1925
Journal of Cell Science 112, 1925-1936 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS4638
Human sweat gland myoepithelial cells express a unique set of cytokeratins
and reveal the potential for alternative epithelial and mesenchymal
differentiation states in culture
Margarete Schön1,*, Jennifer Benwood1, Therese O’Connell-Willstaedt2 and James G. Rheinwald1,2,‡
1Division
of Dermatology/Department of Medicine, Brigham and Women’s Hospital, and 2Division of Cell Growth and Regulation,
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
*Present address: Department of Dermatology, Heinrich-Heine University, Moorenstrasse 5, 40225 Düsseldorf, Germany
‡Author for correspondence (e-mail: [email protected])
Accepted 9 April; published on WWW 26 May 1999
SUMMARY
We have characterized precisely the cytokeratin expression
pattern of sweat gland myoepithelial cells and have
identified conditions for propagating this cell type and
modulating its differentiation in culture. Rare, unstratified
epithelioid colonies were identified in cultures initiated
from several specimens of full-thickness human skin. These
cells divided rapidly in medium containing serum,
epidermal growth factor (EGF), and hydrocortisone, and
maintained a closely packed, epithelioid morphology when
co-cultured with 3T3 feeder cells. Immunocytochemical
and immunoblot analysis disclosed that the cells differed
from keratinocytes in that they were E-cadherin-negative,
vimentin-positive, and expressed an unusual set of
cytokeratins, K5, K7, K14, and K17. When subcultured
without feeder cells, they converted reversibly to a spindle
morphology and ceased K5 and K14 expression. Under
these conditions, EGF deprivation induced flattening,
growth arrest, and expression of α-smooth muscle actin (αsma). Coexpression of keratins and α-sma is a hallmark of
myoepithelial cells, a constituent of secretory glands.
Immunostaining of skin sections revealed that only sweat
gland myoepithelial cells expressed the same pattern of
keratins and α-sma and lack of E-cadherin as the cell type
we had cultured. Interestingly, our immunocytochemical
analysis of ndk, a skin-derived cell line of uncertain
identity, suggests that this line is of myoepithelial origin.
Earlier immunohistochemical studies by others had found
myoepithelial cells to be K7-negative. We tested five K7specific antibodies that can recognize this protein in
western blots and in the assembled keratin filaments of
mesothelial cells. Three of these antibodies did not
recognize the K7 present in myoepithelial cell filaments or
in HeLa cell filaments, indicating that some K7 epitopes are
masked when K7 pairs with K17 instead of with its usual
keratin filament partner, K19.
INTRODUCTION
cell biologic, biochemical, and molecular genetic analysis of
the mechanisms regulating its growth and differentiation.
Much less is known about the regulation of growth, tissue
morphogenesis, and differentiation-related gene expression of
the sweat glands. There are two types of sweat glands, eccrine
and apocrine. Several million eccrine sweat glands having an
estimated aggregate mass of about 100 grams are distributed
over the body surface (reviewed by Sato, 1993). These glands
are comprised of a coiled tube, lying at the border of the dermis
and subcutaneous fat, connected to the surface by a duct that
penetrates the epidermis to permit expulsion of the gland’s
secretory products. The sweat glands can lower body
temperature by producing a film of water on the surface of the
skin that yields evaporative cooling. The secretory portion of
the gland, at the distal end of the coil, transports an isotonic
plasma filtrate into its lumen. A region of the gland farther
along the coil, as it connects to the duct, contains absorptive
cells which selectively remove NaCl such that the sweat is
The outer surface of the body is covered by epithelial tissues
and structures that develop from the embryonic ectoderm. The
epidermis, a stratified squamous epithelium, forms a protective
barrier while the major adnexal structures of the skin, the hair
and the sweat glands, provide the important function of
regulating body temperature. Much has been learned about the
biology of the epidermis and of the hair, including the kinetics
of their renewal, factors that can modulate their growth, their
pattern of cell division under normal and wound response
conditions, and the major structural proteins responsible for
their differentiated functions. The epidermis and the hair have
been amenable to study in detail because these structures are
so large and accessible, easy to examine biochemically and
histologically, and because these tissues continually renew. In
the case of the epidermis, the keratinocyte, which is the cell
type that forms this tissue, can be grown in culture, permitting
Key words: Cultured cell, Eccrine, Apocrine, Keratin, Actin
1926 M. Schön and others
hypotonic when it reaches the surface. Apocrine sweat glands
form at different times and from a different cell lineage than
the eccrine glands. Apocrine glands are formed as part of the
pilosebaceous unit in several restricted regions, primarily in
axillary and pubic skin, and they first become functionally
active at puberty. They secrete sweat and other products (Cohn,
1994) into the pilosebaceous duct next to the hair shaft, rather
than directly onto the epidermal surface. Unlike the epidermis
and the hair follicle, eccrine and apocrine glands do not
regularly renew themselves via cell division and terminal
differentiation.
Histologic and immunohistochemical studies have revealed
that eccrine glands begin forming at 13 weeks of gestation from
clusters of cells that form buds in the embryonic epidermis and
migrate downward as cords into the dermis. Initially, cells in
the gland anlagen appear rather homogenous and express a
combination of keratins and vimentin consistent with their
identity as pluripotent progenitor cells which, as the gland
matures, become restricted to one of three types of
differentiation: duct, secretory, or myoepithelial (Holbrook and
Wolff, 1993; Moll and Moll, 1992). Myoepithelial cells, a cell
type defined by its expression of keratin-type intermediate
filaments and α-smooth muscle actin (Franke et al., 1980;
Norberg et al., 1992), surround and interdigitate with the
secretory coil cells and are thought to provide tensile support
against distention as the gland fills with sweat. These cells may
also contract slightly in response to neurotransmitters, thereby
aiding sweat expulsion (Sato et al., 1979).
Small, short-term cultures of ductal and secretory epithelial
cells can be prepared from outgrowths of eccrine glands
isolated by digesting skin specimens with dispase and
collagenase and microdissecting the adnexal structures (see
Collie et al., 1985; Lee et al., 1986; Jones et al., 1988; Yokozeki
et al., 1990; Bell and Quinton, 1991). The culture of sweat
gland myoepithelial cells has not yet been described, however.
Scientists at a local biotechnology company who were serially
culturing epidermal keratinocytes from skin biopsies to prepare
grafts for burn patients (see O’Connor et al., 1981; Leigh et al.,
1991) brought to our attention the occasional presence in these
cultures of rare epithelial cell colonies of morphology distinct
from that of keratinocytes. We report here the characterization
of these as sweat gland myoepithelial cells and the
identification of conditions promoting their rapid division in
serial culture. We have found that this cell type expresses a
unique pattern of cytokeratin proteins, including coexpression
of the unusual K7/K17 cytokeratin pair, and that modifying
culture conditions induces a reversible transition between
epithelioid and fibroblastoid morphology and pattern of
cytoskeletal protein expression.
MATERIALS AND METHODS
Cells
Full-thickness skin biopsies of undamaged skin obtained from burn
patients (typically from the axilla) were processed by the production
laboratory at Biosurface Technology, Inc. (Cambridge, MA) as the
first step toward expanding the keratinocyte population in culture to
generate epidermal autografts (O’Connor et al., 1981; Leigh et al.,
1991). The skin biopsies were minced into small fragments and stirred
in 0.25% trypsin at 37°C. Released cells were recovered by
centrifugation, resuspended in FAD medium, and co-cultivated with
3T3 feeder cells for ~7 days until cultures were nearly confluent.
These primary cultures then were suspended with trypsin/EDTA and
cryopreserved. Ampules were then thawed and plated under different
conditions to expand the keratinocyte and myoepithelial cell
populations.
