A Preliminary Study of Markers for Human Hair Follicle Melanin Stem Cell
Melanocyte stem cells (MSCs) are derived from the neural crest and serve as the reservoir of melanocytes (MCs). These cells are primarily located in the bulge of hair follicles, where they play a crucial role in the regeneration of melanocytes. During embryonic development, neural crest cells differentiate into melanoblasts (MBs), which migrate through the dermis to the epidermis and into developing hair follicles. Upon entering the hair follicles, some melanoblasts migrate to the hair matrix region and differentiate into mature melanocytes, which produce pigment and transfer it to keratinocytes (KCs). The remaining melanoblasts settle in the follicular region and become melanocyte stem cells, responsible for the continuous regeneration of melanocytes.
The identification of melanocyte stem cells is facilitated by specific markers, including paired box gene 3 (PAX3) and dopachrome tautomerase. PAX3, a transcription factor belonging to the paired box (PAX) family, is crucial for the differentiation, migration, and proliferation of melanoblasts and melanocytes. It is also believed to play a key role in maintaining the undifferentiated state of melanocyte stem cells. Therefore, PAX3 is considered a reliable marker for identifying these cells.
In addition to melanocyte stem cells, hair follicle stem cells (HFSCs) are another type of stem cell found in hair follicles. HFSCs, derived from ectodermal epithelium, have the potential to differentiate into multiple cell types. They can receive melanin from mature melanocytes and generate hair shafts containing melanin. HFSCs are characterized by various molecular markers, including CK15, CK19, and CD34. CD34, an adhesion molecule, is selectively expressed on the surface of stem cells in humans and other mammals, and its expression diminishes as cells mature. Immunohistochemical staining has demonstrated that CD34 is specifically expressed in the bulge region of human hair follicles, making it a useful marker for HFSCs.
This study aimed to investigate the presence of melanocyte stem cells and hair follicle stem cells in mixed skin cells derived from normal human foreskin. The foreskin was chosen due to its high melanocyte content. The skin cells were separated by enzymatic digestion, resulting in a mixture of keratinocytes, melanocytes, fibroblasts, and a small number of skin stem cells from the basal layer of the skin and hair follicles. PAX3 and CD34 were used as markers for melanocyte stem cells and hair follicle stem cells, respectively. Flow cytometry (FCM) was employed to detect the presence of these stem cells in the mixed skin cell population.
The results of the flow cytometry analysis revealed that the percentage of PAX3-positive cells, CD34-positive cells, and PAX3+/CD34+ double-positive cells in the mixed skin cell population was 4.82±0.15%, 5.38±0.21%, and 0.58±0.05%, respectively. After digesting normal human foreskin tissues, the original skin mixed cell samples were obtained, and the PAX3+/CD34+ cells were selected by FCM. The total number of cells in these samples was approximately 8.50±0.65 x 10^7, with the percentage of PAX3+/CD34+ cells being about 0.67±0.15%, and the number of PAX3+/CD34+ cells being approximately 5.70±0.40 x 10^5.
To further characterize the PAX3+/CD34+ cells, the expression of specific melanocyte markers, including tyrosinase-related protein-2 (TRP-2), microphthalmia-associated transcription factor (MITF), Melan-A, tyrosinase (TYR), tyrosinase-1 (TYR-1), and SRY-related HMG-box 10 (SOX10), was analyzed. The results showed that the expression of TRP-2 in the PAX3+/CD34+ cells was similar to that of the control group, which consisted of original melanocytes. However, the expression levels of MITF, Melan-A, TYR, TYR-1, and SOX10 in the PAX3+/CD34+ cells were significantly lower than those in the control group, with expression levels being less than 20% of those in the control group. Immunofluorescence staining confirmed that TRP-2 was expressed on the surface of PAX3+/CD34+ cells, while MITF and Melan-A were not detected.
Based on these findings, it was hypothesized that the PAX3+/CD34+ cells represent a population of cells that express markers of both melanocyte stem cells and hair follicle stem cells. These cells may be transitional cells between melanocyte stem cells and hair follicle stem cells. The expression profile of the PAX3+/CD34+ cells, characterized by the presence of TRP-2 and the absence of MITF and Melan-A, suggests that these cells have the specific characteristics of melanocyte stem cells rather than mature melanocytes.
In conclusion, this study identified a population of PAX3+/CD34+ cells in the mixed skin cell population derived from human foreskin. These cells express markers of both melanocyte stem cells and hair follicle stem cells and exhibit a unique expression profile that distinguishes them from mature melanocytes. The findings suggest that PAX3+/CD34+/TRP-2+/MITF–/Melan-A– could serve as candidate markers for human hair follicle melanin stem cells. Further research is needed to fully understand the role and potential of these cells in melanocyte regeneration and hair follicle biology.
doi.org/10.1097/CM9.0000000000000206
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