1 Laboratoire de Biologie des Cellules Souches Humaines, CNRS-UPR 9045, Institut
André Lwoff, 94800 Villejuif, France
2 L'Oréal, Life Sciences Advanced Research, Centre C. Zviak, 92110
Clichy, France
3 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
02115, USA
* Author for correspondence (e-mail: hatzfeld{at}vjf.cnrs.fr)
Accepted 6 June 2003
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Summary |
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Key words: Human epidermal precursor, Expansion, Feeder layer-free culture, TGF-ß1, High proliferative potential, Reconstructed epidermis, EGF-R
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Introduction |
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Most studies performed to identify cell-surface markers expressed by
primitive keratinocytes have focused on molecules involved in cell adhesion.
It has been reported that early keratinocytes of the basal layer of the
epidermis express the 2ß1 and
3ß1 integrins
(Peltonen et al., 1989
;
Carter et al., 1990
). A high
expression level of the ß1 integrin chain (CD29) has been associated with
the high plating efficiency of primitive human keratinocytes in culture
(Jones and Watt, 1993
;
Jones et al., 1995
), and with
epidermal stem cell functional properties in a murine xenograft model
(Jones et al., 1995
). More
recently, keratinocytes with the greatest proliferative capacity have been
shown to express a high level of the
6 integrin chain (CD49f) and, by
contrast, a low to undetectable level of the transferrin receptor (CD71)
(Li et al., 1998
).
In this study, we have first investigated the possibility of isolating a
cell subpopulation that was enriched for primitive epidermal precursors with
high proliferative potential and maintaining a durable capacity to generate a
pluristratified epidermis throughout expansion. This was achieved by selecting
cells presenting a mitogenic receptorlow cell-surface phenotype, as
we previously described for primitive hematopoietic cells in the HPP-Q working
model. This selection has been performed on the basis of the cell-surface
expression level of the epidermal growth factor receptor (EGFR), which has
largely been described to exert a mitogenic effect on keratinocytes
(Cook et al., 1991). On the
basis of our data suggesting that TGF-ß1 could participate in the control
of hematopoietic progenitor cell immaturity, we then explored the capacity of
TGF-ß1 to control the cell cycling and long-term expansion of immature
keratinocytes. The effect of extremely low, yet physiological, concentrations
of TGF-ß1 (10-30 pg/ml) was analyzed, revealing a hitherto undescribed
property of TGF-ß1. At these low concentrations, TGF-ß1 efficiently
promotes the long-term expansion of undifferentiated epidermal precursor cells
in a feeder layer-free culture condition.
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Materials and Methods |
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Immunofluorescence staining and cell sorting by flow cytometry
For the detection of cell-surface EGF-R, keratinocytes were first incubated
for 15 minutes with rat -globulins (Jackson ImmunoResearch
Laboratories, West Grove, PA), and then for 30 minutes with a non-conjugated
monoclonal anti-human EGF-R mouse IgG2b (EGFR1 clone; Dako,
Glostrup, Denmark) or an isotypic control from the same species (mouse
IgG2b) (Immunotech, Marseille, France). Samples were washed twice
and then incubated for 30 minutes with a rat anti-mouse IgG2a+b-PE
antibody (Becton Dickinson, San Jose, CA). Analyses and cell sorting were
performed using a Vantage Fluorescence Activated Cell Sorter (FACS) (Becton
Dickinson).
Cell-cycle and immuno-phenotypic analyses by laser scanning
cytometry
For cell-cycle analysis, keratinocytes were plated on glass slides and
grown without exogenous TGF-ß1 until formation of multicellular clones.