Pure myoepithelial cell lines were isolated from cultures initiated
from three skin biopsies: BRSO from the axilla of a 13-year-old (yo)
male, CYHI from the axilla of a 28 yo female, and DOLA from the
axilla of a 19 yo male. Mid-lifespan cultures of CYHI and BRSO were
karyotyped by the Dana-Farber Cancer Institute’s clinical
cytogenetics laboratory, using trypsin-Giemsa banding, and were
found to be diploid female and male, respectively. Myoepithelial cells
were also identified in, but were not isolated as pure populations from,
cultures initiated from three other skin biopsies: JAKE from the
abdomen of a 31 yo male, BABU from the axilla of a 28 yo female,
and C5-SkH(my) from the scalp of a >40 yo individual whose gender
was not recorded. The properties of myoepithelial cells in culture were
compared with those of the normal newborn foreskin keratinocyte
strain N (Rheinwald and Beckett, 1980), the normal human foreskin
fibroblast strain S1-F, the human adult peritoneal mesothelial cell
strain LP-9 (Wu et al., 1982), and the human skin-derived cell line
ndk (Adams and Watt, 1988).
Culture conditions
Human myoepithelial cells and keratinocytes were routinely cultured
in ‘FAD medium’, consisting of DME/F12 (Sigma) (1:1 v/v) + 5%
newborn calf serum (Hyclone) + 5 µg/ml insulin, 0.4 µg/ml
hydrocortisone (HC), 10 ng/ml epidermal growth factor (EGF),
1.8×10−4 M adenine, 10−10 M cholera toxin, 2×10−11 M
triiodothryonine, and penicillin/streptomycin (Allen-Hoffmann and
Rheinwald, 1984; Rheinwald, 1989). For use as feeder cells, the
mouse fibroblast cell line 3T3J2 was grown in DME + 10% calf serum
medium, either lethally irradiated with 5,000 R gamma-irradiation
from a cobalt source or treated for 2.5 hours with 3 µg/ml mitomycin
C (Sigma), and plated at ~5×104 cells/cm2 (Rheinwald and Green,
1975). Alternatively, myoepithelial cells were cultured in the absence
of feeder cells in DME/F12 medium supplemented with 10% serum,
hydrocortisone, and EGF. For some experiments, keratinocytes and
myoepithelial cells were grown in Gibco keratinocyte serum-free
medium (ker-sfm) (Life Technologies, Inc., Gaithersburg, MD) (Pirisi
et al., 1987) supplemented with 30 µg/ml bovine pituitary extract, 0.1
ng/ml EGF, penicillin/streptomycin, and additional CaCl2 to bring the
total [Ca2+] to 0.4 mM (Schön and Rheinwald, 1996). Ndk cells
(Adams and Watt, 1988) at 8th passage (kindly provided by F. Watt.,
ICRF, Lincoln’s Inn Fields, London) were cultured in FAD medium.
HeLa cells were cultured in DME/F12 medium supplemented with
10% calf serum. Cells were subcultured by incubation with 0.1%
trypsin/0.02% EDTA in PBS and were cryopreserved in liquid
nitrogen as suspensions in DME/F12 medium supplemented with 10%
calf serum and 10% dimethylsulfoxide.
To compare growth in different medium formulations and in the
presence or absence of specific hormones, growth factors, or growth
inhibitors, cells were plated at 2×103 cells per 9 cm2 well in 6-well
plates in the desired medium, refed every 2 to 4 days, and either
suspended to count or else fixed and stained with Methylene Blue 8
to 10 days after plating. When used, TGF-β1 (R&D Systems) was
added to a concentration of 1 ng/ml (see Rollins et al., 1989) and
phorbol myristyl acetate (PMA) (Sigma) to a concentration of 10−8
M.
Organotypic cultures were prepared as described by Parenteau et
al. (1991) and by Schön and Rheinwald (1996). Briefly, 6.9×104
human fibroblasts (newborn foreskin-derived, strain B038) were
suspended in 3 ml of bovine collagen type I (0.7 mg/ml)
(Organogenesis, Inc., Canton, MA). This suspension then was cast on
top of 1 ml of an acellular collagen gel in six-well tissue culture tray
inserts, the bottoms of which were polycarbonate filters of 3 µm pore
size (Organogenesis, Inc.). In some experiments, myoepithelial cells
Sweat gland myoepithelial cell differentiation 1927
or myoepithelial cells plus fibroblasts were embedded in the collagen
gels. The cells were allowed to contract the collagen gels for 4 days
at 37°C. Keratinocytes and/or myoepithelial cells then were seeded
onto the gels at a density of 2×105 per cm2 of gel surface area and
were cultured submerged for 4 days in DME/F12 medium (3:1 v:v)
supplemented with 0.3% calf serum, 5 µg/ml insulin, 0.4 µg/ml
hydrocortisone, 2×10−11 M triiodothyronine, 5 µg/ml transferrin, 10−4
M ethanolamine, 10−4 M phosphoethanolamine, 5.3×10−8 M
selenious acid, and 1.8×10−4 M adenine. The cultures were then raised
to the air/liquid interface and cultured for 10 more days, after which
they were embedded in OCT compound for cryosectioning.
Two-dimensional gel electrophoresis
Using methods described by Wu et al. (1982), subconfluent cultures
were metabolically labeled with 100 mCi [35S]methionine for 4 hours
and the Triton/high salt-insoluble, cytoskeletal fraction was isolated.
Equal amounts of labeled protein were resolved by non-equilibrium
pH gradient electrophoresis (NEpHGE) in the first dimension and by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the second
dimension. Labeled proteins were detected by autoradiography using
Kodak X-OMAT film.
Antibodies
The following mouse monoclonal antibodies were used for
immunoperoxidase staining and western blotting: keratin 5-specific
antibody AE14 (Lynch et al., 1986) was kindly provided by T.-T. Sun.
Keratin 7-specific antibody OVTL 12/30 (van Niekerk et al., 1991)
was purchased from Biodesign International (Kennebunk, ME).
Keratin 7-specific antibody Ks7.18 (Bartek et al., 1991), keratin 18specific antibody CK2 (Debus et al., 1982), and vimentin-specific
antibody V9 (Osborn et al., 1984) were purchased from BoehringerMannheim Corp. (Indianapolis, IN). Keratin 7-specific monoclonal
antibodies RCK105 (Ramaekers et al., 1983) and CK7 (Tölle et al.,
1985) were kindly provided by F. Ramaekers and M. Osborn,
respectively. Keratin 7-specific antibody LDS-68 (Southgate et al.,
1987), keratin 14-specific antibody CKB1 (Caselitz et al., 1986), and
smooth muscle α-actin-specific antibody 1A4 (Skalli et al., 1986)
were purchased from Sigma (St Louis, MO). Keratin 17-specific
antibody E3 (Troyanovsky et al., 1989) was kindly provided by S. M.
Troyanovsky. Keratin 19-specific antibody Ks19.1 (A53-B/A2)
(Karsten et al., 1985) was purchased from ICN Biomedicals, Inc. Ecadherin-specific antibody E4.6 (Cepek et al., 1994) was kindly
provided by M. B. Brenner.
Immunocytochemical and immunohistochemical staining
Cultures grown in plastic tissue culture plates or multiwell trays were
rinsed briefly in water and fixed for 15 minutes to 4 weeks in cold (−
20°C) methanol. They then were air-dried for 15 minutes and
incubated for 30 minutes each, separated by 5 minute PBS rinses, with
1% normal goat serum, primary mouse monoclonal antibody at
predetermined optimal dilutions, biotinylated goat anti-mouse IgG
secondary antibody, and avidin-biotin peroxidase complex
(immunocytochemical detection reagents purchased from Vector
Laboratories, Burlingame, CA). The cultures were incubated for a
final 5 minutes with the peroxidase substrate 3-amino-9-ethylcarbazole, the enzymatic reaction stopped by rinsing and incubating
for 10 minutes with 10% formalin in PBS. The cultures were then
mounted with a glass coverslip and aqueous mounting medium for
microscopic analysis and photography.
