtained from repeat Cesarean section deliveries and circumcisions were used for the cultures after obtaining approval from by the Institutional Review Board of the Dongguk University Ilsan Hospital (Approval number; 2012-69). The epidermis was separated from the dermis after treatment with 2.4 U/mL of dispase (Roche, Mannheim, Germany) for 1 hour. The epidermal sheets were treated with 0.05% trypsin for 10 minutes to produce a suspension of individual epidermal cells. The cells were suspended in Medium 254 (Cascade Biologics, Portland, OR, USA) supplemented with fetal bovine serum (FBS), bovine pituitary extract, bovine insulin, hydrocortisone, basic fibroblast growth factor (bFGF), bovine transferrin, heparin, and phorbol 12-myristate 13-acetate (Cascade Biologics). The concentration of FBS was 0.5%. When the cells reached 80% confluency, they were detached from the flask and seeded into other culture flasks either in preparation for an experimental procedure, or to maintain and propagate the cell culture. For the following experiments, the concentration of all supplements in the Medium 254 was reduced to 20% (supplement-starved Medium 254), with 0.1% FBS.

Human ADSCs were kindly provided as a gift from Dr. Byungrok Do of Hurim BioCell Inc. (Seoul, South Korea). The cells were cultured in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Life Technology, NY, USA) supplemented with 10% FBS (Gibco/BRL), 100 U/mL of penicillin (Gibco/BRL), and 0.1 mg/mL of streptomycin (Gibco/BRL).

For culture with melanocytes, ADSCs were dissociated with trypsin/EDTA solution and resuspended in low glucose DMEM (Invitrogen). The cells were counted by automatic cell counter, Countess? (Invitrogen) and were plated at 5×10⁴ cells in 25 cm² tissue culture flasks (NUNC, Roskilde, Denmark). Meanwhile, melanocytes were harvested by TrypLE^TM Express (Invitrogen) and then were added in tissue flasks containing ADSCs at a density of 2.5×10⁴ cells. The co-culture cells were cul tured by standard medium of melanocytes, Medium 254 with 1X HMGS (Invitrogen), for two weeks and the medium was generally changed every two days.

8-week-old female BALB/c nude mice (Orient Bio Inc., Korea) and 4-week-old female Sprague-Dawley (SD) rats (Orient Bio Inc., Korea) were utilized in this study. The mice and rats were acclimatized for 1 week for stress relief and pre-feeding purposes. After acclimatization, the animals were anesthetized through intraperitoneal injection of Zoletyl and Lumpen. In case of SD rats, the hair on the dosal lateral back was shaved using an electric shaver. Then, the mice and rats were marked with an approximately 1×1 cm² area, on the dorsal lateral back, using a pen. Dermabrasion was accomplished using a small felt wheel attached to a hand-piece motor (Saeshin Strong 204, Korea) and operated at 5,000 rpm. To improve the grafting efficiency, melanocytes alone, ADSCs alone or cocultures of these cells were treated with fibrinogen solution (Green cross, Korea; 501A13015; 10 mg/ml) and thrombin solution (Green cross; 501A13020; 50 IU/ml) prior to the application of the dermabraded area. After 5 minutes, the area was covered with a Tegaderm^TM Film.

Mice were euthanized and the skin of the dermabraded area was removed and placed in 10% buffered formalin solution, and embedded in paraffin blocks. The paraffin-embedded skin samples were sectioned to 4 μm thickness, and then stained with hematoxylin and eosin (H&E). The cell density was expressed as the number of cells per 5 high-power fields (×400) for each section.

The statistical analysis of the experimental data was performed using the Student’s t-test. The results were expressed as the mean ± S.D. A p-value <0.05 was considered significant.

Based on the in vitro finding that 2.5×103 melanocytes/mL and 1.5×10⁴ ADSCs/mL (mitomycin C-treated) were required to be seeded for coculturing (Kim et al., 2012), 1:1 and 1:10 cell mixtures of melanocytes to ADSCs were preliminary examined for grafting in nude mice. Because the result was sig nificantly better with the 1:1 cell mixture (data not shown), tra nsplantation of cultured cell mixture to nude mice was performed using ratios of melanocytes to ADSCs as 1:3, 1:1, and 3:1. A group of melanocyte monocultures and that of ADSCs were used as positive and negative controls, respectively. The number of melanocytes was examined in each group composed of 4 mice weekly for 4 weeks. Light microscopic examination in skin specimens stained with H&E suggested that the number of melanocytes was not increased in all groups, until 3-week post-grafting. After 3 weeks, the melanocyte number was increased, particularly in the groups of 1:1 and 1:3 mixtures (Fig. 1A, B). Although cultured cells were grafted on the skin after removal of epidermis by dermabrasion alone, melanocytes were detected in epidermis, upper dermis, and middermis at 1-week and 2-week post-transplantation, respectively (Fig. 1C). No inflammatory cells infiltrated the epidermis and dermis (Fig.1A, C).

It was uncertain whether transplantation of cocultured melanocytes and ADSCs could result in an outcome similar to the transplantation of each of the cultured cell (Fig.1A, B). However, grafting of melanocytes and ADSCs cocultured for 2 weeks at ratios of 1:1, 1:2, and 1:3 showed more melanocytes at the ratios of 1:1 and 1:2 (data not shown). Therefore, the number of melanocytes was examined on grafting the cocultured cells at the ratios of 1:1 and 1:2 in nude mice for 3 weeks. A group of melanocyte monocultures and that of ADSCs were used as positive and negative controls, respectively. The number of mice in each group was 5. The number of melanocytes was significantly increased by grafting of cocultured cells from 1-week to 3-week post-examination (Fig. 2A). No significant differences in melanocyte number were detected between the cocultures at 1:1 and 1:2 (Fig. 2A).

To examine an appropriate duration of cocultures, melanocytes and ADSCs were cocultured at the fixed ratio of 1:2, for 1 and 2 weeks. The cells were then transplanted to nude mice. A group of melanocyte monocultures and that of ADSCs were used as positive and negative controls, respectively. The number of mice in each group was 5. The number of melanocytes, determined by microscopy, at 2-week post-transplantation, was significantly increased in the group grafted with cocultured cells. No significant difference was observed between the 1-week and 2-week durations, although the melanocyte number increased a little more in 2-week cocultures (Fig. 2B). No melanocytes were detected in other tissues or organs (Fig. 2C).

No significant effect of the culture duration was observed on the melanocyte number in nude mice (Fig. 2B). Therefore, melanocytes and ADSCs cocultured at the fixed ratio of 1:2 for 2 weeks were grafted in SD rats, and the melanocyte number was examined at 1-week and 2-week post-grafting. A group of melanocyte monocultures and that of ADSCs were used as positive and negative controls, respectively. The number of rats in each group was 5. Similar to the result in nude mice, the number of melanocytes was significantly increased in the group grafted with cocultured cells at 1-week and 2-week post-grafting (Fig. 3).

To examine the lasting duration of grafted cells in nude mice skin, melanocytes and ADSCs cocultured for 1 week at the 1:2 ratio were grafted in nude mice, and melanocyte number was subsequently examined at a 2-week interval, up to 12 weeks. A group of melanocyte monocultures and that of ADSCs were used as positive and negative controls, respectively. The number of mice in each group was 5. The number of melanocytes was increased up to 4 weeks after the grafting of either cocultured cells or monocultured melanocytes, despite more cells in the group of cocultures. The melanocyte number at 6-week post-grafting was not so low as compared to that at 2-week and 4-week post-grafting, however, it was suddenly decreased thereafter, without remaining detectable melanocytes at 8-week of grafting, or later (Fig. 4).