ARTICLE
Auteur(s) : Md Saiful Islam1, Huanmin Zhou2
1College of Animal Science and Medicine. Inner
Mongolia Agricultural University, No. 306 # Zhao Wu Da Road, Huhhot
010018, Inner Mongolia, People’s Republic of China
2Department of Bio-engineering, Inner Mongolia
Agricultural University, No. 306 # Zhao Wu Da Road, Huhhot 010018,
Inner Mongolia, People’s Republic of China
accepté le 21 Mars 2007
The epidermis is the outermost layer of the body and is in direct
contact with the external environment. It is constantly renewed,
and consists of keratinocytes showing variable degrees of
differentiation. Cell kinetic analyses of epidermal turnover can be
used to divide keratinocytes into stem cells, transient amplifying
(TA) cells, and post mitotic differentiating cells [14, 29].
Epidermal stem cells, like other adult stem cells, are best defined
by their capacity to self-renew and to generate large amounts of
tissue for an extended period of time or even a life time.
Transient-amplifying cells replicate with higher frequencies than
stem cells but have limited proliferative potentials. They further
differentiate, detach from the basement membrane, and migrate
toward the epidermis surface. Terminally differentiated cells
subsequently die and form external layers of cornified cells [1,
10, 12, 17, 19]. Recent studies have described two topographical
sites of epidermal stem cell location. The first is in the upper
regions of the outer root sheath of the hair follicle (the
so-called bulge region), and the second is the basal layer of the
interfollicular epidermis [13, 29]. There is compelling evidence
that stem cells with differing epithelial lineage potentials are
present at several locations in the skin: for example, the basal
layer of the epidermis contains unipotent epidermal stem cells
whereas the upper region (the bulge) of the hair follicle contains
multipotent stem cells.Isolation and identification of putative
epidermal stem cells has been carried out before. In earlier
studies, it has been found that in vitro three types of colony,
holoclone, paraclone and meroclone, could be formed because of the
different amplification potentials of human keratinocytes [1].
Holoclones are thought to be derived from epidermal stem cells,
while the other two types of clone are from TA cells. Epidermal
stem cells could be selected using rapid substrate attachment.
Collagen type IV, an extracellular matrix protein, is the ligand of
β1 integrin which showed high levels of expression in
epidermal stem cells and thus collagen IV may be a possible
candidate for the selection of epidermal stem cells [2, 9-11]. One
of the controversies in the in-depth study of skin stem cells is
related to the existence of specific phenotypic markers. Many
attempts have been made to identify a universal epidermal stem cell
marker; no specific markers have been commonly accepted. Recent
research has identified putative epidermal stem cell markers, that
relate to their slow-cycling, clonogenic traits and specific
localization, but no unequivocal tag is yet available. The
potential candidate skin stem cells markers include β1 integrin,
keratin 15, keratin 19, CD71, transcription factor p63 and CD34
[15]. Although several characteristics such as capacity of
self-renewal and multipotency have been defined for the epidermal
stem cells, the determination of a specific marker or combination
of markers that would assist in their direct identification within
the epidermis is still the focus of much interest.In this study, we
isolated a cell population as putative epidermal stem cells by
rapid attachment on collagen IV substrate from cultured goat fetal
epidermal keratinocytes in vitro. The selected population was
cultured and characterized by colony formation efficiency (CFE),
morphological study and the expression of molecular markers.
Materials and methods
Fibroblast culture as feeder cell
In the study, we used fetuses of 12-16 weeks of gestation. The goat
fetal dermal fibroblasts were obtained from a fetal foreskin biopsy
and cultured by a previously published method [5]. The skin sample
was minced into small pieces (1 mm3) and 10-15
small pieces were placed into a 60 mm cell culture plate.