TGF-ß1 (R&D Systems, Abingdon, UK) was then added to the medium at
concentrations ranging from 10 to 3000 pg/ml. Samples were processed 24 hours
later for cell-cycle analyses. Cells were fixed and permeabilized in acetone
for 30 minutes at -20°C. They were then treated with DNase-free RNase A
(Boehringer) for 20 minutes at 37°C, and incubated for at least 15 minutes
at room temperature with 20 mg/ml propidium iodide (Sigma). The distribution
of the keratinocytes into the G0/G1 and
S+G2/M phases of the cell cycle was analyzed in each condition
using a Laser Scanning Cytometer (LSC) (CompuCyte, Cambridge, MA). Acquisition
and data analysis were performed using Wincyte software (CompuCyte). For the
analysis of 6 and ß1 integrin expression, keratinocytes from
cultures initiated with Adh+++EGF-R-/+ cells, previously
cultured with or without 10 pg/ml TGF-ß1 during four successive passages,
were plated on glass slides and grown for three additional days in the same
culture conditions. Cells were then fixed in methanol for 5 minutes at
-20°C. Prior to
6 or ß1 integrin immunostaining, cells were
washed in PBS containing 0.2% BSA (PBS/BSA), then incubated respectively with
rat or mouse
-globulins (Jackson ImmunoResearch Laboratories) for 15
minutes, and then with a FITC-conjugated monoclonal anti-human integrin
6 chain (CD49f) rat IgG2a (GoH3 clone; Pharmingen, San
Diego, CA), or a FITC-conjugated monoclonal anti-human integrin ß1 chain
(CD29) mouse IgG2a (K20 clone; Immunotech) for 30 minutes. For Smad
phosphorylation studies, keratinocytes from cultures initiated with
Adh+++EGF-R-/+ cells were plated on glass slides and
grown for 72 hours with or without 10 pg/ml TGF-ß1. After fixation in
methanol for 5 minutes at -20°C, samples were washed twice in PBS/BSA, and
then successively incubated for 15 minutes with irrelevant chicken Ig (Jackson
ImmunoResearch Laboratories), for 30 minutes with non-conjugated polyclonal
rabbit anti-human phosphorylated (p)-Smad1 (Ser463/Ser465) antibodies
(sc-12353-R; Santa Cruz Biotechnology, Santa Cruz, CA) or non-conjugated
polyclonal rabbit anti-human p-Smad2 and 3 (Ser433/Ser435) antibodies
(sc-11769-R; Santa Cruz Biotechnology), and then for 30 minutes with Alexa
Fluor488-conjugated chicken anti-rabbit IgG (Molecular Probes, Eugene, OR).
Appropriate negative controls were used to determine background signals. The
percentage of positive cells, as well as the median value of fluorescence
(arbitrary units, a.u.) measured at the level of the population, were
evaluated in each culture condition by LSC.
Long-term expansion assays
Cultures were carried out on plastic substrates (25 cm2 Falcon)
in a serum-free medium containing 100 pg/ml EGF, 5 µg/ml insulin, 0.5
µg/ml hydrocortisone, 0.4% (v/v) bovine pituitary extract (KGM Bullet Kit;
BioWhittaker, Clonetics, San Diego, CA). Cultures were initiated with selected
populations of keratinocytes plated at 2400 cells/cm2. After
reaching no more than 70-80% confluence, expanded keratinocytes were detached
by trypsinization (Boehringer), counted and replated at 2400
cells/cm2. Cultures were continually passaged until the growth
capacity of the cells was exhausted. Culture medium supplemented or not with
the specified concentrations of recombinant human TGF-ß1 (R&D
Systems) was completely renewed three times per week. The cumulated total cell
outputs were calculated assuming that all the cells from the previous passage
had been replated. For each cell population and growth factor condition
studied, cultures were performed in quadruplicate. At each step, cell
viability was evaluated by trypan blue exclusion. Statistical analyses were
performed using the Student's t test.
Reconstructed epidermis
Substrate
De-epidermized human dermis (DED) was prepared according to the technique
described by Régnier et al.
(Régnier et al., 1981).
Briefly, split-thickness human skin, obtained at plastic surgery, was floated
in magnesium/calcium-free phosphate-buffered saline at 37°C for 10 days.
