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Regulation of adhesion molecules and basic fibroblast growth factor 2 in non-segmental vitiligo-derived primary melanocytes

Published:November 03, 2022DOI:https://doi.org/10.1016/j.jdermsci.2022.10.005
      Non-segmental vitiligo patients reportedly have a significantly higher rate of relapse than patients with segmental vitiligo due to the poor proliferation and quality of the autologous melanocytes [
      • Kawakami T.
      Surgical procedures and innovative approaches for vitiligo regenerative treatment and melanocytorrhagy.
      ]. Primary melanocytes isolated from non-depigmented skin of the forearm in two Japanese patients with non-segmental vitiligo were difficult to grow in relation to culture maintenance. Adding Rho-associated protein kinase (ROCK) inhibitor to the culture medium dramatically increased the yield of pure melanocytes (Fig. 1A). ROCK inhibitor can enhance the efficiency and yield of human primary melanocyte in culture. We established a human non-segmental vitiligo-derived primary melanocyte-keratinocyte co-culture system according to our previous experiments [
      • Kawakami T.
      • Komatsu T.
      • Yokoyama K.
      • Iwama E.
      • Dong Y.
      Establishment of co-culture of human induced pluripotent stem (iPS) cell-derived melanocytes and keratinocytes in vitro.
      ]. The study protocol was approved by the ethics committee of Tohoku Medical and Pharmaceutical University (2021–2–160).
      Fig. 1
      Fig. 1(A) Human primary melanocytes from non-depigmented skin in a patient with non-segmental vitiligo and enhanced by ROCK inhibitor, Primary melanocytes isolated from non-depigmented skin of the forearm of a 71-year-old Japanese female patient and a 72-year-old Japanese male patient with a one-week history of non-segmental vitiligo using ROCK inhibitor. ROCK inhibitor enhanced melanocyte growth during culture. Melanocytes had bipolar or tripolar dendrites, slight discrepancy in size, smaller cell body and strong light refraction of cytoplasm after ROCK inhibitor addition., Scale bar = 100 µm(B) RT-qPCR analysis of melanocyte-specific markers in vitiligo-derived primary melanocytes and co-culture of human primary melanocytes and normal human adult keratinocytes, RT-qPCR analysis of vitiligo-derived primary melanocytes revealed significantly decreased mRNA levels of MITF compared to normal melanocytes. Tyrosinase, TYRP1, and SCF mRNA expression levels in vitiligo-derived primary melanocytes were similar to normal melanocytes (upper graphs). In contrast, mRNA expression levels of SCF were significantly higher in co-culture of vitiligo-derived primary melanocytes and normal keratinocytes than in co-culture of normal melanocytes and normal keratinocyte (lower graphs). MITF, tyrosinase, and TYRP1 mRNA expression levels in co-culture of vitiligo-derived primary melanocytes and normal keratinocytes were similar to co-culture of normal melanocytes and normal keratinocytes. Black bars indicate normal melanocytes only and white bars indicate the others. (C) RT-qPCR analysis of melanocyte related adhesion molecules in vitiligo-derived primary melanocytes and co-culture of human primary melanocytes and normal human adult keratinocytes RT-qPCR analysis of melanocyte related adhesion molecules (E-cadherin, DDR1, GPNMB, and HSD17β1) in vitiligo-derived primary melanocytes revealed significantly decreased mRNA levels compared to normal melanocytes (upper graphs). In contrast, mRNA expression levels of melanocyte related adhesion molecules were significantly higher in co-culture of vitiligo-derived primary melanocytes and normal keratinocytes than in co-culture of normal melanocytes and normal keratinocytes (lower graphs). Black bars indicate normal melanocytes only and white bars indicate the others.
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