4 mL of fibroblast growth medium [Dulbecco’s Modified Eagle’s
Medium (DMEM) (Gibco) containing 10% Fetal Bovine Serum (FBS)
(TBD), 25 mM HEPES (Sigma) and 100 U/mL Penicillin (Sigma),
100 μg/ml Streptomycin (Sigma)] were added to culture and incubated
at 37 °C in the presence of 5% CO2 in a cell
culture incubator. At early confluence, fibroblasts were detached
by trypsin-EDTA (Sigma) (0.1% trypsin and 0.02% EDTA in PBS),
expanded in 60 mm culture plates and subcultured at a final
seeding density of about 105 viable cells per mL.
Keratinocyte culture
Normal keratinocyte cell suspensions were prepared from fetal skin
tissue derived from sacrificed pregnant goats. Keratinocyte culture
and serial cultivation were performed using the methods previously
described [20]. Skin from the abdomen and back of fetuses was
removed by using surgical scissors and a scalpel with a surgical
blade. After removing fat and membranous materials, the skin piece
was sliced into 0.5 cm wide strips, immersed in 0.125% trypsin
solution and placed at 4 °C overnight. After overnight
incubation, the epidermis was scraped from the dermis with a
scalpel and placed into a sterilized Ehrlenmayer flask with a
sterilized magnetic mixing bar containing keratinocyte growth
medium [Three parts of DMEM plus one part of Ham’s F12 medium
(Gibco) containing 10% FBS, 20 mM HEPES, 100 U/mL Penicillin, 100
μg/mL Streptomycin, 5 μg/mL Transferrin (Sigma), 5 μg/mL Insulin
(Sigma), 1.4 ng/mL Triiodothyronine (Sigma), 24 μg/mL Adenine
(Sigma), 0.4 μg/mL Hydrocortisone (Sigma), 10 ng/mL Epidermal
growth factor (EGF) (Sigma)] to completely cover the tissues and
mixed on a magnetic stirrer with a medium speed at room temperature
for 1 hour. The cell suspensions were filtered through a sterile
mesh (100 μm). Cells were resuspended in keratinocyte growth
medium and cultured on mitomycin C (Roche) treated 24 h old
fibroblast feeder layer at a final seeding cell density of
approximately 105 viable cells per ml. The cultures were
grown in a humidified 5% CO2 atmosphere at 37 °C.
Keratinocytes were passaged at 80% confluence after removing the
feeder cells with 0.02% EDTA.
Clonal analysis
Clonal analysis was performed as described previously [19] with
slight modifications. 25-30 cells from secondary keratinocyte
cultured masses were plated onto 60 mm culture plates. After
10 days, individual large colonies were identified under the
microscope. Selected individual enlarging colonies were then
detached by cloning cylinder (Sigma) as described previously [4],
and each was then transferred to a new plate already containing
feeder cells. The plate was fixed and stained with 1% rhodamine B
after 12-14 days of culture. Each clone was scored according to its
size and whether its progeny was progressively growing or aborted.
Determination of number of cell generations
Number of cell generations was determined as described previously
[22]. It was calculated using the following formula: x =
3.322 log N/No, where N equals the total number of cells
obtained at each passage and No equals the number of clonogenic
cells. Clonogenic cells were calculated from the colony-forming
efficiency data, which were determined separately in parallel
dishes at the time of cell passage. Colony forming efficiency was
evaluated as the ratio of the number of colonies to the number of
inoculated cells.
Separation of putative epidermal stem cells by adherence on
type IV collagen
Culture media
Medium-I
Keratinocyte growth medium
Medium-II
Medium-II was composed of three parts of DMEM (chelex-treated) plus
one part of Ham’s F12 medium (chelex-treated) containing 5% FBS
(chelex treated), 20 mM HEPES, 100 U/mL Penicillin, 100 µg/mL
Streptomycin, 5 µg/mL Transferrin, 5 µg/mL Insulin, 1.4 ng/mL
Triiodothyronine, 24 µg/mL Adenine, 0.4 µg/mL Hydrocortisone, 10
ng/mL EGF and 0.05 mM CaCl2.
