To the Editor: Melanocyte stem cells (MSCs), derived from the neural crest, function as the repository of melanocytes (MCs). Currently, most scholars suggest that MSCs mainly exist in the bulge of hair follicles. At the end of the embryo, the neural crest cells differentiate into melanoblasts (MBs), which migrate through the dermis to the epidermis and into the developing hair follicles. When the MBs enter the hair follicles, some of them migrate to the hairy mother region and differentiate into mature MCs, producing the pigment and passing it to the keratinocytes (KCs). The others are settled in the follicular and become MSCs, which are responsible for the regeneration of MCs. The labelled marker of MSCs includes paired box gene 3 (PAX3) and dopachrome tautomerase.PAX3, as a kind of important transcription factor and belonging to the paired box (PAX) family, plays a key role in the differentiation, migration, and proliferation of MB and MCs. Some scholars consider that PAX3 plays a key role in maintaining the undifferentiated molecular genetic mechanism of MSCs. So PAX3 could be used as a marker of MSCs. In addition to MSC, another kind of stem cell called hair follicle stem cell (HFSC) is also found in hair follicles. HFSCs, derived from ectodermal epithelium, have the potential of differentiation in multiple directions; they could accept melanin from mature MCs and generate hair shaft which contains melanin. HFSCs have various molecular markers, including CK15, CK19, CD34, and others. As a kind of adhesion molecule, CD34 is selectively expressed on the surface of stem cell of human and other mammalian, and gradually diminishes with the maturity of the cell. In 2010, some scholars used immunohistochemical staining of CD34 to label hair follicle skin tissue and demonstrated that CD34 had a positive expression with good specificity in the bulge of human hair follicle. Therefore, in this study, CD34 was used as the marker of HFSCs.
This study was approved by the Institutional Research Ethics Committee of Shanghai General Hospital. Informed consents were obtained from all patients before the enrollment in this study. As foreskin tissue contains more MCs, we separated the skin cells from normal human foreskin by enzymatic digestion in this study. In theory, these cells include large numbers of KCs, MCs, partial fibroblasts, and a small amount of skin stem cells from basal layer of the skin and hair follicles. PAX3 and CD34 were regarded as markers of MSCs and HFSCs, respectively, and then we detected whether MSCs and HFSCs exist in the original mixed skin cells by flow cytometry (FCM). In view of the study that the hair follicle melanin stem cells and the HFSCs share a cell nest, we tried to detect whether there was a certain kind of cell in the original hybrid cells that expressed markers of HFSCs and MSCs at the same time. If it exists, does the cell express the specific markers of MCs such as tyrosinase-2 (TYR-2), Melan-A, and microphthalmia-associated transcription factor (MITF), and whether they have the expression characteristics of MCs or MSCs?
The percentage of cells was detected by FCM. The result showed the percentage of PAX3+, CD34+ and PAX3+/CD34+ was 4.82 ± 0.15%, 5.38 ± 0.21%, and 0.58 ± 0.05%, respectively [Figure 1]. After digesting normal human foreskin tissues (n = 9), the original skin mixed cell samples were obtained, and the PAX3+/CD34+ cells in these samples were selected by FCM. The result of FCM revealed total number of cells in these samples was about (8.50 ± 0.65) × 107, the percentage of PAX3+/CD34+ cells in these samples was about 0.67 ± 0.15%, and the number of PAX3+/CD34+ cells in these samples was about (5.70 ± 0.40) × 105. The original MCs from human were set as control group (n = 9), PAX3+/CD34+ cells detected by FCM in the original skin mixed cells were set as PAX3+/CD34+ group. The relative expression of tyrosinase related protein-2 (TRP-2), MITF, Melan-A, TYR, TYR-1, and SRY-related HMG-box 10 (SOX10) were detected in PAX3+/CD34+ and control groups. The result showed that there was no significant difference in expression of TRP-2 between PAX3+/CD34+ and control groups (1.06 ± 0.20 and 0.91 ± 0.11, P > 0.05). The expression of MITF in PAX3+/CD34+ group was 0.15 ± 0.03, which was obvious lower than control group (0.93 ± 0.10, P < 0.05). Similarly, there were significant differences in the expression of Melan-A, TYR, TYR-1, and SOX10 between PAX3+/CD34+ and control groups (0.13 ± 0.04 vs. 0.83 ± 0.16, 0.16 ± 0.02 vs. 0.90 ± 0.15, 0.15 ± 0.03 vs. 0.87 ± 0.20, 0.10 ± 0.03 vs. 0.92 ± 0.20, all P < 0.05). Therefore, the expression of TRP-2 in PAX3+/CD34+ group was similar to the control group, while the expression levels of the MITF, Melan-A, TYR, TYR-1, and SOX10 were almost less than 20% of the control group. Immunofluorescence showed that TRP-2 in PAX3+/CD34+ cells was stained red positive. Melan-A and MITF in PAX3+/CD34+ cells were stained negative and no fluorescence.
In this study, we examined the PAX3+ cells and CD34+ cells in the original hybrid cells by FCM, namely MSCs and HFSCs. And CD34+/PAX3+ cells also did exist in the primary skin mixture, accounting for 0.53%, which was about one eighth of the PAX3+ cells and one tenth of the CD34+ cells. In addition, we selected PAX3+/CD34+ cells in original skin mixed cells, which accounted for 0.67 ± 0.15% of the mixed cells. We hypothesized that this part of the cells expressed both the marker of MSCs and HFSCs, CD34+ cells may be hair follicle melanin stem cells [Table 1].
In this study, we further examined whether PAX3+/CD34+ cells in the original skin cells expressed specific markers of MCs. Results showed that the expression of TRP-2 in PAX3+/CD34+ cells was similar to that of control group, while the MITF, Melan-A, TYR, TYR-1, and SOX10 were rarely expressed, which meant that these PAX3+/CD34+ might not have the similar characters as mature MCs. By immunofluorescence assay, we confirmed that the TRP-2 was expressed on the surface of PAX3+/CD34+ cells. Nevertheless, MITF and Melan-A were hardly ever expressed on cells’ surface. Therefore, we deemed that this kind of cell has the particular expression characteristics of MSCs. Accordingly, we considered that PAX3+/CD34+/TRP-2+/MITF–/Melan-A– could be the candidate marker for human hair follicle melanin stem cells. We suggested that this kind of cell might belong to the transition cells of MSCs and HFSCs.
This work was supported by a grant from the National Natural Science Foundation of China (No. 81703140).
Conflicts of interest
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