Dedifferentiation of human epidermal melanocytes in vitro by long-term trypsinization.

Human epidermal melanocytes can be induced to form melanocyte spheroids and revert to immature characteristics by long-term trypsinization (LTT). To further explore the biological characteristics of melanocytes after LTT and to study the underlying mechanism. Melanocytes were subjected to long-term (2 h) trypsinization in this study, after which their viability, proliferation and autophagy were characterized. The expression of melanocyte markers [human melanoma black45 (HMB45), tyrosinase (TYR) and Nestin] was detected and relative expression levels of mRNAs encoding TYR, Nestin, c-Kit and microphthalmia-associated transcription factor (MITF) were determined. After LTT, more short spindle-shaped melanocytes appeared and viability assays demonstrated that most melanocytes survived that treatment but had decreased proliferation rates compared to the untreated controls. There was a significant increase in autophagy of melanocytes after LTT and the expression of TYR was decreased compared with untreated control melanocytes. There were no significant differences in the expression of HMB45 or Nestin between the two groups. Compared with untreated melanocytes, levels of message ribonucleic acid (mRNAs) encoding TYR, c-Kit and MITF were decreased after LTT, while Nestin mRNA levels were increased. These results clarified that Long-term treatment with trypsin causes the dedifferentiation of mature epidermal melanocytes in vitro.

An unusual case of secondary syphilis misdiagnosed as allergic dermatitis for 2 years.

Dermatologists should be aware that the clinical manifestations of syphilis are very complex and changeable. Unilaterally distributed skin lesions and painless lip ulcers may also be the clinical manifestations of secondary syphilis.

The tolerance of human epidermal cells to trypsinization in vitro.

To characterize the tolerance of different types of human epidermal cells to trypsinization in vitro and develop a new method to separate and purify melanocytes according to their tolerance to trypsinization. Epidermal cells were obtained by separating the epidermis from human foreskins. Some of those cells were used for routine culture, and then were subjected to differential trypsin digestion. The remaining epidermal cells were resuspended in a 0.25% trypsin solution and then were neutralized by the addition of bovine serum at different time points. Immunofluorescence staining of HMB45, K15 and vimentin was used to identify melanocytes, keratinocytes and fibroblasts, respectively. We found that Keratinocytes, melanocytes and fibroblasts are primary cells obtained from conventional cultures of human skin. Purified keratinocytes and melanocytes can be obtained by conventional differential trypsin digestion, but fibroblasts in the melanocyte population quickly gain a survival advantage after passage. With longer trypsin digestion times, the number of adherent cells decreased, the time required for cell attachment increased, and the proportion of melanocytes increased. There were no obvious keratinocytes in cell populations obtained after 12 h of trypsinization of epidermal cells, and more short spindle-shaped melanocytes appeared, all of which were HMB45-positive. In conclusion, the tolerance of human epidermal melanocytes to trypsinization in vitro was better than epidermal keratinocytes, and that property can be used to purify melanocytes and was better than traditional differential trypsin digestion. The morphology of cells that survived the long-term trypsin digestion changed and they had good proliferative activity, but seemed to be more immature.

Characterization of Induced Pluripotent Stem Cells from Human Epidermal Melanocytes by Transduction with Two Combinations of Transcription Factors.

OBJECTIVE: In order to generate induced Pluripotent Stem Cells (iPSCs) more efficiently, it is crucial to identify somatic cells that are easily accessible and possibly require fewer factors for conversion into iPSCs. METHODS: Human epidermal melanocytes were transduced with lentiviral vectors carrying 3 transcription factors (OCT-4, KLF-4 and c-MYC, 3F) or 4 transcription factors (OCT-4, KLF-4, c-MYC and SOX-2, 4F). Once the clones had formed, assays related to stem cell pluripotency, including alkaline phosphatase staining, DNA methylation levels, expression of stem cell markers and ultrastructure analysis were carried out. The iPSCs obtained were then induced to differentiate into the cells representing the three embryonic layers in vitro. RESULTS: Seven days after the transduction of epidermal melanocytes with 3F or 4F, clones were formed that were positive for alkaline phosphatase staining. Fluorescent staining with antibodies against OCT-4 and SOX-2 was strongly positive, and the cells showed a high nucleus-cytoplasm ratio and active karyokinesis. No melanosomes were found in the cytoplasm by ultrastructural analysis. There were obvious differences in DNA methylation levels between the cloned cells and their parental cells. However, there was not a significant difference between 3F or 4F transfected clonal cells. Meanwhile, the iPSCs successfully differentiated into the three germ layer cells in vitro. CONCLUSION: Human epidermal melanocytes do not require ectopic SOX-2 expression for conversion into iPSCs, and may serve as an alternative source for deriving patient-specific iPSCs with fewer genetic elements.

Generation of Induced Pluripotent Stem Cells from Human Epidermal Keratinocytes.

Induced pluripotent stem cells (iPSCs) play an important role in cell replacement therapy. Several studies have shown that keratinocytes are promising reprogrammed cells. We easily and efficiently enriched epidermal stem cells by attaching them for a limited time in culture dishes. Individual epidermal cells enriched in stem cells, which showed strong immunostaining for K15, were obtained and generated iPSCs within 10 days after transfection with lentiviruses encoding 4 transcription factors (OCT4, SOX2, KLF4, and NANOG). Immunofluorescent staining showed that those iPSCs expressed SOX2, OCT4, NANOG, and SSEA3 (a specific marker of embryonic stem cells). The embryoid bodies generated from those iPSCs stained positively for OCT4 and NANOG and also with the CDy1 dye that is specific for stem cells. When the iPSCs were subcutaneously injected into 4-week-old BALB/c nude mice, teratoma developed at the inoculation site. The iPSCs also demonstrated reduced DNA methylation compared with the original cells and could be induced to differentiate into adipocytes (mesodermal), hepatocytes (endodermal), and neural cells (ectodermal) in vitro. Our research provides an easy and efficient method for producing iPSCs from keratinocytes, which has important applications in cell replacement therapy.