A total of 171 biopsy samples of the facial skin and the scalp which were randomly selected from the pathologic archives of the Department of Dermatology, Kyungpook National University Hospital, were used for the study. H&E staining of all samples (blocks of paraffin-embedded tissues) were reviewed retrospectively, and 103 samples with SGs were identified. Among them, 54 slides were already stained with the antiserum to S-100 (Dako, Ely, UK), 7 with antiserum to human melanoma black-45 (HMB-45; Dako), 11 with Fontana-Masson (F-M) stain (Junsei Chemical Co., Ltd., Tokyo, Japan), 4 with antiserum to CD1a (Novocastra, Newcastle, UK), 7 with antiserum to c-kit (Dako, Glostrup, Denmark), 10 with antiserum to microphthalmia-associated transcription factor (MITF; Leica Biosystems, Newcastle, UK), and 10 with antiserum to tyrosinase (Thermo Scientific, Fremont, CA, USA). All tissue sections contained epidermal melanocytes and Langerhans cells, which served as internal positive controls (Fig. 1).

Facial skin and scalp biopsy specimens were obtained from two adult Korean males and post-fixed for 2 hours at 4℃ in 2% osmium tetroxide, and then dehydrated in graded alcohol and embedded in epoxy-embedding resin for transmission electron microscopy (TEM). Semi-thin sections of approximately 0.5-µm thickness were obtained using an ultramicrotome and stained with 0.75% toluidine blue; they were then examined under a light microscope. Ultra-thin sections were stained using a conventional method involving staining with 4% uranyl acetate for 10 to 15 minutes, followed by Reynold's lead citrate for 10 to 15 minutes. The specimens were observed under TEM (LEO/Zeiss, Oberkochen, Germany).

Human SGs were isolated from dissected hair follicles under a binocular microscope, and transferred to a tissue culture dish. The cells were maintained in Dulbecco modified Eagle medium at 37℃ in a humidified atmosphere of 5% CO₂. Five days later, the medium was discarded and melanocyte growth medium (MGM) was added. The MGM was composed of MCDB-154 medium supplemented with 0.2% (v/v) bovine pituitary extract, 0.5% (v/v) fetal bovine serum, 5 µg ml^-1 bovine insulin, 5 µg ml^-1 bovine transferrin, 3 ng ml^-1 basic fibroblast growth factor, 0.18 µg ml^-1 hydrocortisone, 3 µg ml^-1 heparin, 10 ng ml^-1 phorbol 12-myristate 13-acetate (Cascade Biologics, Portland, OR, USA), 10 nmol L^-1stem cell factor (Sigma, St. Louis, MO, USA), 100 U ml^-1 penicillin (Gibco BRL, Grand Island, NY, USA), and 100 µg ml^-1 streptomycin (Gibco BRL), 0.25 µg ml^-1 fungizone (Gibco BRL). The medium was changed once every three days.

S-100-positive cells were observed mainly at the periphery of SGs in 34 out of 54 specimens (63%) (Fig. 2A). Several cells, including Langerhans cells and melanocytes, showed a positive reaction to S-100. Therefore, immunohistochemistry with an antibody to HMB-45 and F-M stain was performed in order to prove melanogenic activity and melanin production in S-100-positive cells. All specimens showed negative staining for HMB-45 (seven specimens) and F-M (11 specimens). Two specimens, however, stained positively for CD1a, which is a specific indicator of Langerhans cells (Fig. 2B). In addition, c-kit-positive cells were observed in SGs in 4 specimens (Fig. 2C), and MITF-positive cells (Fig. 2D) and tyrosinase positive cells were observed in 2 specimens (Fig. 2E). These findings suggest that both differentiated melanocytes and Langerhans cells exist in human SGs.

TEM images representing a series of observations made from different sections demonstrated the presence of melanocytes in the suprabasal portion of SGs. Sebaceous melanocytes were large and triangular or polygonal in shape, and the plasma membrane was devoid of tonofilaments and desmosomes. They were located between undifferentiated peripheral cells containing no lipid droplets. The nuclei had a slightly irregular contour with shallow indentations. These melanocytes showed fewer melanin-containing granules than the melanocytes in the basal surface of the epidermis. However, the individually distributed melanosomes in the suprabasal melanocytes were larger compared to those of the epidermal melanocytes. The melanin granules did not migrate to the surrounding sebocytes (Fig. 3A, B).

We isolated melanocytes from SGs through cell culture. The sebaceous melanocytes appeared to be triangular or polygonal in shape. In cells cultured with MGM, the sebaceous melanocytes spread, developed long processes, and became bipolar, tripolar, or dendritic. The sebaceous melanocytes had morphologically homogeneous, slender cell bodies with few-dendrites (less than 4 dendrites per cell) (Fig. 3C, D).