Optimization of the method for the culture of melanocyte precursors from hair follicles and their activation by 1,25-dihydroxyvitamin D3
Open Access
- 6 August 2013
- journal article
- Published by Spandidos Publications in Experimental and Therapeutic Medicine
- Vol. 6 (4), 967-972
- https://doi.org/10.3892/etm.2013.1252
Abstract
The melanocytes in vitiligo repigmentation are derived predominantly from melanocyte precursors (MPs) present in the outer root sheath (ORS) of hair follicles. The methods currently used for culturing MPs are unstable, and the cultured cells have the capacity to produce melanin. These factors are problematic when conducting in vitro studies to investigate the mechanism of repigmentation. Although 1,25‑dihydroxyvitamin D3 (VID) has been demonstrated to be highly effective in the treatment of vitiligo in the clinic, its precise mode of action has yet to be elucidated. In the present study, the method for the culture of MPs from the ORS of hair follicles was optimized and the ability of VID to activate MPs was investigated. The results suggested that the MPs cultured using the optimized method mainly exhibited bipolar morphology. The cells proliferated well and were negative for 3,4-dihydroxy‑L‑phenylalanine (DOPA) staining. Transmission electron microscopy revealed that the cytoplasm of the MPs contained numerous stage I and stage II melanosomes; however, stage III and IV melanosomes were not observed. Following VID treatment, the MPs showed increased dendritic morphology, the cells stained positive for DOPA and stage III and IV melanosomes appeared in the cells. Western blotting revealed that microphthalmia‑associated transcription factor (MITF), tyrosinase (TYR), tyrosinase‑related protein‑1 (TRP‑1) and TRP‑2 were expressed in the MPs and that VID increased the expression levels of MITF, TYR and TRP‑1. However, the levels of MITF, TYR and TRP‑1 in the MPs prior to and following VID treatment were significantly lower compared with those in cultured epidermal melanocytes, while the levels of TRP‑2 in these three groups were not significantly different. Subsequent to VID treatment, the TYR activity in the MPs increased significantly, as did the corresponding melanin levels. In conclusion, the present study successfully optimized the method for MP culture. The MPs demonstrated no significant TYR activity or melanin synthesis; therefore, the MP cultures exhibited the features of MPs in vivo. In addition, VID significantly promoted the differentiation of MPs.Keywords
This publication has 18 references indexed in Scilit:
- Prostaglandin D2 production in FM55 melanoma cells is regulated by α-melanocyte-stimulating hormone and is not related to melanin productionExperimental Dermatology, 2009
- Isolation and Culture of Amelanotic Melanocytes from Human Hair FolliclesPigment Cell Research, 2004
- Human Melanoblasts in Culture: Expression of BRN2 and Synergistic Regulation by Fibroblast Growth Factor-2, Stem Cell Factor, and Endothelin-3Journal of Investigative Dermatology, 2003
- Differentiation of Murine Melanocyte Precursors Induced by 1,25-Dihydroxyvitamin D3 Is Associated with the Stimulation of Endothelin B Receptor ExpressionJournal of Investigative Dermatology, 2002
- Correlation between the number of melanosomes, tyrosinase mrna levels, and tyrosinase activity in cultured murine melanoma cells in response to various melanogenesis regulatory agentsJournal of Cellular Physiology, 1995
- Isolation and Long-Term Culture of Human Hair-Follicle MelanocytesJournal of Investigative Dermatology, 1995
- Hormonal effects of vitamin D3 on epidermal melanocytesJournal of Cellular Physiology, 1988
- Vitamin D, its Precursors, and Metabolites do not Affect Melanization of Cultured Human MelanocytesJournal of Investigative Dermatology, 1988
- Stimulation of Human Melanocytes by Vitamin D3 Possibly Mediates Skin Pigmentation After Sun ExposureJournal of Investigative Dermatology, 1988
- Stimulation of melanogenesis in cultured melanoma cells by calciferolsFEBS Letters, 1974