For immunohistochemical staining, tissue samples or organotypic
cultures were embedded in OCT compound (Miles Inc., Elkhart, IN)
and snap-frozen in liquid nitrogen. Five µm cryostat sections were
acetone-fixed and stained by the ABC-immunoperoxidase method
(reagents from Vector Labs) as described above.
Western blotting
Equal amounts (10 µg/lane) of Triton/high salt-insoluble protein (the
‘cytoskeletal fraction’) of cultured cells, prepared as described by Wu
et al. (1982), were fractionated by SDS-PAGE and electrotransferred
to nitrocellulose filter paper. These blots were first incubated for 1
hour in a blocking buffer consisting of 50 mM Tris, 200 mM NaCl,
0.05% Tween, and 10% nonfat powdered milk (pH 7.5), and then in
a PBS solution of primary antibody for 30 minutes at room
temperature. A peroxidase-conjugated, goat anti-mouse secondary
antibody (Promega, Madison, WI) then was applied and antigenantibody complexes detected by chemiluminescence using the ECL
kit from Amersham, Inc.
Fluorescence-activated cell scanning (FACS)
Cells were detached from culture dishes using 0.1% trypsin/0.02%
EDTA in PBS, fixed with 1% paraformaldehyde in PBS,
permeabilized with 0.3% saponin (Sigma), and rinsed in PBS. The
cells then were incubated in suspension with saturating amounts of
primary antibody or with isotype-matched, non-immune mouse IgG,
followed by FITC-conjugated goat anti-mouse IgG. Finally, cells were
rinsed twice in PBS and analyzed in a FACScan apparatus (BectonDickinson) using Cell Quest Software (BD Immunocytometry
Systems).
RESULTS
Isolation of morphologically distinctive epithelial
cell lines from some human skin-derived
keratinocyte cultures
Rare colonies of morphology different from that of typical
epidermal keratinocyte colonies (Fig. 1a,c,d) were apparent in
some early passage cultures initiated from cells disaggregated
by trypsin from full-thickness human skin biopsies and plated
in the 3T3 feeder layer system (Rheinwald and Green, 1975;
Rheinwald, 1989). These ‘variant’ colonies were tightly
packed but unstratified and intercellular spaces appeared wider
and more refractile when viewed with phase contrast optics.
Immunocytochemical staining disclosed that, unlike
keratinocytes, the variant cells did not express E-cadherin (Fig.
1b) or P-cadherin (data not shown).
Pure populations of this morphologically variant cell type
were isolated from early passage mixed (predominantly
keratinocyte) skin cell cultures from three different donors. In
one case, CYHI, keratinocytes became nonproliferative by the
end of the third passage, much earlier than usual, thereby
permitting the variant cells to prevail and take over the
population. From two other donors, BRSO and DOLA,
colonies of variant morphology were identified in third passage
cultures, isolated with cloning cylinders, and passaged
subsequently as pure populations. The three variant cell lines,
named CYHI, BRSO, and DOLA for the initials of their
donors, were indistinguishable from one another
morphologically. Their distinctive, non-keratinocyte colony
morphology (Fig. 1c) remained constant during serial passage
with 3T3 fibroblast feeder cells in FAD medium until they
senesced after 45-60 population doublings.
BRSO, DOLA, and CYHI cells, unlike epidermal
keratinocytes, grew just as rapidly (Td~1 day) in FAD medium
without fibroblast feeder cells, even from very low density
platings (e.g. <100 cells/cm2). Under these conditions,
however, the cells adopted a very different morphology,
quickly losing contact with neighboring cells in growing
colonies and becoming spindly or stellate (Fig. 1e), a
‘fibroblastoid’ morphology which, nevertheless, was easily
1928 M. Schön and others
Fig. 1. Identification of a rare,
morphologically distinct epithelial
cell type growing in epidermal
keratinocyte cultures initiated from
full thickness biopsies of human
skin. (a) Third passage culture of
BABU skin cells growing in FAD
medium + 3T3 feeder cells, showing
an epidermal keratinocyte colony at
left merging with an unstratified
epithelial cell colony at right,
indicated by arrowhead. The large
cells in the upper right corner are
3T3 feeder cells. (b) Another region
of the same culture as in a
immunostained for E-cadherin,
showing an E-cadherin-negative
epithelial cell colony at right,
indicated by arrowhead, bordered by
E-cadherin-positive keratinocytes.
(c) 6th passage (~35 cumulative
population doublings) culture of
DOLA that had been serially
subcultured in FAD medium + 3T3
feeder cells, showing long-term
retention of the morphology
characteristic of DOLA, BRSO, and
CYHI cells when grown under these
conditions. (d) Second passage
culture of C5-SkH skin cells
growing in the same conditions as c,
showing a pure keratinocyte colony
of typical morphology bordered by
3T3 feeder cells. (e) 8th passage
culture of BRSO (~45 cumulative
population doublings) that had been
grown for three passages in FAD
medium without 3T3 feeder cells,
showing the characteristic spindly and stellate morphology that BRSO, DOLA, and CYHI cells adopt when cultured under these conditions.
(f) 6th passage DOLA cells immunostained for α-smooth muscle actin after having been cultured for 7 days in DME/F12 medium
supplemented with serum and HC but without EGF, showing α-sma expression by a majority of the cells, ranging from low to high levels
(arrowhead points to one of the several completely negative cells in the field). Bar, 200 µm, for all panels.
distinguishable from that of the longer and more slender
human dermal fibroblast. Karyotype analysis of BRSO and
CYHI disclosed that they were diploid, consistent with the
conclusion that they are a normal constituent cell of skin and
not an abnormal keratinocyte variant that had arisen during
growth in culture.
The pattern of cytoskeletal protein expression by
the ‘variant’ cell type in culture is consistent with
their identity as myoepithelial cells
Seeking to identify this novel skin cell type, we used 2-D gel
electrophoresis to analyze the intermediate filament proteins
the cells express in culture (Fig. 2a-c). DOLA (and BRSO, not
shown) expressed keratins K5, K6 (minor), K7, K14, and K16
or K17 (the latter two keratins are not resolved in this system)
as well as high levels of vimentin. Western blot analysis with
keratin subunit-specific monoclonal antibodies confirmed the
expression by DOLA and BRSO cells of K5, K7, K17, and
vimentin (Fig. 3d) and also of K14 (data not shown). The
expression of K7 and high levels of vimentin by DOLA
cells (Fig. 2a) distinguished these cells from epidermal
keratinocytes (Fig. 2b). The expression by DOLA cells of K7
in the absence of any other simple epithelial keratin
distinguished them from mesothelial cells (Fig. 2c) as well as
from all other cell types characterized to date (see Wu et al.,
1982; Moll et al., 1982). Indeed, coexpression of K7 and K17
is very unusual, K19 being the apparent partner for K7 in
keratin filament assembly in all normal epithelial cell types
studied to date (Moll et al., 1982).
When near-confluent DOLA or BRSO cultures growing in
the absence of 3T3 feeder cells were refed with medium
lacking EGF, the cells soon stopped dividing and adopted a
more flattened morphology, some developing prominent
cytoplasmic stress fibers. Many of these flat, growth-arrested
cells were found to express smooth muscle α-actin (Figs 1f,
2f), reminiscent of the expression of this actin isoform by
growth-arrested myoepithelial cells cultured from mammary
gland (Peterson and van Deurs, 1988; O’Hare et al., 1991).