Medium-III
It was made up of three parts of DMEM (Chelex treated) plus one
part of Ham’s F12 medium (Chelex treated) containing 9% FBS (Chelex
treated), 100 U/mL Penicillin, 100 μg/mL Streptomycin, 4 ng/ml EGF,
0.05 mM CaCl2 and 50% Fibroblast conditioned medium
(mentioned below).
Preparation of chelex treated DMEM, F-12 and FBS to remove free
Ca2+
To deplete the calcium ions in DMEM, F-12 and FBS, the medium and
FBS were incubated with chelating resin chelex 100 (Sigma)
overnight at 4 °C. Chelex resin was washed with distilled
water before use.
Preparation of conditioned medium
Fibroblasts (3-5 passages) in their normal growth medium were
allowed to grow to about 60% confluence. 30 mL of the low
Ca2+ medium (mentioned below) were added after washing
the cell layer with PBS. Conditioned medium were collected after
two days and stored at –20 °C. After collection, fibroblasts
were cultured in their normal growth medium for at least two days
to allow the cells to recover before they could be used for
conditioned collection again. Before use, the conditioned medium
was thawed at 4 °C overnight, centrifuged at 5,000 g for
10-15 minutes at 4 °C and filtered using a filter with
0.2 μm size.
Preparation of low Ca2+ medium
Low Ca2+ medium consists of 3 parts of DMEM (Chelex
treated) plus one part of Ham’s F12 medium (Chelex treated)
containing 9% FBS (Chelex treated), 100 U/mL Penicillin, 100 μg/mL
Streptomycin, and 0.05 mM CaCl2.
Preparation of collagen IV coated plate
1 mL of collagen solution containing 100 μg/mL (5
μg/cm2) of collagen type IV in dH2O was
placed in 35 mm culture plate in order to ensure the solution
was spread uniformly over the surface of the plate for 4 °C
overnight. Coated culture plates were rinsed three times with PBS
or culture medium to neutralize the acidity before seeding the
cells.
Seeding the cells on type IV collagen coated plates
Cell suspension, obtained by detaching keratinocytes in cultures,
were seeded 105 cells/mL in the collagen IV coated plate
to allow the cells to attach in the cell culture incubator with 5%
CO2 at 37 °C. After 10 min, unattached cells
were removed by a gentle wash of the cell layer with the medium and
2 mL of growth medium were added to the cells. Cells were
cultured for 4 days before replacing with a fresh growth medium.
Thereafter, every third day the medium was changed.
Immunofluorescence staining
At 50% confluence, cells were fixed with 2% formaldehyde in PBS and
permeabilized with of 0.5% Triton X-100 in PBS. After three washes
in PBS, the cells were incubated with primary antibody for
overnight at 4 °C. After washing with PBS three times, FITC
conjugated secondary antibody (Southern Biotechnology) was added
and incubated for one hour at room temperature. Cells were examined
under a fluorescent and confocal microscope. The anti-keratin 15
(Clone LHK 15), anti-keratin 19 (Clone A53-B/A2.26), Anti p63
(Clone 4A4), Anti CD71 (Clone 10F11), Anti involucrin (Clone SY5)
and Anti Keratin 10 (Clone LHP1) monoclonal antibodies were used in
the immunofluorescence staining at dilutions suggested by the
suppliers. All antibodies were purchased from Lab Vision
Corporation.
Results
Cultivation of keratinocytes on fibroblast feeder layers
When a skin specimen was placed onto a culture plate, the initial
spindle shaped fibroblast monolayer formed around the tissue
explants after 2-3 days. After culturing for 8-9 days, the
fibroblasts reached confluence (figure 1A). Keratinocytes
were maintained in culture with mitomycin C treated fetal skin
fibroblasts as a feeder layer and consistently formed colonies
(figure 1B, C and
D). Keratinocytes started to grow slowly on the feeder
layer and first colonies were observed after 5-7 days in primary
culture. With successive passages, cells grew with greater
frequency. Cell growth speeded up once the cell density became
greater. The shapes and appearances of most colonies in the primary
cultured mass, as well as in subsequent culture, were typical of
epithelial cells. With successive passages, the culture reached
confluence within 7-8 days after plating. Keratinocytes were
serially cultivated up to 8 passages (about 48 ± 5 cumulative cell
doublings) without showing signs of replicative senescence (figure 2).