Thereafter, the epidermis was separated from the dermis. Dermal cells were
killed by serial freezing and thawing, and the cell-free dermis was stored at
-20°C until use.
Reconstruction of an epidermis
Human foreskin keratinocytes cultured as described previously were seeded
at different passages onto the DED and cultured for 6 days in Dulbecco's
modified Eagle medium/Ham F12 (Invitrogen), containing 10% fetal calf serum
(Invitrogen), 10 ng/ml EGF (BD Biosciences, USA), 0.4 µg/ml hydrocortisone
(Sigma), 10-6 M isoproterenol (Sigma), 5 µg/ml transferrin
(Sigma), 2x10-9 M triiodothyronine (Sigma),
1.8x10-4 M adenine (Sigma) and 5 µg/ml insulin (Sigma).
Thereafter, the cultures were raised to the air-liquid interface, and were
continued in the absence of isoproterenol, transferrin, triiothyronine and
adenine. Histological examination of the reconstructed epidermis was performed
after 7 days of culture.
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Results and Discussion |
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First enrichment step: selection of keratinocytes with high adhesion
capacity (Adh+++)
An initial enrichment step was based on the knowledge that maturing basal
keratinocytes lose their capacity to adhere to extracellular matrix components
(Adams and Watt, 1990). The
basis of this method is provided by the work of Jones and Watt, who
demonstrated that the most primitive keratinocytes with characteristics of
stem cells (high colony-forming efficiency and long-term proliferative
potentiel) adhered most rapidly to type IV collagen, whereas later
keratinocyte populations (transit amplifying cells and post-mitotic
differentiated keratinocytes) adhered more slowly
(Jones and Watt, 1993
). Cells
with the highest adhesion capacity (Adh+++) were selected here on a
substrate coated with type I collagen, thus eliminating most of the
post-mitotic mature keratinocytes. Morphological observation of the two cell
populations obtained after adhesion-based separation indicated that the
selected Adh+++ population is homogenously composed of small-sized
undifferentiated cells of less than 12 µm in diameter, whereas the
non-selected Adh-/+ population is largely heterogenous, mainly
composed of differentiating keratinocytes increasing in size. The
Adh+++ population represented only 10.4% of the total keratinocytes
obtained from neonatal foreskins, but was significantly enriched with
clonogenic cells that effectively contribute to culture initiation. In a
short-term assay, 2.3% of selected Adh+++ keratinocytes possessed a
clone-forming ability, whereas only 0.2% of the Adh-/+
keratinocytes were clonogenic (n>10 independent samples). This is
in agreement with a previous study showing an inverse correlation between the
size of keratinocytes and their clonogenic potential (Barrandon et al.,
1985).
Comparison of the long-term proliferative potential of these populations
confirmed that Adh+++ keratinocytes were more primitive than
Adh-/+ keratinocytes (Fig.
1A). The estimated output for one plated Adh+++
keratinocyte was at least 100-fold higher than that of unfractionated
keratinocytes. By contrast, Adh-/+ keratinocytes expressed only a
limited proliferative potential, giving rise to 500-fold fewer cells than
a similar number of unfractionated keratinocytes. In the typical experiment
shown in Fig. 1A, mean
estimated outputs from one plated keratinocyte were
109-1010 for Adh+++ cells,
106-107 for unfractionated cells, and
103-104 for Adh-/+ cells, corresponding
respectively to 31-32 population doublings (PDs), 23-24 PDs, and 12-13 PDs
(unfractionated versus Adh+++ or versus Adh-/+ cells;
P<0.01, n>5 experiments). It has been reported that,
in post-confluent sheets of cultured human keratinocytes, a process of
autoregulation adjusts the frequency of primitive cells independently of their
initial frequency in the culture (Jones et
al., 1995
). The results presented in
Fig. 1A show that an initial
enrichment in primitive cells at the onset of culture results in an increased
cumulated expansion throughout several successive passages, which on the
contrary suggests no significant autoregulation of primitive cell frequency.