These authors had also reported that mammary gland
myoepithelial cells are dependent on EGF for growth in
culture, consistent with the possibility that the cells we had
cultured from skin were myoepithelial cells, which are a
Sweat gland myoepithelial cell differentiation 1929
Fig. 2. Distinctive pattern of structural proteins expressed by the
novel skin-derived cell type. (a-c) Cytoskeletal extracts from DOLA
cells (a), epidermal keratinocyte strain N (b), and mesothelial cell
strain LP-9 (c) were analyzed by two-dimensional gel
electrophoresis, with non-equilibrium pH gradient electrophoretic
separation in the horizontal dimension (more basic proteins left,
more acidic proteins right) followed by SDS-polyacrylamide gel
electrophoretic separation in the vertical dimension (larger proteins
top, smaller proteins bottom). Asterisks identify the position of
keratin subunits that were not completely dissociated from their
filament partners during the initial isoelectric focusing step (see Wu
et al., 1982; Moll et al., 1982; Franke et al., 1983). (Keratins K16 and
K17 are not resolved in this system, so the presence of K17 must be
determined by immunologic methods (see d). (d-f) Western blot
detection of structural proteins expressed (the protein specifically
recognized by the respective antibody in each extract is shown at
right). (d) Lane 1: BRSO; lane 2: DOLA; lane 3: epidermal
keratinocyte strain N; lane 4: mesothelial cell strain LP-9. The
antibody used to detect K7 in this experiment was RCK105.
(e) Lanes 1 and 2: DOLA; lanes 3 and 4: epidermal keratinocyte
strain N. The cells in lanes 1 and 3 were cultured in FAD medium
with 3T3 feeder cells, and the cells in lanes 2 and 4 were cultured in
Gibco ker-sfm medium. (f) Lane 1: cultured bovine aortic smooth
muscle cells; lane 2: EGF-deprived culture of BRSO; lane 3: EGFdeprived culture of DOLA; lane 4: epidermal keratinocyte strain N.
For each antibody the relative electrophoretic mobility of the main
immunostained band, compared with co-electrophoresed molecular
mass standards, was as expected for the respective protein.
constituent of the secretory coils of eccrine and apocrine sweat
glands (reviewed by Sato, 1993; Holbrook and Wolff, 1993).
Masking of some K7 epitopes in myoepithelial cell
keratin filaments, correlated with K7/K17
coexpression
Arguing against a myoepithelial identity, however, were the
results of a comprehensive immunohistochemical analysis of
Fig. 3. Keratin K7 in the filaments of intact DOLA cells is not
recognized by some K7-specific antibodies that recognize this keratin
in the filaments of mesothelial cells. (a,b) DOLA cells; (c,d) LP-9
mesothelial cells. (a and c) Immunostained with K7-specific antibody
OVTL 12/30. (b and d) Immunostained with K7-specific antibody
Ks7. 18. (e) Western blot of LP-9 (lane 1) and DOLA (lane 2) protein
extracts, showing that the Ks7. 18 antibody recognizes K7 in the
SDS-PAGE fractionated extracts of both cell lines. (Asterisk in e
indicates a lower molecular mass polypeptide recognized by the
antibody in the LP-9 extract, which may be a K7 proteolytic
degradation product.) Bar, 200 µm, for a-d.
keratins expressed during human sweat gland development
(Moll and Moll, 1992), which had concluded that secretory coil
cells express K7, but not K17, and that myoepithelial cells
express K17, but not K7. That study employed the K7-specific
antibody CK7 (Tölle et al., 1985), an antibody which had been
found previously not to immunostain mammary gland
myoepithelial cells (Fischer et al., 1987). We sought to resolve
this question by comparing the immunocytochemical staining
characteristics of five independently derived, K7-specific
monoclonal antibodies on DOLA and BRSO cells and on two
other cell lines that express K7, the primary human mesothelial
cell line LP-9 and HeLa cells. As shown in Fig. 3 and
summarized in Table 1, all five antibodies immunostained the
keratin filaments of mesothelial cells and four out of four of
this set that were tested by western blotting all recognized
electrophoretically-separated K7 protein, both from DOLA
and from LP-9. Three of the five antibodies, however, Ks7.18
(Bartek et al., 1991), CK7 (Tölle et al., 1985), and LDS-68
1930 M. Schön and others
Table 1. Masking of epitopes recognized by some keratin
K7-specific antibodies in keratin filaments of myoepithelial
cells, correlated with expression of K17 as a potential
pairing partner
Mesothelial
cell
Myoepithelial
cell
HeLa
Immunostained by
K7 antibody:
RCK 105
OVTL 12/30
CK7
KS7.18
LDS-68
+
+
+
+
+
+
+
−
−
−
+
+
−
−
−
Keratins expressed
by this cell type:
K5
K7
K8
K14
K17
K18
K19
−
+
+
−
−
+
+
+
+
−
+
+
−
−
−
+
+
−
+
+
−, ±
(Southgate et al., 1987), did not immunostain DOLA cells (Fig.
3), BRSO cells, or HeLa cells (data not shown). The HeLa cell
line is unusual in that it expresses K7, K8, K17, K18, and only
trace amounts of K19 (Moll et al., 1983). As summarized in
Table 1, the results we observed are consistent with the
conclusion that the epitope(s) on K7 recognized by many K7specific monoclonal antibodies is masked in cells in which K7
pairs with K17 in filament formation instead of with its more
common partner, K19.
Identification of K7+/K17+ myoepithelial cells in
sweat glands of human skin in vivo
We used a K7-specific antibody, OVTL 12/30 (van Niekerk et
al., 1991), that could recognize this keratin in the filaments of
DOLA cells to stain cryosections of human scalp skin. As
shown in Fig. 4 and summarized in Table 2, of all the cell types
and structures in skin, only the myoepithelial cell component
of sweat gland secretory coils exhibited the same pattern of
structural protein expression as DOLA and BRSO cells in
culture, namely, K5/K14, K7/K17, vimentin, α-smooth muscle
actin, and an absence of E-cadherin. The cells we have cultured
are therefore almost certainly normal sweat gland
myoepithelial cells.
Fig. 4. The pattern of structural protein expression of eccrine sweat gland myoepithelial cells detected immunohistochemically in vivo is
identical to that of DOLA and BRSO cells in culture. Cryostat sections of human scalp skin were immunostained for the following proteins: (a
and c) K7, (b and d) K17, (e) K14, (f) vimentin, (g) α-smooth muscle actin, and (h) E-cadherin. (a and b) S indicates a sebaceous gland, H
indicates a hair follicle, and E indicates an eccrine sweat gland. (d-f) Asterisks indicate the luminal, secretory cell layers of eccrine gland
secretory coils. (d,f,g,h) Open arrowheads indicate the ductal portions of eccrine glands. (h) The arrows indicate the outer, unstained
myoepithelial cell layer of the eccrine gland secretory coil. Bars: 100 µm (a and b); 50 µm (c-h).
Sweat gland myoepithelial cell differentiation 1931
Table 2. Immunohistochemical detection of filament and junctional proteins in the epithelial structures of human skin
Structure
Epidermis
Hair follicle
Sebaceous gland
Eccrine duct
Eccrine secretory
Eccrine myoepithelium
K5
K7
K8
K14
K17
K19
Vim
α-s.m.
actin
E-cad
+
+
+
+
−
+
−
+
+
−
+
+
−
−
−
−
+
−
+
+
+
+
−
+
−
+
+
−
−
+
−
+
−
+
+
−
−
−
−
−
−
+
−
−
−
−
−
+
+
+
+
+
+
−
Reversible modulation of myoepithelial cell
morphology and keratin expression under different
conditions of culture
The epithelioid-to-fibroblastoid transition of myoepithelial
cells when cultured in the absence of 3T3 feeder cells,
described above, proved to be accompanied by marked changes
in keratin protein expression (Fig. 5). In the ‘fibroblastoid’
state, K5 and K14 content decreased in most cells to
undetectable levels, K7 increased, and K17 remained about the
same. We found that this altered morphology and keratin
expression pattern was rapidly reversible; when DOLA or
BRSO cells that had been serially passaged for ten doublings
or more in the absence of 3T3 feeder cells were then replated
with feeder cells, they converted back to an epithelioid
morphology within several days and re-expressed K5 and K14,
while reducing their level of K7. This ‘switching’ of keratin
expression was quantitated by FACScan analysis (Fig. 6). We
do not yet know the mechanism by which the presence of 3T3
fibroblast feeder cells exerts this marked phenotypic change in
myoepithelial cells. Preliminary experiments disclosed that co-
cultivation with irradiated human dermal fibroblasts also
induced conversion of morphologically fibroblastoid
myoepithelial cells back to an epithelioid phenotype, but
feeding morphologically fibroblastoid myoepithelial cells with
medium conditioned by a confluent culture of 3T3 cells did not
induce epithelioid conversion. Interestingly, DOLA cells
transduced to express the HPV16 E6 and E7 oncoproteins did
not arrest growth or induce expression of α-smooth muscle
actin in the absence of EGF and also did not convert to
epithelioid morphology when cultured with 3T3 feeder cells
(data not shown).