Clonogenic ability of keratinocytes
When small numbers of keratinocytes (25-30 cells/plate) were
inoculated, 5-17 single colonies were detected in culture after 10
days. Twelve enlarging single colonies were selected in different
culture plates, detached after 10 days, and each was transferred to
a new 60 mm culture plate (200 viable cells/plate). The
culture was fixed 12-14 days later and used to classify the clonal
type on the basis of colony size and whether their progeny were
progressively growing or aborted. Holoclones, meroclones, and
paraclones were detected after subcloning (table
1, figure
3). Table 1 shows that 965
clones from 12 individual colonies were analyzed. The majority of
the clones were classified as meroclone.
Table 1 Different types of clone
|
Original clone No.
|
Subclones
|
|
Total number of subclones/200 cells seeded
|
%CFE
|
No. of each type of clone
|
|
Holoclone
|
Meroclone
|
Paraclone
|
|
1
|
59
|
29.5
|
10
|
32
|
17
|
|
2
|
67
|
33.5
|
8
|
48
|
11
|
|
3
|
93
|
46.5
|
21
|
49
|
23
|
|
4
|
43
|
21.5
|
17
|
17
|
9
|
|
5
|
118
|
59
|
23
|
58
|
37
|
|
6
|
84
|
42
|
6
|
38
|
40
|
|
7
|
87
|
43.5
|
18
|
46
|
23
|
|
8
|
94
|
47
|
17
|
42
|
35
|
|
9
|
113
|
56.5
|
21
|
73
|
19
|
|
10
|
72
|
36
|
11
|
48
|
13
|
|
11
|
74
|
37
|
26
|
34
|
14
|
|
12
|
61
|
30.5
|
14
|
31
|
16
|
Putative epidermal stem cell selection by adherence on collagen
IV substrate
After seeding in a collagen type IV coated plate, ~ 10-20% of the
total amount of cells remained attached after 10 minutes
incubation. The cells grew slowly at first and after 5-6 days began
to grow quickly, taking 8-10 days to reach confluent. By day 4-5,
colonies appeared, sized about a few cells, and the cells were
flattened (figure
4). They were replated in the same conditions and showed
the same proliferative potential in subsequent culture. After 3-4
passsages, cells were serially cultivated in non-coated plate. The
high Ca2+ condition culture reached confluent more
rapidly than low calcium conditions (medium II, & Medium III).
Cellular growth in coated plate was not so clonal as compared with
the keratinocytes on the feeder layer. Generally they grew
dispersedly in the culture plate and showed the same morphology in
serial culture. In this study, the cultures were propagated
serially up to 12th passage in high Ca2+
condition and 6th passage in low Ca2+
conditions.
Colony formation efficiency
When 1000 cells from three different medium cultures (Medium I,
Medium II and Medium III condition) were seeded onto a 100 mm
cell culture plate containing feeder cells, they could form
colonies and the CFE of 18.05 ± 1.38%, 22.02 ± 1.79% and 20.22 ±
1.27% were determined in three different culture conditions;
respectively. Only colonies > 32 cells were scored. The CFE of
cell populations in low calcium conditions (Medium II and Medium
III) were significantly higher than those of the cell populations
in high Ca2+ conditions (Medium I) (P < 0.05). This
observation may be related to stemness of selected cells.