This difference might be explained by the fact that, in the long-term culture
experiments presented here, cells were systematically passaged before reaching
confluence in order to limit, as far as possible, any regulation of cell fate
linked to contact inhibition and homeostasis.
|
Second enrichment step: sorting of Adh+++ epidermal
precursor cells presenting a low level of EGF-R expression
(Adh+++EGF-Rlow)
In a second enrichment step, we separated by FACS the Adh+++
population into four subpopulations of equal size, with increasing levels of
cell-surface expression of EGF-R from Adh+++EGF-R-/+ to
Adh+++EGF-R++++ (sorting gates are shown in
Fig. 1B). Keratinocytes with
the greatest proliferative potential were mostly included in the
Adh+++EGF-R-/+ subpopulation (20% of
Adh+++ cells,
2% of total keratinocytes).
Adh+++EGF-R-/+ (Adh+++EGF-Rlow)
cells provided the highest cumulative expansion in long-term cultures compared
with Adh+++EGF-R++++
(Adh+++EGFRhigh) cells (P<0.01, n=5
experiments). In the representative experiment shown in
Fig. 1C, the mean output from
one plated Adh+++EGF-R-/+ keratinocyte was
1012-1013 (41-42 PDs, 13 successive passages).
Adh+++EGF-R++ and Adh+++EGF-R+++
keratinocytes promoted a less-efficient expansion: cell outputs of
109-1010 and 105-106 (respectively
30-31 and 17-18 PDs, 12 and 9 successive passages). The
Adh+++EGF-R++++ subpopulation showed the lowest
proliferative capacity with a cell output of 102-103
(9-10 PDs, 8 successive passages).
Cell sorting based on the EGF-Rlow phenotype, as in the HPP-Q
working model, is shown here to be effective for selection, within the
Adh+++ population, of the primitive keratinocytes possessing the
greatest expansion potential in long-term culture. It is important to note
that EGF-R is not a specific marker of the epidermal stem cell compartment.
This tyrosine kinase receptor is widely expressed in the basal and suprabasal
layers of the epidermis, regulating not only primitive epidermal cell cycling,
but also commitment and terminal differentiation of later keratinocytes
(Peus et al., 1998). The
starting population used here to sort EGF-Rlow epidermal precursors
consisted of Adh+++ cells and not of unfractionated keratinocytes.
Indeed, it appeared more appropriate to work on a cell population depleted in
more mature keratinocyte populations, in which the level of EGF-R cell-surface
expression is not related to stem cell cycling.
Capacity of primitive Adh+++EGF-Rlow epidermal
precursors to generate a pluristratified epidermis
Another feature of Adh+++EGF-R-/+
(Adh+++EGF-Rlow) keratinocytes that was evaluated was
their organogenic potential. Subpopulations of keratinocytes
(Adh+++EGF-Rhigh or
Adh+++EGF-Rlow) were passaged up to seven times and then
seeded onto a dermal substrate. At early passages (p4), keratinocytes
initially expressing either a high or a low level of EGF-R were able to form
an epidermis (Fig. 1D). In both
cases, the characteristic epidermal differentiation pattern was observed: (1)
a basal layer containing polygonal cells oriented perpendicular to the
underlying dermis; (2) 3-4 layers of spinous cells; (3) 4-6 layers of granular
cells characterized by the presence of keratohyalin granules; and (4)
anucleated, flattened cornified cells forming a compact stratum corneum. At
later passages (p7), only keratinocytes displaying the highest proliferative
capacity, namely the Adh+++EGF-Rlow subpopulation, were
still able to form an epidermis (Fig.
1D), demonstrating a greater capacity of this subpopulation to
maintain their organogenic potential throughout in vitro expansion.