Behavior of myoepithelial cells cultured with
keratinocytes and fibroblasts in Type I collagen
organotypic cultures
In light of the remarkable morphologic plasticity and
modulation and switching of keratin expression patterns under
different conditions of conventional culture, we attempted to
study their differentiation potential in three-dimensional,
‘organotypic’ culture under conditions that would permit them
Fig. 5. The feeder fibroblast-modulated morphologic transition of myoepithelial cells is accompanied by alterations in keratin expression.
DOLA cells were cultured in FAD medium for eight days with (a-e) or without (f-j) 3T3 feeder cells, such that they adopted an epithelioid or
fibroblastoid morphology, respectively. They were then immunostained for the indicated proteins: (a,f) keratin K5; (b,g) keratin K14; (c,h)
keratin K7 (antibody OVTL 12/30); (d,i) keratin K17; (e,j) vimentin. Bar, 100 µm, for all panels.
1932 M. Schön and others
Fig. 6. Quantitative assessment of
modulated keratin expression by
cultured myoepithelial cells by FACS
analysis. DOLA cells were cultured as
described in Fig. 5 and their expression
of intermediate filament proteins was
assessed by FACS (middle and bottom
rows). Epidermal keratinocytes (strain
N) were analyzed for comparison (top
row). The numbers in each panel
indicate the mean fluorescence intensity
of the peak.
to interact with a Type I collagen gel matrix and with other
skin cell types. Myoepithelial cells were co-cultured in
organotypic culture with either epidermal keratinocytes,
dermal fibroblasts, or both (Fig. 7). When plated on the surface
of a Type I collagen gel that contained dermal fibroblasts and
cultured at the air-liquid interface, myoepithelial cells formed
a single cell layer, very different from the stratified,
differentiated epidermal tissue structure formed by
keratinocytes (Fig. 7a,b). When embedded in a collagen gel
with dermal fibroblasts, myoepithelial cells remained as single
cells or formed small aggregates aligned along collagen fibers
but did not form complex structures, such as ducts, discernible
at the level of light microscopy. When plated as a mixed cell
suspension with keratinocytes on the surface of organotypic
cultures, myoepithelial cells tended to sort into clusters,
preferentially aggregating with one another on the collagen
Fig. 7. Myoepithelial cells in organotypic culture
preferentially aggregate but do not stratify or form
obvious glandular structures. DOLA cells were cultured
at the air-liquid interface either on top of a Type I
collagen gel or within the gel, either alone or together
with epidermal keratinocytes or with dermal fibroblasts in
relative proportions indicated below each panel of the
figure. Cryostat sections of the cultures were
immunostained for keratin 7 to identify myoepithelial
cells (a and c) or for keratin 17 to identify both
keratinocytes and myoepithelial cells (b). Arrows at left
and right indicate the surface epithelial/collagen gel
interface. Bar, 100 µm, for all panels.
layer. They also formed close cell-cell attachments with
surrounding keratinocytes such that they moved upward with
them to the level of the granular layer when the keratinocytes
formed a stratified, keratinized epithelium (Fig. 7c). However,
there was no evidence of pore formation similar to the
acrosyringium formed by eccrine sweat gland duct cells as they
penetrate the epidermis in vivo. Myoepithelial cells embedded
alone within a collagen gel adopted a spindle shaped
morphology identifiable by phase microscopy and contracted
the gel, similar to the behavior of dermal fibroblasts (data not
shown).
Identification of proliferative myoepithelial cells as
rare constituents in cultures of other skin biopsies
We sought to identify and culture myoepithelial cells from
eight additional full-thickness skin biopsies in order to estimate
Sweat gland myoepithelial cell differentiation 1933
the population density of proliferative myoepithelial cells in
skin and to attempt to generate additional primary lines of this
cell type. Rare colonies of myoepithelial cells were identified
in second and third passage cultures initiated from three of
these biopsies, JAKE, BABU, and C5-SkH (see Fig. 1a-c). At
the time of their first detection, colonies of myoepithelial cells
were present in these three cultures at only about 1/10001/10,000 the frequency of keratinocyte colonies and at only
about 1/10-1/100 the frequency of fibroblast colonies. We were
able to confirm the identity of myoepithelial cells by
immunostaining cultures enriched in these cells, generated by
isolating regions of cultures containing colonies of
characteristic myoepithelial morphology. However, we were
unable to obtain pure myoepithelial cell cultures free from
keratinocyte and/or fibroblast contamination before senescence
limited their growth.
We sought to identify selective conditions for obtaining pure
populations of myoepithelial cells from mixed skin cell
cultures by comparing the growth rates of myoepithelial cells
with those of epidermal keratinocytes and dermal fibroblasts in
various medium formulations and in the presence of several
keratinocyte growth inhibitors. BRSO and DOLA grew with
similar doubling times in a variety of culture medium
formulations, including DME/F12 and M199, supplemented
with calf serum, EGF, and HC, and also in Gibco ker-sfm
supplemented with EGF, HC, and either bovine pituitary
extract or calf serum. Similar to keratinocytes and unlike
fibroblasts, BRSO and DOLA cells were strongly inhibited by
TGF-β and by PMA (data not shown). We have not yet
identified conditions that are simultaneously permissive for
Fig. 8. Ndk cells express structural proteins
consistent with a myoepithelial origin. Ndk cells at
8th passage were cultured in multiwell plates for 8
days in FAD medium. A phase contrast view of the
cells is shown in a. Cells were immunostained for
the following proteins: (b) keratin K17; (c) keratin
K7 (antibody OVTL 12/30); (d) keratin K7
(antibody Ks7. 18); (e) keratin K19; (f) α-smooth
muscle actin (the cells in this well had been refed
with medium lacking EGF for the final five days
before fixation). Bar, 200 µm, for all panels.
myoepithelial cell growth and selective against both
keratinocytes and fibroblasts.
The ndk cell line exhibits characteristics of
myoepithelial cells
The origin and identity of the ‘ndk’ line, a human skin-derived
cell line with unusual properties (Adams and Watt, 1988), has
remained unresolved. Ndk was isolated from newborn foreskin
as a population of morphologically distinctive cells that grew
during serial passage in the 3T3 feeder layer system and
possessed a longer, albeit finite, replicative lifespan than the
keratinocytes that had predominated in the first several
passages. The ndk cells were reported to have a slight
karyotypic abnormality, to express keratins similar by
electrophoretic analysis to those of epidermal keratinocytes,
and to be unable to form cornified envelopes. As shown in Fig.
8, our immunocytochemical analysis disclosed that ndk cells
express α-sma when deprived of EGF and they express K17
and K7, the latter recognizable in intact cells by the OVTL
12/30 but not by the Ks7.18 antibody. These results suggest that
ndk is of myoepithelial origin and is not a non-differentiating,
mutational variant that arose from a normal epidermal
keratinocyte of the donor. The ndk cells exhibited some
differences from our BRSO and DOLA myoepithelial cell
lines, perhaps as a result of their chromosome abnormality or
because they were near replicative senescence when we
examined them. They did not convert to an epithelioid
morphology when plated with 3T3 cells, a small fraction of the
cells in the population expressed K19, and a similar fraction
were immunostained by the Ks7.18 antibody. Whether the
1934 M. Schön and others
Ks7.18+ cells were the same cells that were K19+ in the ndk
culture was not determined, but it seems likely that at least
some K7 in the K19+ cells paired with K19 instead of K17 to
form filaments, exposing the epitope recognized by the Ks7.18
antibody.
