One of the most effective and safe therapeutic methods for treating vitiligo, mixed autologous keratinocytes (KCs) and melanocytes (MCs) cultures have been used for autologous cell transplantation. However, the present transplantation method is faced with a problem that may require a large amount of skin tissue and keratinocytes have limited culture potency. We have found previously that human adipose derived stromal cells (hASCs) from aspirated fat tissue could be used in place of KCs and sufficient amounts of hASCs for transplantation could be obtained by small amount of aspirated fat tissue. The present investigation was determined the effect of ASCs on ex vivo expansion MCs for transplantation. In addition, we examined for a preservation conditions of MCs which have reported low recovery rates and a slowdown in growth after cryopreservation. Various conditions including ASCs ratio, incubation period, and additive materials for MCs cultivation was determined to improve the expansion ability of MCs. The growth rate of MCs colony was elevated 6.85 folds compared the previous conditions. These MCs showed a specific expression of immature melanocyte protein, Trp-2, but did not express the mature melanocyte proteins and markers (c-kit, CD133, and etc.) of mesenchymal stem cells that represents in ASCs feeder. Results in cryopreservation experiments were determined a preservation medium for MCs showing an increased recovery rates after thawing. The characteristics of MCs after cryopreservation using a designed medium were indicated consistent morphology and immunophenotype. In conclusion, ASCs as a feeder could be used in place of keratinocytes for ex vivo expansion of MCs. For clinical trial for vitiligo patients, efficiency experiments in preclinical state should be followed.

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. They are widely distributed in the human diet through crops, beans, fruits, vegetables and red wine. The specific health effects that anthocyanins might have in vivo are not known, although there are several possibilities related to obesity, cardiovascular disease, and cancer. In this study we used human subcutaneous adipose mesenchymal stem cells (hADSC) and mouse subcutaneous adipose mesenchymal stem cells (mADSC) to evaluate the effects of anthocyanins. And we examined the effect of cell activity and adipocyte differentiation by Cyanidin-3-O-glucoside (C3G), Delphinidin-3-ß-D-glucoside (D3G) that are among the anthocyanin family and black soybean extract. Using MTT assay method, we tested cellular metabolic activity. In mADSC, cell activity is significantly decreased by C3G and D3G (50 uM, 100 uM, and 200 uM), and black soybean extract (100 ug/ml and 200 ug/ml). In hADSC, cell activity is significantly decreased only by C3G (50 uM, 100 uM and 200 uM) unlike in mADSC. Cell activity is significantly increased of 100 uM D3G and black soybean extract (50 ug/ml, 100 ug/ml and 200 ug/ml). In mADSC, 50 uM C3G promoted differentiation into adipocyte but no effect in other concentration. D3G suppressed the differentiation of mADSC at 100 uM and 200 uM. 50 ug/ml black soybean extract promoted differentiation of mADSC, but 200 ug/ml black soybean extract suppressed differentiation. In hADSC, 50 uM, 100 uM and 200 uM C3G suppressed differentiation. 100 uM D3G promoted differentiation into adipocyte, but 200uM D3G suppressed it. Black soybean extract suppressed the differentiation into adipocyte at 50 ug/ml, 100 ug/ml and 200 ug/ml. These data showed that the responsibility to the C3G and D3G were different between hADSC and mADSC. Interestingly the responsibility to the black soybean extract was similar between hADSC and mADSC. Based on them, it is suggested that there are species-specificity to the cellular responsibility to the anthocyanins in subcutaneous ADSC.

Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and 2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

최근 골수와 혈액으로 유래된 중간엽 줄기세포와 비슷한 능력을 가지는 것으로 알려진 지방 유래 중간엽줄기세포가 새로운 세포 치료제로 떠오르고 있다. 하지만 줄기세포를 이용하여 치료하려는 질병은 나이가 들어감에 따라 발병하는 퇴행성 질환들이 대부분인데, 노화가 진행됨에 따라 줄기세포의 능력이 차이가 있다고 알려져 있다. 이에 본 연구에서는 노화가 일어남에 따라 발생되는 신경성 질환을 자가 유래 지방 중간엽 줄기세포를 이용하여 치료함에 있어서 노화가 진행됨에