Promotion of epidermal precursor cell amplification by low
TGF-ß1 concentrations
The cytokine TGF-ß1 has been described to have a pleiotropic effect on
hematopoietic CD34+ stem and progenitor cells by either maintaining
them in quiescence (Fortunel et al.,
1998; Hatzfeld et al.,
1991
; Fortunel et al.,
2000a
; Fortunel et al.,
2000b
), or by preventing differentiation and apoptosis
(Batard et al., 2000
;
Pierelli et al., 2000
).
However, the effect of TGF-ß1 on long-term expansion of progenitor cells
has not yet been demonstrated in a mammalian system. The isolation and
characterization of the Adh+++EGF-Rlow keratinocyte
epidermal precursors revealed a novel function of TGF-ß1. It appears to
act as a long-term expansion-promoting factor for human primitive
keratinocytes. Whereas the concentrations previously used are 10-1000-fold
higher, our results demonstrated that, when present at extremely low but
physiological concentrations (10-30 pg/ml), TGF-ß1 efficiently increased
primitive keratinocyte expansion.
In long-term cultures initiated with Adh+++ keratinocytes
(Fig. 2A shows one typical
experiment), the mean estimated output for a single plated cell was
108-109 in the control condition, and
1011-1012 when 10 pg/ml TGF-ß1 was added to the
medium. Calculations made to cumulate the results from independent cultures
indicated that this represents a mean increase from 28-29 to 37-38 PDs
(P<0.01, n=5 experiments). By contrast, 10 pg/ml
TGF-ß1 did not increase amplification of Adh-/+ keratinocytes
more than tenfold (from 104 to
105;
Fig. 2A), which is about
1000-fold less than the effect observed on Adh+++ cells (from
108 to
1012;
Fig. 2A). These results suggest
that this effect of TGF-ß1 concerns mainly the most primitive cells. The
slight increase in Adh-/+ keratinocyte expansion might be due to
the fact that the adhesion-based enrichment procedure was not effective in
removing 100% of the Adh+++ cells from the Adh-/+
population.
|
An inhibition of keratinocyte proliferation by TGF-ß1 was observed in
our culture system when high, but nevertheless physiological, concentrations
(=100 pg/ml) were used (Fig.
2A,B). A significant reduction of the percentage of keratinocytes
in S+G2/M phase of the cell cycle was observed 24 hours after
addition of TGF-ß1 at concentrations of 100 pg/ml
(Fig. 3A). It is important to
note that this growth-inhibitory effect appeared to be reversible. Indeed,
keratinocytes initially cultured for 30 days with an inhibiting concentration
of TGF-ß1 started to divide as soon as the addition of the factor was
stopped, and subsequently they did not show any reduced capacity to
proliferate and expand (Fig.
3B). Moreover, the proliferation of keratinocytes initially
cultured for 21 days without exogenous TGF-ß1 could be transiently
inhibited by TGF-ß1 (
100 pg/ml) for 20 days, without any alteration
of their subsequent long-term expansion potential
(Fig. 3C). These observations
confirm those of a previous study, showing that exposure for 48 hours to
TGF-ß1 mediates a reversible growth arrest and does not alter the
clonogenic capacity of keratinocytes
(Shipley et al., 1986
), and
suggest that TGF-ß1 does not induce apoptosis of stem and progenitor
cells, even at high physiological concentrations.
|
The promotion of epidermal precursor amplification by low TGF-ß1
concentrations (10-30 pg/ml) was particularly impressive in cultures initiated
with primitive Adh+++EGFRlow cells. Indeed, in the
experiment shown in Fig. 2B,
the mean output for one Adh+++EGF-Rlow plated cell was
1011-1012 in the control condition and this value
reached 1016-1017 in the cultures treated with 10 pg/ml
TGF-ß1, representing an increase from 38-39 to 55-56 PDs. Given that the
initial cloning efficiency of Adh+++EGFRlow
keratinocytes is less than 2% (results not shown), and making the
assumption that only clonogenic cells contribute to the total cell output, we
estimated that each clonogenic cell in this subpopulation can generate more
than
1018 keratinocytes (
60 PDs). One feature of the most
primitive epidermal cells is their smaller size in comparison with more mature
keratinocytes committed to differentiation
(Barrandon and Green, 1985
). As
shown in Fig. 2C, a low
concentration of TGF-ß1 not only increased the cumulative keratinocyte
expansion, but also prolonged the production of clones composed of small,
undifferentiated keratinocytes. It is also important to note that
TGF-ß1-mediated expansion did not alter the organogenic potential of
epidermal precursor cells, which showed a continuous capacity to form an
epidermis equivalent to that of cells cultured without a low TGF-ß1
concentration (results not shown).