DISCUSSION
Having begun with the objective of determining the identify of
a novel cell type that occasionally appears in human epidermal
keratinocyte cultures, we have identified cell type-specific
markers and have determined permissive culture conditions for
sweat gland myoepithelial cells. The in vitro growth
requirements of skin myoepithelial cells, most notably their
dependence upon EGF and HC in addition to mitogens present
in serum or pituitary extract, are consistent with the results of
earlier studies of human mammary gland myoepithelial cells
in short-term or mixed cell cultures (Peterson and van Deurs,
1988; O’Hare et al., 1991) and are similar to those previously
identified for two simple (as distinguished from stratified)
epithelial cell types, mesothelial cells and kidney tubule
epithelial cells (Connell and Rheinwald, 1983; Rheinwald and
O’Connell, 1985).
Our study discovered the expression of cytokeratin K7 by
myoepithelial cells, which had not been detected previously
(Moll and Moll, 1992) using an antibody that recognizes this
keratin in other cell types, including the secretory coil cells of
the sweat gland. Comparing the keratin expression patterns and
immunocytochemical staining characteristics of cultured
myoepithelial cells, mesothelial cells, and HeLa cells led us to
the conclusion that when cells express K7 and K17 but not
K19, K7 pairs with K17 in filament formation and results in
masking of epitopes recognized by several commonly used K7specific monoclonal antibodies. K7/K17 coexpression is very
unusual. Immunohistologic detection of K7/K17 pairing in
cells, accomplished by using an appropriate set of K7 and K17
antibodies as we have reported here, could be used
diagnostically to aid in identifying normal and neoplastic cells
of myoepithelial origin in skin and other tissues.
The extraordinary degree of morphologic and
differentiative plasticity of the myoepithelial cell in culture
was unexpected, considering the limited ability of sweat
glands to regenerate in developmentally mature skin. The
cells responded to the presence of serum, when in the absence
of fibroblast feeder cells, with a pronounced elongation to a
spindle shape accompanied by an altered pattern of keratin
expression. We do not know whether there is a precise
analogue of this situation in vivo, although structural
disruption and exposure to serum factors during the several
days following deep incisional or abrasive skin wounding
could elicit in myoepithelial cells some of the changes we
observed in culture. The plasticity and considerable
proliferative potential of myoepithelial cells is consistent
with a potential role for this cell type in skin wound healing
in addition to its structural function in the intact sweat gland.
Myoepithelial cells may contract and remodel collagen fibers
in granulation and scar tissue, a possibility supported by the
fact that this cell type can contract a Type I collagen gel in
culture.
The ability to switch between an epithelial and mesenchymal
cell morphology and pattern of protein expression occurs as
part of the morphogenesis of many tissues during development
but it is a very uncommon property among somatic cell types
in the adult. Mesothelial cells and renal cortical tubule
epithelial cells are two other cell types that can undergo a
similar epithelial-mesenchymal conversion in culture and,
presumably, under certain conditions in vivo (Connell and
Rheinwald, 1983; LaRocca and Rheinwald, 1984; Rheinwald
and O’Connell, 1985). We found that myoepithelial cells stably
transfected to express the HPV16 E6 and E7 proteins were
unable to arrest growth and express α-sma when cultured in
the absence of EGF and also were unable to convert to an
epithelial phenotype when co-cultured with 3T3 feeder cells.
This indicates that the ability to modulate expression of both
of these features of myoepithelial cell differentiation requires
normal p53 and/or pRB dependent cell cycle regulation. The
myoepithelial cell’s ‘epithelial/mesenchymal’ switching
differs from that of the mesothelial and kidney tubule cell in
that the myoepithelial cell continues to express two keratins,
K7 and K17, in its morphologically fibroblastoid state, whereas
mesothelial cells and kidney tubule epithelial cells become
completely keratin-negative.
The ndk cell line, isolated as a morphologic variant from an
epidermal keratinocyte culture by Adams and Watt (1988), has
proved to express distinctive myoepithelial markers, K7/K17
and α-sma, suggesting that this cell line is not a differentiation
defective keratinocyte mutant, as had been proposed initially.
The late passage ndk cells we examined exhibited slight
differences from our three diploid primary myoepithelial cell
lines, however. The ndk cells did not reacquire an epithelioid
morphology when cultured with 3T3 feeder cells and a small
proportion of the cells expressed K19. This may be a result of
the chromosome 1 abnormality identified in this cell line
(Adams and Watt, 1988).
Both the apocrine and eccrine sweat glands of human skin
contain myoepithelial cells. The cells we cultured were derived
from full-thickness skin specimens, most of which were from
axilla, so our myoepithelial cell lines could be of either eccrine
or apocrine origin. We were unable to obtain specimens of
human skin from which the relatively rare apocrine glands
could be identified in sections for immunohistochemical
analysis. Ultrastructural examination has identified
myoepithelial cells in the secretory coil of apocrine glands as
well as eccrine glands (for example, see Saga and Takahashi,
1992). Although the developmental formation of these two
types of sweat glands is different, they form at different times
from separate cell lineages in the primitive epidermis, the
function of myoepithelial cells in all glands that contain them
appears to be the same. Myoepithelial cells from eccrine,
apocrine, and even mammary, salivary, and lacrymal glands
would, therefore, be expected to have very similar patterns of
gene expression and regulation. Possible differences among
them might include differential responsiveness to reproductive
hormones and neurotransmitters which regulate the function of
some of these glands and may affect the myoepithelial cells
directly.
The stringent growth requirement of myoepithelial cells for
EGF and HC, even in the presence of high concentrations of
serum, is also a characteristic of human mesothelial cells and
kidney tubule epithelial cells (Connell and Rheinwald, 1983;
Rheinwald and O’Connell, 1985). However, in contrast to the
Sweat gland myoepithelial cell differentiation 1935
latter cell types, myoepithelial cells are inhibited by TGF-β and
by PMA, resembling the keratinocyte in this respect. Further
research on the biology of this cell type will be aided greatly
by identifying a culture medium formulation that promotes
myoepithelial cell proliferation while completely or
substantially preventing keratinocyte and fibroblast
proliferation, thereby permitting routine initiation of primary
myoepithelial cell lines from full-thickness skin biopsies. Rare
proliferative myoepithelial cells in skin biopsies could
potentially be selected out from the large population of
epidermal keratinocytes by culturing in serum-supplemented
Gibco ker-sfm medium without 3T3 feeder cells. However, any
dermal fibroblasts also present in such cultures can eventually
outgrow the myoepithelial cells in this and all other medium
formulations we have tested to date. Enriched myoepithelial
cell populations have been obtained from mammary gland cell
suspensions by antibody-based cell sorting, exploiting the
expression of the CALLA antigen by mammary gland
myoepithelial cells in vivo (O’Hare et al., 1991; Gomm et al.,
1995). Although myoepithelial cells are present at several
orders of magnitude lower proportion of the total cell
population in skin than in the mammary gland, such a strategy
may aid the isolation of pure sweat gland myoepithelial cell
populations.
The repair and regeneration capacity of skin adnexal
structures and the regulatory mechanisms involved are subjects
of great interest, in part for the potential applicability of this
knowledge to developing improved transplantation and
pharmacologic strategies for restoring skin damaged by burns.
