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.

Human mesenchymal stem cells are known that multipotent stromal cells have the ability to divide asymmetrically, differentiate into many tissue types, and modulate cellular fate or function. Previous reports have proved that direct or indirect effects of mesenchymal stem cells in damaged cells or tissue were able to contribute to regenerative remodeling. One of incurable diseases, vitiligo is a depigmenting skin disorder resulting from the loss of melanocytes in the epidermis. Although vitiligo is a common disorder with a frequency of 0.1~2% in population, it still remains incurable and recurrent. Up to now, various treatment methods has been available for vitiligo therapy. Especially, transplantation of melanocytes (MCs) cultured with keratinocytes (KCs) is well-known therapy in clinic. We have recently reported functional role of adipose-derived stromal cells (ASCs) could assist MCs growth and maintenance of immature MCs. Therefore, the present study investigated whether the influence of ASCs may be elevated a transplantation yield of MCs in vivo. Transplantation was accomplished by syringe injection or grafting after dermabrasion. The procedure of dermabrasion is a mechanically invasive skin planning method and may be to help settle adequate location of transplanted cells to therapy. To improve an efficacy of cell transplantation, various additives or conditions of ratio were compared in vivo. These data was concluded that mixture of MCs and ASCs in the determined condition was improved engraftments of melanocytes for patients with vitiligo.

Glycogen synthase kinase 3 beta (GSK3β)의 선택적 저해제인 Kenpaullone가 B16 멜라노마 및 사람의 멜라노 사이트에 미치는 멜라닌 합성능을 조사하였다. Kepaullone은 B16 멜라노마 및 사람의 멜라노사이트에 대하여 세포증식에는 영향이 없는 범위 내에서 농도 의존적으로 멜라닌 합성을 촉진시켰다. B16 멜라노마 세포에 Kenpaullone을 첨가 48 h 후 tyrosinase 활성이 증가하였으며, 농도별 처리에 대하여 tyrosinase 단백질의 발현 및 tyrosinase mRNA양이 증가함을 관찰하였다. 결론적으로 Kenpaullone는 B16 멜라노마 세포에서 tyrosinase 효소의 발현을 증가시켜 멜라닌 합성을 촉진하는 것으로 판단되어진다. 따라서 GSK3β 저해제가 멜라닌 합성을 촉진시키는 결과는 백반증과 같은 저색소관련 질병의 치료제 개발의 가능성을 갖고 있는 소재로서 응용가능하리라고 판단되어진다.