
	Version PDF

Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors

European Journal of Dermatology. Volume 18, Number 5, 518-23, September-October 2008, Investigative report

DOI : 10.1684/ejd.2008.0485

Summary

Author(s) : Maho Kanoh, Yasuyuki Amoh, Yuichi Sato, Kensei Katsuoka , Department of Dermatology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa, 228-8555, Japan, Department of Molecular Diagnostics, Kitasato University School of Allied Health Sciences, Sagamihara 228-8555, Japan.

Summary : Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34\; a middle part that was CD34-positive and K15-negative\; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.

Keywords : basal cell carcinoma, keratin 15, nestin, squamous cell carcinoma, stem cell, trichilemmoma

Pictures

ARTICLE

Auteur(s) : Maho Kanoh¹, Yasuyuki Amoh¹, Yuichi Sato², Kensei Katsuoka¹

¹Department of Dermatology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa, 228-8555, Japan
²Department of Molecular Diagnostics, Kitasato University School of Allied Health Sciences, Sagamihara 228-8555, Japan

accepté le 16 Avril 2008

The hair follicle undergoes dynamic cycling between growth (anagen), regression (catagen), and resting (telogen) phases. Stem cells located in the hair follicle bulge area give rise to the follicle structures during each anagen phase [1-4]. Nestin, a marker of neural stem cells, is expressed in the bulge area stem cells of the mouse hair follicle. In mice, these stem cells give rise to the outer-root sheath and the nestin-expressing interfollicular vascular network [5-9]. Nestin-expressing stem cells isolated from the mouse hair follicle bulge areas that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. These pluripotent nestin-expressing stem cells are positive for the stem cell marker CD34 and negative for K15, suggesting that they are in a relatively undifferentiated state. The apparently primitive state of nestin-expressing stem cells is compatible with their pluripotency [6-10]. Many recent reports have suggested that various types of cutaneous tumors originate from hair follicles and epidermal stem cells [11, 12].

In this study, we examined immunohistochemically the expression and precise localization of the hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles, as well as epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC).

Materials and methods

Preparation of normal skin and tumor tissue specimens

For immunohistochemical analysis, tissue samples were obtained from surgically resected normal scalp skin; 11 trichilemmomas, including 1 case of desmoplastic type; 26 cases of BCC (multifocal superficial type [n = 5], nodular [n = 8], morphemic-like [n = 6], and micronodular [n = 7]); and 16 SCCs (well differentiated [n = 6], moderately differentiated [n = 5], and poorly differentiated [n = 5]) of the skin. All experiments were performed in accordance with the Helsinki guidelines, in compliance with national regulations for the experimental use of human material. They were routinely fixed in 10% formalin and embedded in paraffin. In all samples, both immunohistochemical stainings were performed on serial sections.

Immunocytochemical staining of cultured cells

Immunocytochemical staining was performed on NHEK cells derived from normal human epidermal keratinocytes (Kurabo, Osaka, Japan), TL-1 cells derived from trichilemmoma, and SCC cells derived from SCC tumors. TL-1 and SCC cell lines were established in our laboratory [13, 14] and were maintained in Dulbecco’s Modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (ICN biomedicals, Aurora, OH) and 50 μg mL^–1 gentamicin (Invitrogen, Carlsbad, CA). NHEK cells were grown in the serum-free keratinocyte growth media (HuMedia-KB2, Kurabo) supplemented with 0.4% bovine pituitary extract, 10 μg mL^–1 bovine insulin, 0.5 μg mL^–1 hydrocortisone, 0.1 ng mL^–1 recombinant human epidermal growth factor, 50 μg mL^–1 gentamicin, and 50 ng mL^–1 amphotericin-B (Kurabo).

Cells were incubated on Lab-Tek Chamber Slides (Nunc, Rochester, NY) overnight at 37 °C in a 5% CO₂ incubator. The cells were washed with Dulbecco’s phosphate buffered saline (PBS^-) and then fixed with IHC Zinc Fixative (BD Biosciences, Franklin Lakes, NJ) for 60 min at room temperature. Next, the cells were washed with PBS^- and air-dried. Immunocytochemical staining for nestin, K15, and CD34 was detected by the dextran polymer method (ChemMate Envision, Dako, Carpinteria, CA). Briefly, after eliminating endogenous peroxidase activity by treatment with 3% hydrogen peroxide for 5 min at room temperature, the slides were incubated with 5% fetal calf serum in PBS^- for 5 min at room temperature to block nonspecific protein binding. The following primary antibodies were used: anti-nestin polyclonal (1:200; Chemicon, Temecula, CA), anti-K15 monoclonal (clone LHK15, 1:100; Lab Vision, Fremont, CA), and anti-CD34 monoclonals NU-4A1 (1:100; Nichirei, Tokyo, Japan) and QBEnd/10 (1:200; Lab Vision). The slides were incubated with each antibody for 60 min and then incubated with ChemMate Envision for 30 min at room temperature. Finally, immunoreactive proteins were visualized with 8-amino-9-ethylcarbazole solution (Dako) and counterstained with Mayer’s hematoxylin (Wako Pure Chemical, Osaka, Japan).