The epidermis can be restored permanently on large areas of
full thickness wounds by applying sheets of cultured
autologous epidermal keratinocytes to residual dermal or
granulation tissue (O’Connor et al., 1981; Leigh et al., 1991)
but regenerating the very complex structures of the
pilosebaceous unit and the eccrine sweat gland presents a much
greater challenge (reviewed by Martin, 1997). The patterns and
density of these adnexae in the normal adult skin are
determined and the structures themselves formed during midgestation development. Pluripotent progenitor cells, which in
the embryonic epidermis migrate downward, replicate, and
ultimately differentiate into the three cell types of the mature
eccrine sweat gland, are unlikely to be present in adult skin.
After a partial thickness wound, the eccrine sweat duct can
reestablish its connection through the regenerated epidermis to
the skin surface. Duct cells, therefore, must retain both
replicative potential and the ability to form their appropriate
three dimensional structure, presumably using the deeper,
undamaged part of the duct both as a source of stem cells and
as a template. In full thickness wounds, however, both the duct
and the secretory coil of the sweat gland are destroyed.
Although the surface of such a wound may become
reepithelialized by keratinocytes migrating from the periphery
of the wound and differentiating to reestablish the epidermis,
sweat glands do not regenerate.
Cells of the sweat gland secretory coil normally undergo
very little turnover and replacement (for example, see
Morimoto and Saga, 1995). The type and extent of damage and
disruption from which this structure can recover by repairing
itself and reestablishing connection with remnants of the duct
is unknown, however, and the proliferative potential of the
myoepithelial cells is consistent with regeneration potential.
Our initial attempt to assess the histogenic potential of
myoepithelial cells in vitro, using a Type I collagen matrix
organotypic culture system with dermal fibroblasts and
epidermal keratinocytes, was inconclusive. Culturing
myoepithelial cells in the presence of basement membrane
collagens and laminins, sweat gland duct and secretory cells,
and soluble morphogenetic proteins such members of the BMP
family would provide a more comprehensive in vitro
assessment of the histogenic potential of this cell type. The
development of methods for identifying and culturing
myoepithelial cells which we have reported here, combined
with methods for initiating cultures of eccrine duct and
secretory cells (Collie et al., 1985; Lee et al., 1986; Jones et
al., 1988; Pedersen, 1989) provides a starting point for
investigations aimed at understanding the regenerative
potential of the sweat gland and at transplanting cultured cells
to restore these structures in damaged skin.
We thank J. Sigler, O. Kehinde, S. Schaffer, A. Schermer, and R.
Tubo of Biosurface Technology, Inc. for providing cryopreserved
ampules of early passage cells cultured from human skin biopsies and
for providing several pure or enriched cell populations of cells which
we subsequently characterized as myoepithelial cells. We thank Dr R.
Tantravahi for karyotyping the CYHI and BRSO cell lines, Dr N.
Parenteau for providing materials for and advice on preparing
organotypic cultures, and Dr F. Watt for providing the ndk cell line.
We also thank Drs T.-T. Sun, S. M. Troyanovsky, M. Osborn, and F.
Ramaeckers for providing keratin antibodies. This work was
supported by Skin Disease Research Center grant # P30 AR42689
from the N.I.H. to T.S. Kupper, by grants from Biosurface
Technology, Inc. and from Organogenesis, Inc. to J.G.R., and by a
postdoctoral fellowship from the Deutsche Forschungsgemeinschaft
to M.S. Some of the data reported here were presented at the 1995
meeting of the Society for Investigative Dermatology and were
published in abstract form (O’Connell-Willstaedt, T., Schön, M. and
Rheinwald, J. G. (1995) J. Invest. Dermatol. 104, 641).
REFERENCES
Adams, J. C. and Watt, F. M. (1988). An unusual strain of human
keratinocytes which do not stratify or undergo terminal differentiation in
culture. J. Cell Biol. 107, 1927-1938.
Allen-Hoffmann, B. L. and Rheinwald, J. G. (1984). Polycyclic aromatic
hydrocarbon mutagenesis of human epidermal keratinocytes in culture.
Proc. Nat. Acad. Sci. USA 81, 7802-7806.
Bartek, J., Vojtesek, B., Staskova, Z., Bartkova, J., Kerekes, Z., Rejthat,
A. and Kovarik, J. (1991). A series of 14 new monoclonal antibodies to
keratins: characterization and value in diagnostic histopathology. J. Pathol.
164, 215-224.
Bell, C. L. and Quinton, P. M. (1991). Effects of media buffer systems on
growth and electrophysiologic characteristics of cultured sweat duct cells.
In Vitro Cell Dev. Biol. 27, 47-54.
Caselitz, J., Walther, B., Wustrow, J., Seifert, G., Weber, K. and Osborn,
M. (1986). A monoclonal antibody that detects myoepithelial cells in
exocrine glands, basal cells in other epithelia and basal and suprabasal cells
in certain hyperplastic tissues. Virchows Arch A Pathol. Anat. Histopathol.
409, 725-738.
Cepek, K. L., Shaw, S. K., Parker, C. M., Russell, G. J., Morrow, J. S.,
Rimm, D. L. and Brenner, M. B. (1994). Adhesion between epithelial cells
and T lymphocytes mediated by E-cadherin and the αEβ7 integrin. Nature
372, 190-193.
Cohn, B. A. (1994). In search of human skin pheromones. Arch Dermatol.
130, 1048-1051.
Collie, G., Buchwald, M., Harper, P. and Riordan, J. R. (1985). Culture of
sweat gland epithelial cells from normal individuals and patients with cystic
fibrosis. In Vitro Cell Dev Biol. 21, 597-602.
Connell, N. D. and Rheinwald, J. G. (1983). Regulation of the cytoskeleton
1936 M. Schön and others
in mesothelial cells: reversible loss of keratin and increase in vimentin
during rapid growth in culture. Cell 34, 245-253.
Debus, E., Weber, K. and Osborn, M. (1982). Monoclonal cytokeratin
antibodies that distinguish simple from stratified squamous epithelia:
Characterisation on human tissues. EMBO J. 1, 1641-1647.
Fischer, H. P., Altmannsberger, M., Weber, K. and Osborn, M. (1987).
Keratin polypeptides in malignant epithelial liver tumors. Differential
diagnostic and histogenic aspects. Am. J. Pathol. 127, 530-537.
Franke, W., Schmid, E., Reudenstein, C., Appelhans, B., Osborn, M.,
Weber, K. and Keenan, T. W. (1980). Intermediate-sized filaments of
prekeratin type in myoepithelial cells. J. Cell Biol. 84, 633-654.
Franke, W. W., Schiller, D. L., Hatzfield, M. and Winter, S. (1983). Protein
complexes of intermediate-sized filaments: melting of cytokeratin
complexes in urea reveals different polypeptide separation characteristics.
Proc. Nat. Acad. Sci. USA 80, 7113-7117.
Gomm, J. J., Browne, P. J., Coope, R. C., Liu, Q. Y., Buluwela, L. and
Coombes, R. C. (1995). Isolation of pure populations of epithelial and
myoepithelial cells from the normal human mammary gland using
immunomagnetic separation with dynabeads. Anal. Biochem. 226, 91-99.
Holbrook, K. A. and Wolff, K. (1993). The structure and development of
skin. In Dermatology in General Medicine (ed. T. B. Fitzpatrick, A. Z. Eisen,
K. Wolff, I. M. Freedberg and K. F. Austen), pp. 97-145. McGraw-Hill, New
York.
Jones, C. J., Bell, C. L. and Quinton, P. M. (1988). Different physiological
signatures of sweat gland secretory and duct cells in culture. Am. J. Physiol.
255, C102-C111.
Karsten, U., Papsdorf, G., Roloff, G., Stolley, P., Abel, H., Walther, I. and
Weiss, H. (1985). Monoclonal anti-cytokeratin antibody from a hybridoma
clone generated by electrofusion. Eur. J. Cancer Clin. Oncol. 21, 733-740.
LaRocca, P. J. and Rheinwald, J. G. (1984). Coexpression of simple
epithelial keratins and vimentin by human mesothelium and mesothelioma
in vivo and in culture. Cancer Res. 44, 2991-2999.