Morphology
When supported by feeder cells, three different colony types could
be recognized at 10-12 days of culture. Progressively growing
keratinocyte colonies, which are believed to be derived from stem
cells, contained a homogenous population of small cells, but small
colonies with a low proliferative potential displayed a population
of greatly differentiated morphology. A layer consisting of large
flattened cells covered the small cells of highly proliferative
colonies when the culture became overconfluent. Selected cells on
collagen IV were also maintained homogeneously in culture although
a few irregular sized cells with differentiated morphology appeared
with them (figure
4F). These differences were verified by a higher
magnification of microscopy (figure 5), which
demonstrated that the small cells showed a large nuclear to
cytoplasmic ratio but larger and irregular cell populations showed
a smaller nuclear to cytoplasmic ratio. This result indicated that
cultured cells most likely represented the stem cell population.
Molecular marker expression
In order to evaluate the cultured cells, we examined the expression
of some previously reported markers in keratinocyte stem cells by
immunofluorescence. Marker expression has been evaluated at the
second selection of secondary keratinocytes on collagen IV
substrate (table 2, figure 6). p63 was strongly
expressed in the selected population. Keratin 19 and keratin 15
positive cells were also detected in this cell population. CD71
expressing cells were found in culture as single cells. These
results also proved that cultured cells showed the characteristic
of stem cells. Larger and irregularly sized cells with
differentiated morphology were observed in the selected population
and these were initially rare but their proportions were greater in
later cultures. In this case, expression of suprabasal
differentiation markers, keratin 10 and involucrin was examined in
the same selected population. Involucrin expression especially
focused on larger and irregularly sized cells. Keratin 10
expression was also associated with this type of cell.
Table 2 Markers expression in collagen IV selected
population
|
Markers
|
% positive cells (Mean ± S.d.)
|
|
Medium I
|
Medium II
|
Medium III
|
|
p63
|
53 ± 10.32
|
57.6 ± 9.69
|
51.8 ± 8.11
|
|
Keratin 19
|
27.25 ± 5.56
|
21.5 ± 5.97
|
19.25 ± 4.27
|
|
Keratin 15
|
21.25 ± 5.56
|
16.75 ± 4.57
|
19 ± 3.91
|
|
CD71
|
< 1
|
Not detected
|
< 1
|
|
Involucrin
|
4.75 ± 2.87
|
2.25 ± 1.89
|
2 ± 1.41
|
|
Keratin 10
|
< 1
|
Not detected
|
Not detected
|
Discussion
In this study, we report the development of a procedure for
isolation and serial propagation of keratinocytes from fetal goat
skin. The system uses isolated epidermal keratinocytes seeded on
mitomycin C treated fetal skin fibroblasts. For convenience, we
plated the keratinocytes on a 24 h old feeder layer although
if desired, the feeder cells could be plated together or in advance
of the keratinocytes, as they are at their optimum 48h after
seeding [25]. Feeder layers of treated fibroblasts helped the
clonal growth of keratinocytes without contamination of other cell
types [26]. With the support of feeder cells, keratinocytes could
be maintained in serial culture by using a serum containing growth
medium supplemented with several chemically defined compounds that
enhance the keratinocyte lifespan. However, keratinocytes can also
grow without feeders in serum-free culture media [3, 16, 32]. By
clonal analysis, previously described [1, 17, 19, 22] three types
of clones have been recognized in keratinoyte culture. Holoclones
are thought to be derived from epidermal stem cells, while the
other two types, meroclones and paraclones, are generated by
transient amplifying cells and an intermediate type of cell which
is a reservoir of transient amplifying cells, respectively.
The clonogenic kertinocyte culture was enriched in stem cells by
plating on an artificial dermal matrix formed by a collagen layer.
Enriched cells were maintained in three different growth medium
conditions and this system had been successfully applied for
growing cell populations in serial culture. However, the culture
presented irregular and larger sized cells together with homogenous
cell populations. In high Ca2+ conditions, cell growth
continued as a monolayer in cultures that had reached confluence,
and in time, some cells were forced out of the plane of the
monolayer and increased their size. Virtually all these deplaced
cells are terminally differentiated cells [30]. Involucrin and
keratin 10 expression were associated with these cells, which could
be distinguished from the smaller population by their morphology.