Effects of low TGF-ß1 concentrations on keratinocytes at the
molecular level
TGF-ß intracellular signaling is mediated through Smad, the mammalian
homolog of the Drosophila Mothers against dpp (Mad). This cascade
involves Smad1, Smad2 and Smad3 as direct targets of the type I TGF-ß
serine/threonine kinase receptor family
(Abdollah et al., 1997;
Liu et al., 1997
;
Souchelnytskyi et al., 1997
;
Chen et al., 1999) (reviewed by Moustakas
et al., 2001
). To determine whether concentrations of TGF-ß1
as low as 10 pg/ml are able to activate the Smad pathway, we next analyzed the
degree of Smad1 and Smad2/3 phosphorylation in cultures of keratinocytes,
initiated with Adh+++EGF-R-/+ cells, and grown with or
without 10 pg/ml TGF-ß1. Phosphorylated forms of Smads, p-Smad1
(Ser463/Ser465) and p-Smad2 and 3 (Ser433/Ser435), were quantified and
compared in each culture condition (Fig.
4). Results indicate that a treatment with 10 pg/m TGF-ß1 is
sufficient to increase Smad phosphorylation. In the representative control
experiment shown, 47.2% and 26.1% of the keratinocytes were detected as
positive cells for the presence of p-Smad1 and p-Smad2/3 respectively. These
percentages reached respectively 76.3% and 54.3% in the presence of 10 pg/ml
TGF-ß1. Similarly, the median values of p-Smad1 and p-Smad2/3
fluorescence were respectively increased from 5.2x106 and
3.2x106 arbitrary units (a.u.), in the control condition, to
7.6x106 and 5.8x106 a.u. in
TGF-ß1-supplemented culture.
|
A high expression of 6 (CD49f) and ß1 (CD29) integrin chains is
known to be associated with an immature state of keratinocytes
(Jones et al., 1995
;
Li et al., 1998
). To
investigate further the promotion of long-term amplification of epidermal
precursors by low TGF-ß1 concentrations, we analyzed and compared the
expression of these cell-surface markers in long-term cultures initiated with
Adh+++EGF-R-/+ cells and performed with or without 10
pg/ml TGF-ß1. Typical labeling distributions obtained from cultures at
passage 4 are presented in Fig. 5
(A-F). In the control culture, 53.1% and 66.1% of the
keratinocytes were detected as positive cells for the expression of
6
and ß1 integrins respectively (Fig.
5C,D). These percentages reached respectively 92.4% and 83.4% in
the presence of 10 pg/ml TGF-ß1 (Fig.
5E,F). Similarly, the median values of
6 and ß1
integrin fluorescence were respectively increased from
9.5x106 and 11.3x106 a.u. in the control
condition (Fig. 5C,D) to
16.7x106 and 16.5x106 a.u. in
TGF-ß1-supplemented culture (Fig.
5E,F), indicating a higher degree of immaturity in this optimized
culture condition.
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Conclusions |
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The involvement of TGF-ß1 signaling in the control of the homeostasis
of the epidermis has been confirmed in vivo in various transgenic mouse models
(Cui et al., 1995;
Wang et al., 1997
;
Amendt et al., 1998
), and
suggested in humans by clinical observations showing that a dysregulation of
the TGF-ß1 signaling cascade is often associated with the malignant
conversion of skin keratinocytes (Lange et
al., 1999
). However, although these in vivo observations confirmed
the inhibition of keratinocyte cell cycling described in vitro with high
concentrations of TGF-ß1, they do not provide any information about the
positive effect of TGF-ß1 on immature keratinocyte expansion, as
demonstrated in this in vitro study with very low concentrations (10-30
pg/ml).
