Optimization of the method for the culture of melanocyte precursors from hair follicles and their activation by 1,25-dihydroxyvitamin D3

Wang,Daguang; Xu,Xihui; Ma,Huijun; Yue,Xuezhuang; Li,Chengrang; Zhu,Wenyuan

doi:10.3892/etm.2013.1252

October 2013 Volume 6 Issue 4

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October 2013 Volume 6 Issue 4

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Article

Optimization of the method for the culture of melanocyte precursors from hair follicles and their activation by 1,25-dihydroxyvitamin D3

Authors:
- Daguang Wang
- Xihui Xu
- Huijun Ma
- Xuezhuang Yue
- Chengrang Li
- Wenyuan Zhu
View Affiliations / Copyright

Affiliations: Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China, Department of Hematology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
Pages: 967-972
|
Published online on: August 6, 2013

https://doi.org/10.3892/etm.2013.1252
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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.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Optimization of the method for the culture of melanocyte precursors from hair follicles and their activation by 1,25-dihydroxyvitamin D3

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