Immunohistochemical staining of normal skin and tumor tissues

Immunohistochemical staining was performed on the paraffin sections of skin from normal scalp, trichilemmoma, BCC, and SCC tissues. The primary antibodies and immunohistochemical procedures were as described above (see “Immunocytochemical staining of cultured cells”). In the case of nestin staining, the paraffin sections were heated for 10 min in 10 mM citrate buffer (pH 6.0) in a microwave oven, for antigen retrieval. Double-immunohistochemical staining for nestin and K15 in paraffin-embedded tissue sections was performed by an indirect method using fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (1:10; Chemicon) and tetramethylrhodamine isothiocyanate-conjugated swine anti-rabbit IgG (1:80; Nordic Immunological Laboratories, Tilburg, Netherlands).

Results

Expression of nestin, K15, and CD34 in normal epidermis and hair follicle

Immunohistochemical staining showed that, in normal skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34 (figure 1). The hair follicle cells below the sebaceous glands were strongly positive for nestin, negative or partially positive for K15, and negative for CD34. The upper part of the outer-root sheath cells was partially positive for nestin, strongly positive for K15, and negative for CD34. Some of the basal sebocytes expressed nestin (figure 2). The middle part of the outer-root sheath cells was positive for CD34 and negative for K15. The lower part of the outer-root sheath cells was positive for K15 and negative for CD34 (figure 3).

Expression of nestin, K15, and CD34 in cultured cells

Nestin immunoreactivity in TL-1 cells was stronger than in NHEK and SCC cells (figure 4). K15 immunoreactivity was observed in the cytoplasm of NHEK, TL-1, and SCC cell lines. The staining for K15 was stronger in TL-1 and NHEK cells than in SCC cells. No CD34 immunoreactivity was observed in any of the cell lines.

Expression of nestin, K15, and CD34 in trichilemmoma, BCC, and SCC

In all trichilemmoma tissues, nestin expression was observed in tumor cells. All BCC tissues were negative for nestin. The tumor cells in 13 of 16 SCC tissues were negative for nestin. All trichilemmoma tissues were partially positive for K15. Of 26 BCC tissues, 17 were K15-positive. K15 was observed in part of the tumor cells in 10 out of 16 SCC tissues. The tumor cells in SCC tissues were negative for nestin and partially expressed K15 (figure 5, table 1). CD34 immunoreactivity was not observed in any of the tissues except for one case of desmoplastic trichilemmoma.
Table 1

		Nestin	K15	CD34
BCC	1	–	+	–
2	–	+	–
3	–	+	–
4	–	+	–
5	–	–	–
6	–	–	–
7	–	+	–
8	–	+	–
9	–	–	–
10	–	*	–
11	–	*	–
12	–	*	–
13	–	+	–
14	–	–	–
15	–	+	–
16	–	*	–
17	–	–	–
18	–	–	–
19	–	*	–
20	–	+	–
21	–	–	–
22	–	–	–
23	–	+	–
24	–	–	–
25	–	+	–
26	–	+	–
TL	1	+	*	–
2	+	*	–
3	+	*	–
4	+	*	–
5	+	*	–
6	+	*	–
7	+	*	–
8	+	*	–
9	+	*	–
10	+	*	–
11	+	*	+
SCC	1	–	*	–
2	–	*	–
3	*	–	–
4	–	*	–
5	–	–	–
6	*	*	–
7	–	*	–
8	–	*	–
9	–	–	–
10	–	*	–
11	–	–	–
12	–	–	–
13	*	*	–
14	–	*	–
15	–	–	–
16	–	*	–

Discussion

Nestin, a marker of neural stem cells, is expressed in bulge area stem cells of the hair follicle. Studies in transgenic mice expressing nestin-green fluorescent proteins show that the nestin-expressing hair follicle stem cells give rise to the outer-root sheath and the nestin-expressing interfollicular vascular network [5-9]. We recently reported that nestin-expressing hair follicle stem cells isolated from mice are in a relatively undifferentiated state and are pluripotent [6-10].