Lee, C. M., Carpenter, F., Coaker, T. and Kealey, T. (1986). The primary
culture of epithelia from the secretory coil and collecting duct of normal
human and cystic fibrotic eccrine sweat glands. J. Cell Sci. 83, 103-118.
Leigh, I. M., McKay, I., Carver, N., Navsaria, H. and Green, C. (1991).
Skin equivalents and cultured skin: from the petri dish to the patient. Wounds
3, 141-148.
Lemullois, M., Rossignol, B. and Mauduit, P. (1996). Immunolocalization
of myoepithelial cells in isolated acini of rat exorbital lacrimal gland:
cellular distribution of muscarinic receptors. Biol. Cell 86, 175-181.
Lynch, M. H., O'Guin, W. M., Hardy, C., Mak, L. and Sun, T.-T. (1986).
Acidic and basic hair/nail (‘hard’) keratins: their co-localization in upper
cortical and cuticle cells of the human hair follicle and their relationship to
‘soft’ keratins. J. Cell Biol. 181, 2593-2606.
Martin, P. (1997). Wound healing–aiming for perfect skin regeneration.
Science 276, 75-81.
Moll, I. and Moll, R. (1992). Changes of expression of intermediate filament
proteins during ontogenesis of eccrine sweat glands. J. Invest. Dermatol. 98,
777-785.
Moll, R., Franke, W. W. and Schiller, D. L. (1982). The catalog of human
cytokeratins: Patterns of expression in normal epithelia, tumors and cultured
cells. Cell 31, 11-24.
Moll, R., Krepler, R. and Franke, W. W. (1983). Complex cytokeratin
polypeptide patterns observed in certain human carcinomas. Differentiation
23, 256-269.
Morimoto, Y. and Saga, K. (1995). Proliferating cells in human eccrine and
apocrine sweat glands. J. Histochem. Cytochem. 43, 1217-1221.
Norberg, L., Dardick, I., Leung, R., Burford-Mason, A. and Rippstein, P.
(1992). Immunogold localization of actin and cytokeratin filaments in
myoepithelium of human parotid salivary gland. Ultrastruct Pathol. 16, 555568.
O’Connor, N. E., Mulliken, J. B., Banks-Schlegel, S., Kehinde, O. and
Green, H. (1981). Grafting of burns with cultured epithelium prepared from
autologous epidermal cells. The Lancet 10, 75-78.
O’Hare, M. J., Ormerod, M. G., Monaghan, P., Lane, E. B. and Gusterson,
B. A. (1991). Characterization in vitro of luminal and myoepithelial cells
isolated from the human mammary gland by cell sorting. Differentiation 46,
209-221.
Osborn, M., Debus, E. and Weber, K. (1984). Monoclonal antibodies specific
for vimentin. Eur. J. Cell Biol. 34, 137-143.
Parenteau, N. L., Nolte, C. M., Bilbo, P., Rosenberg, M., Wilkins, L. M.,
Johnson, E. W., Watson, S., Mason, V. S. and Bell, E. (1991). Epidermis
generated in vitro: Practical considerations and applications. J. Cell.
Biochem. 45, 245-251.
Pedersen, P. S. (1989). Human sweat duct cells in primary culture. Basic
bioelectric properties of cultures derived from normals and patients with
cystic fibrosis. In Vitro Cell Dev. Biol. 25, 342-352.
Peterson, O. W. and van Deurs, B. (1988). Growth factor control of
myoepithelial-cell differentiation in cultures of human mammary gland.
Differentiation 39, 197-215.
Pirisi, L., Yasumoto, S., Feller, M., Doniger, J. and DiPaolo, J. A. (1987).
Transformation of human fibroblasts and keratinocytes with human
papillomavirus type 16 DNA. J. Virol. 61, 1061-1066.
Ramaekers, F., Huysmans, A., Moesker, O., Kant, A., Jap, P., Herman, C.
and Vooijs, P. (1983). Monoclonal antibody to keratin filaments, specific
for glandular epithelia and their tumors. Lab. Invest. 49, 353-361.
Rheinwald, J. G. and Green, H. (1975). Serial cultivation of strains of human
epidermal keratinocytes: The formation of keratinizing colonies from single
cells. Cell 6, 331-344.
Rheinwald, J. G. and Beckett, M. A. (1980). Defective terminal
differentiation in culture as a consistent and selectable character of
malignant human keratinocytes. Cell 22, 629-632.
Rheinwald, J. G. and O'Connell, T. M. (1985). Intermediate filament
proteins as distinguishing markers of cell type and differentiated state in
cultured human urinary tract epithelia. Ann. NY Acad. Sci. 455, 259-267.
Rheinwald, J. G. (1989). Methods for clonal growth and serial cultivation of
normal human epidermal keratinocytes and mesothelial cells. In Cell
Growth and Division: A Practical Approach (ed. R. Baserga), pp. 81-94.
IRL Press, Oxford.
Rollins, B. J., O'Connell, T. M., Bennett, G., Burton, L. E., Stiles, C. D.
and Rheinwald, J. G. (1989). Environment-dependent growth inhibition of
human epidermal keratinocytes by recombinant human transforming growth
factor-β. J. Cell. Physiol. 139, 455-462.
Saga, K. and Takahashi, M. (1992). Immunoelectron microscopic
localization of epidermal growth factor in eccrine and apocrine sweat
glands. J. Histochem. Cytochem. 40, 241-249.
Sato, K., Nishiyama, A. and Kobayashi, M. (1979). Mechanical properties
and functions of the myoepithelium in the eccrine sweat gland. Am. J.
Physiol. 237, C177-C184.
Sato, K. (1993). Biology of the eccrine sweat gland. In Dermatology in
General Medicine (ed. T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M.
Freedberg and K. F. Austen), pp. 221-241. McGraw-Hill, New York.
Schön, M. and Rheinwald, J. G. (1996). A limited role for retinoic acid and
retinoic acid receptors RARα and RARβ in regulating keratin 19 expression
and keratinization in oral and epidermal keratinocytes. J. Invest. Dermatol.
107, 428-438.
Skalli, O., Ropraz, P., Treciak, A., Benzonana, G., Gillessen, D. and
Gabbiani, G. (1986). A monoclonal antibody against α-smooth muscle
actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103, 27872796.
Southgate, J., Williams, H. K., Trejdosiewicz, L. K. and Hodges, G. M.
(1987). Primary culture of human oral epithelial cells. Growth requirements
and expression of differentiated characteristics. Lab. Invest. 56, 211-223.
Tölle, H.-G., Weber, K. and Osborn, M. (1985). Microinjection of
monoclonal antibodies specific for one intermediate filament protein in cells
containing multiple keratins allow insight into the composition of particular
10 nm filaments. Eur. J. Cell Biol. 38, 234-244.
Troyanovsky, S. M., Guelstein, V. I., Tchipysheva, T. A., Krutovskikh, V.
A. and Bannikov, G. A. (1989). Patterns of expression of keratin 17 in
human epithelia: dependency on cell position. J. Cell Sci. 93, 419-426.
van Niekerk, C. C., Jap, P. H., Ramaekers, F. C., van de Molengraft, F.
and Poels, L. G. (1991). Immunohistochemical demonstration of keratin
7 in routinely fixed paraffin-embedded human tissues. J. Pathol. 165, 145152.
Wu, Y.-J., Parker, L. M., Binder, N. E., Beckett, M. A., Sinard, J. H.,
Griffith, C. T. and Rheinwald, J. G. (1982). The mesothelial keratins: A
new family of cytoskeletal proteins identified in cultured mesothelial cells
and nonkeratinizing epithelia. Cell 31, 693-703.
Yokozeki, H., Saga, K., Sato, F. and Sato, K. (1990). Pharmacological
responsiveness of dissociated native and cultured eccrine secretory coil cells.
Am. J. Physiol. 258 (Reg Int Comp Physiol 27), R1355-R1362.