Analysis of how obtaining melanocytes by magnetic cell separation contributes to autoepidermal transplantation technology in treating leucoderma.

Abstract

Leucoderma, a depigmentation of the skin, is a common disease in humans, and has been observed in cattle, horses and buffalo as well. To analyze the correlation between melanin stem cells and the differentiation and proliferation of melanocytes (MCs). Magnetic cell separation was used to separate melan-A+ cells and PAX3+ cells, which were cultured in vitro. The L-DOPA staining was used to observe cell morphology; Cell Counting Kit-8 (CCK8) was used to determine the cell proliferation rate; and flow cytometry (FCM) was used to determine cell cycle changes. The relative mRNA levels of melanocyte-inducing transcription factor (MITF), dopachrome tautomerase (DCT) and melan-A in cells were determined with reverse-transcription polymerase chain reaction (RT-PCR). The number of MC dendrites increased and extended continually during in vitro culture following magnetic cell separation. The proportion of positive L-DOPA staining cells increased from a baseline 40.70% to 82.03%, and the cell proliferation rate increased from 335.0% at D3 to 1577.4% at D20. The results of FCM showed that the cell proportion at the G1 stage in the D20 group was significantly lower than the D3 group; the cell proportion at the G2/M stage also decreased significantly. The expression of MITF and melan-A increased as the culture time increased, while the expression of DCT decreased. The number of MC stem cells decreased and mature MCs increased gradually, indicating that MC stem cells can gradually differentiate into mature MCs during in vitro culture following magnetic cell separation.