The growth of differentiated cells was minimized in culture by
using low calcium concentration medium. Reducing the level of
Ca2+ in culture medium prevents the culture from
stratifying but does not prevent cell division [6, 30]. In low
calcium conditions (< 0.06 mM), the cells fail to form
desmosomal interconnections and are spaced out as a monolayer.
Although the keratinocytes fail to stratify, they commence terminal
differentiation when calcium levels are restored (to 1.2 mM)
[3].
Generally, epidermal stem cells have a high clonogenic ability
and proliferative potential although they are slow to enter a
proliferative phase. Cells from progressively growing keratinocyte
colonies showed high colony formation efficiency (21%-59%) and
subsequently, collagen enriched cells also showed high clonogenic
potentiality when co-cultured with feeder cells. However, it
appears that the CFE of our selected cells was lower than in other
observations in which FACS had been applied for sorting stem cells
[7, 8, 10].
It is believed that genuine stem cells are smaller in size and
possess a large nucleus and small cytoplasm. Morphological studies
of selected cells revealed the same criteria. Keratinocyte cultures
showed that expanding colonies (holoclones) were generated by the
small cells but the interior of the colony contained larger and
flattened cells, which implies that an upper,
terminally-differentiating layer may cover the monolayer of the
small cell population [1].
Keratinocytes with characteristics of stem cells can be isolated
on the basis of choosing specific surface molecular markers and
separating the basal keratinocyte population containing epidermal
stem cells from the rest. Our choice to assess p63, keratin 15,
keratin 19 and CD71 expression as keratinocyte stem cell markers
were based upon different studies demonstrating, by means of the
immunofluorescence microscopy, techniques that allow the
identification of marker expressions in the cultured cells. In this
study, all these marker expressions were observed in the selected
cells although their expression level was different from one
another. Transcription factor p63, a homologue of p53, associated
with stem cells and definitely distinguished them from their TA
progeny in stratified squamous epithelia. p63 is specifically known
to be expressed in stem cells of human epidermis and limbal
epithelium [21], and its expression pattern in this study is
similar to other observations [24]. Keratin 19 was expressed in the
hair follicle, and was absent at the interfollicular epidermis at
hairy sites when checking the expression in mouse and human skin.
In contrast, at glabrous sites, K19-positive-cells were found in
deep epidermal rete ridges. There was more keratin 19 expression in
newborn than older foreskin, which correlated with the keratinocyte
culture lifespan. This observation suggests that keratin 19 is a
valuable marker, or at least a co-marker, for skin stem cells [18].
Recently, it has also been reported that the expression of the
intermediate filament keratin gene, K15 is restricted to cells at
the bulge region of the human hair follicle; however, later work
has proved that keratin 15 is present in the basal layer of
stratified epidermis as well as the entire outer root sheath of the
human hair follicle [23, 28, 31]. Immunohistological study
demonstrated the differential expression of CD71 in the hair
follicle. The interfollicular epidermis and upper portion of the
hair follicle, including the sebaceous gland, exhibited relatively
bright CD71 staining, with the strongest expression detected in the
bulb region at the base of hair follicles [27].
Our studies provide the means of characterizing putative
epidermal stem cells in animal fetal keratinocyte culture models.
Further study is needed to investigate the relationship between
stem cells and the panel of marker expressions in goat skin.
Finally, the accessibility of skin makes keratinocyte stem cells an
ideal vehicle for genetic manipulation and gene therapy. The
ability to identify and isolate these cells represents an important
prerequisite for the development of these approaches.
Acknowledgement
The study was supported by a grand from Natural Science Foundation
of China (NSFC) (No. 30260077). Conflict of interest: none.
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