Previously, Poblet et al. [15] reported that the anagen follicles contain CD34 immunoreactivity in the outer root sheath. They found different patterns of staining were found for CD34 and K15 in the anagen follicles: CD34-positive cells were located in the outer root sheath below the attachment zone of the arrector pili muscle, whereas K15-positive cells were located in the outer root sheath above the attachment zone of the arrector pili muscle. We obtained similar results. In normal skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The upper part of the outer-root sheath cells was K15-positive and CD34-negative, the middle part was CD34-positive and K15-negative, and the lower part was K15-positive and CD34-negative. On this basis, we suspected that nestin is expressed by immature sebocytes in the sebaceous gland.

Recently, Jih et al. [11]. and Kanitakis et al. [12] suggested that follicular tumors are related to hair follicle stem cells in the bulge. On the basis of immunohistochemical staining with antibodies to various keratin polypeptides, they demonstrated that BCC cells have phenotypes resembling either bulge or matrix follicular cells. They suggested that K15 is a characteristic marker within pilar tumors. In the study by Jih et al. [11], 27% of BCCs expressed K15.

In the present study, all trichilemmomas expressed nestin, whereas all BCCs were nestin-negative. Moreover, 17 out of 26 (65%) BCCs expressed K15. Poblet et al. [16] reported that trichilemmomas showed a strong and diffuse pattern of staining for CD34. Although we tried to stain the trichilemmoma with two different CD34 antibodies, we did not find any CD34-positive cells except for one case of desmoplastic trichilemmoma. These results suggest that trichilemmomas originate from the nestin-partially positive/K15-positive/CD34-negative outer-root sheath cells, BCC tumor cells from the more mature nestin- negative/CD34-negative/K15-positive outer-root sheath cells, and SCCs from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis (figure 6).

Acknowledgements

Grant support: This work was supported by the Ishidsu Shun Memorial Scholarship (to M.K.) and the Uehara Memorial Foundation (to Y.A.).

References

1 Hoffman RM. The hair follicle as a gene therapy target. Nat Biotechnol 2000; 18: 20-1.

2 Oshima H, Rochat A, Kedzia C, et al. Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 2001; 104: 233-45.

3 Cotsarelis G, Sun T-T, Lavker RM. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 1990; 61(7): 1329-37.

4 Taylor G, Lehrer MS, Jensen PJ, et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000; 102: 451-61.

5 Li L, Mignone J, Yang M, et al. Nestin expression in hair follicle sheath progenitor cells. Proc Natl Acad Sci USA 2003; 100: 9958-61.

6 Amoh Y, Li L, Yang M, et al. Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells. Proc Natl Acad Sci USA 2004; 101: 13291-5.

7 Amoh Y, Li L, Katsuoka K, et al. Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons. Proc Natl Acad Sci USA 2005; 102: 5530-4.

8 Amoh Y, Li L, Campillo R, et al. Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves. Proc Natl Acad Sci USA 2005; 102: 17734-8.

9 Amoh Y, Li L, Katsuoka K, Hoffman RM. Chemotherapy targets the hair-follicle vascular network but not the stem cells. J Invest Dermatol 2007; 127(1): 11-5.

10 Yu H, Fang D, Kumar SM, et al. Isolation of a novel population of multipotent adult stem cells from human hair follicles. Am J Pathol 2006; 168(6): 1879-88.

11 Jih DM, Lyle S, Elenitsas R, et al. Cytokeratin 15 expression in trichoepitheliomas and a subset of basal cell carcinomas suggests they originate from hair follicle stem cells. J Cutan Pathol 1999; 26: 113-8.

12 Kanitakis J, Bourchany D, Faure M, Claudy A. Expression of the hair stem cell-specific keratin 15 in pilar tumors of the skin. Eur J Dermatol 1999; 9(5): 363-5.

13 Kanzaki T, Eto H, Umezawa A, et al. Morphological, biological, and biochemical characteristics of a benign human trichilemmoma cell line in vivo and in vitro. Cancer Res 1981; 41: 2468-75.

14 Kameyama K, Tone T, Eto H, et al. Recombinant gamma interferon induces HLA-DR expression on squamous cell carcinoma, trichilemmoma, adenocarcinoma cell lines, and cultured human keratinocytes. Arch Dermatol Res 1987; 279: 161-6.

15 Poblet E, Jiménez F, Godínez JM, et al. The immunohistochemical expression of CD34 in human hair follicles: a comparative study with the bulge marker CK15. Clin Exp Dermatol 2006; 31(6): 807-12.

16 Poblet E, Jiménez F, Rocamora A. QBEND/10 (anti-CD34 antibody) in external root sheath cells and follicular tumors. J Cutan Pathol 1994: 224-8.