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.).
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