The potent biphasic effect of TGF-ß1 previously reported for the
development of hematopoietic progenitor cells
(Fortunel et al., 2000a) and
osteoclast-like cells (Shinar and Rodan,
1990
) for in vitro angiogenesis
(Pepper et al., 1993
) and
mammary gland ductal morphogenesis
(Soriano et al., 1996
) is here
clearly demonstrated for human skin precursor cells. Functional studies may be
applied in such culture systems to elucidate further the complex regulatory
network involved in the exquisitely dose-dependent response of cells to
TGF-ß1. In addition to the various effectors regulating
TGF-ß1-induced cell-cycle arrest in keratinocytes
(Hannon and Beach, 1994
;
Ivarone and Massagué,
1999
), it will be interesting to focus on genes whose expression
may be linked to the maintenance of the epidermal stem cell pool, such as
p21(WAF1/Cip1) (Topley et al.,
1999
), 14-3-3
(Dellambra et al., 2000
), P63
(Pellegrini et al., 2000
),
c-Myc (Waikel et al., 2001
),
ß-catenin (Zhu and Watt,
1999
; Huelsken et al.,
2001
), genes involved in delta-notch signaling
(Lowell et al., 2000
), and the
transcriptional regulators Tcf3 and Lef1 (Merril et al., 2001).
We describe here the capacity of low TGF-ß1 concentrations to promote
long-term expansion of undifferentiated human epidermal precursors, an effect
that was suggested, but not demonstrated, in the hematopoietic system.
However, much remains to be done to characterize this function at the
molecular level. In an avian system, it has been reported that TGF-ß1 is
capable of sustaining erythrocytic progenitor cell proliferation and
self-renewal, and that this effect occurs through a cooperation between the
TGF-ß and TGF- receptors via the Mek-Map kinase pathway
(Gandrillon et al., 1999
). In
the human hematopoietic system, TGF-ß1 could participate in the
maintenance of a pool of primitive and undifferentiated progenitors in part by
upmodulating the immaturity marker, CD34, on cycling cells
(Batard et al., 2000
;
Pierelli et al., 2000
).
TGF-ß1 has not yet been demonstrated to regulate directly the expression
of the mucin-like protein CD34 in normal hematopoietic stem/progenitor cells,
but this has been demonstrated in the pluripotent erythroleukemia cell line
TF-1. Marone et al., have shown that TGF-ß1 transcriptionally activates
CD34 and then prevents differentiation of TF-1 cells, by acting independently
through Smad, TAK1 and p38 pathways
(Marone et al., 2002
). The
downstream component of the Wnt signaling cascade, ß-catenin, appears to
be an important molecule especially in epidermal stem cells
(Zhu and Watt, 1999
;
Huelsken et al., 2001
). Thus,
as described in the early amphibian embryo during the formation of Spemann's
organizer (Nishita et al.,
2000
), a possible interaction between Wnt and TGF-ß signaling
should be investigated in the case of epidermal precursors treated with the
low TGF-ß1 concentrations described here to promote expansion.
One important clinical application of this work is the possibility of
improving the culture systems currently used to amplify keratinocytes for skin
grafting (Ronfard et al.,
2000) by the addition of the TGF-ß1 concentrations described
here to optimize the long-term expansion of immature keratinocytes.
Furthermore, the general principle applied here in purifying primitive cell
subpopulations could be used as an approach to purify precursor cells from
other somatic tissues. Similarly, since the manipulation of TGF-ß1
appears to be effective in permitting in vitro expansion of primitive
epidermal cells, it would be of interest to investigate whether similar use
and manipulation of TGF-ß1 might be applied in the expansion of other
adult tissue precursors.
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Acknowledgments |
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