First published online September 2, 2003
doi: 10.1242/10.1242/jcs.00702
Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-ß1 concentrations
Nicolas O. Fortunel1,
Jacques A. Hatzfeld1,
Pierre-Antoine Rosemary1,
Corinne Ferraris2,
Marie-Noëlle Monier1,
Valérie Haydont1,
Joanna Longuet2,
Benoit Brethon1,
Bing Lim3,
Isabelle Castiel2,
Rainer Schmidt2 and
Antoinette Hatzfeld1,*
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
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Fig. 1. Characterization of the Adh+++EGF-R-/+ epidermal stem
cell subpopulation. (A) Basal keratinocytes were separated on the basis of
their adhesion properties. The cell population designated as Adh-/+
is composed of keratinocytes with low adhesion capacity, and the
Adh+++ population is composed of keratinocytes with high adhesion
capacity. Cells of each population were studied for their long-term expansion
potential. Expansion curves are expressed as a cumulated cell output. Data
represent means±s.d. of four replicate cultures from one typical
experiment. (B) Cells of the Adh+++ population were labeled to
analyze their level of EGF-R cell-surface expression by flow cytometry.
Sorting gates were defined to isolate four subpopulations: the
Adh+++EGF-R-/+ subpopulation contains the 20% of the
Adh+++ keratinocytes presenting the lowest level of EGF-R
expression; the Adh+++EGF-R++++ subpopulation contains
the 20% of the Adh+++ keratinocytes presenting the highest level of
EGF-R expression; the Adh+++EGF-R++ and
Adh+++EGF-R+++ subpopulations each contained the 20% of
the Adh+++ keratinocytes presenting intermediate levels of EGF-R
expression. (C) The long-term proliferative potential of
Adh+++EGF-R-/+, Adh+++EGFR++,
Adh+++EGF-R+++ and Adh+++EGF-R++++
keratinocytes were compared. Data represent means±s.d. of four
replicate cultures from one typical experiment. (D) Capacity of cell
subpopulations, sorted according to the level of cell-surface EGF-R
expression, to generate a reconstructed epidermis. Selected keratinocytes of
the Adh+++EGF-R-/+ and
Adh+++EGF-R++++ subpopulations were expanded in defined
culture conditions, and then seeded on to a dermal substrate at an early
passage (p4) and a late passage (p7) to evaluate their capacity to produce a
pluristratified epidermis. Histological preparations shown are from one
typical experiment.
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Fig. 2. Dose-response effect of TGF-ß1 on the proliferation and expansion of
keratinocytes. (A) Long-term cultures were initiated with keratinocytes of the
Adh+++ or Adh-/+ populations, and maintained with or
without addition of exogenous TGF-ß1 at a concentration of 10, 30, 100 or
300 pg/ml throughout the culture period, as specified. Results are expressed
as a cumulated cell output. (B) Long-term cultures were initiated with sorted
keratinocytes of the Adh+++EGF-R-/+ subpopulation, and
maintained in the presence or absence of exogenous TGF-ß1 at a
concentration of 10 or 100 pg/ml throughout the culture period. Results are
expressed as a cumulated cell output. Data represent means±s.d. of four
replicate cultures from one typical experiment. (C) Long-term cultures were
initiated with sorted keratinocytes of the
Adh+++EGF-R-/+ subpopulation and maintained in the
presence or absence of TGF-ß1 at the concentration that promotes
self-renewal of immature keratinocytes (10 pg/ml). Typical morphology of the
cells obtained in these two conditions after 85 and 95 days of culture is
illustrated in Fig. 2C.
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Fig. 3. Reversibility of the inhibitory effect of high TGF-ß1 concentrations
on keratinocyte cell cycling. (A) Cells of the Adh+++ population
initially grown on slides without TGF-ß1 were cultured for 24 hours with
this factor added to the medium at a concentration of 0, 10, 30, 100, 300,
1000 or 3000 pg/ml. Samples were then processed for cell-cycle analysis by
Laser Scanning Cytometry (LSC). Data from one typical experiment are shown
(image of fluorescent nuclear DNA staining and cell-cycle analyses). (B)
Long-term cultures were initiated with keratinocytes of the Adh+++
population and maintained in the presence of 100 pg/ml exogenous TGF-ß1
up to day 30. Half of the cultures were continued from day 30 without addition
of TGF-ß1 and the rest were continued in the presence of 100 pg/ml
exogenous TGF-ß1. (C) Long-term cultures were initiated with
keratinocytes of the Adh+++ population and maintained up to day 21
without addition of TGF-ß1. Half of the cultures were continued from day
21 to day 41 in the presence of 300 pg/ml exogenous TGF-ß1, and then from
day 41 without addition of TGF-ß1. The other half were continued from day
21 without addition of TGF-ß1. Data represent means±s.d. of four
replicate cultures from a typical experiment.
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Fig. 4. Effect of a low TGF-ß1 concentration on Smad1 and Smad2/3
phosphorylation. 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. The degree of Smad1
and Smad2/3 phosphorylation (presence of p-Smad1 and p-Smad2 and 3) was then
evaluated in each condition by immunofluorescence and compared by Laser
Scanning Cytometry (LSC) analysis. Results from one representative experiment
are presented (n=3). Data are expressed as percentages of positive
cells and median values of fluorescence (arbitrary units, a.u.) detected in
the two culture conditions.
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© The Company of Biologists Ltd 2003
6 (CD49f) and ß1
(CD29) integrin expression by keratinocytes. Long-term cultures were initiated
with Adh+++EGF-R-/+ cells and performed with or without
10 pg/ml TGF-ß1. Cells at passage 4 were plated on glass slides, grown
for 72 hours, and then processed for immunofluorescence. Expression of
integrins was analyzed by Laser Scanning Cytometry (LSC) and compared in the
two culture conditions. (A) Isotypic control corresponding to