p63 identifies keratinocyte stem cells
Graziella Pellegrini*†, Elena Dellambra*†, Osvaldo Golisano*†, Enrica Martinelli*, Ivana Fantozzi*, Sergio Bondanza*,
Diego Ponzin‡, Frank McKeon§, and Michele De Luca*¶
*Laboratory of Tissue Engineering IDI, Istituto Dermopatico dell’Immacolata, 00040 Rome, Italy;‡Fondazione Banca degli Occhi del Veneto, 30174 Venice,
Italy; and§Department of Cell Biology, Harvard Medical School, Boston, MA 02115
Communicated by Howard Green, Harvard Medical School, Boston, MA, January 19, 2001 (received for review November 15, 2000)
The proliferative compartment of stratified squamous epithelia
consists of stem and transient amplifying (TA) keratinocytes. Some
polypeptides are more abundant in putative epidermal stem cells
than in TA cells, but no polypeptide confined to the stem cells has
yet been identified. Here we show that the p63 transcription
factor, a p53 homologue essential for regenerative proliferation in
epithelial development, distinguishes human keratinocyte stem
cells from their TA progeny. Within the cornea, nuclear p63 is
expressed by the basal cells of the limbal epithelium, but not by TA
cells covering the corneal surface. Human keratinocyte stem and
TA cells when isolated in culture give rise to holoclones and
paraclones, respectively. We show by clonal analysis that p63 is
abundantly expressed by epidermal and limbal holoclones, but is
undetectable in paraclones. TA keratinocytes, immediately after
their withdrawal from the stem cell compartment (meroclones),
have greatly reduced p63, even though they possess very appre-
ciable proliferative capacity. Clonal evolution (i.e., generation of
TA cells from precursor stem cells) is promoted by the sigma
isoform of the 14-3-3 family of proteins. Keratinocytes whose
14-3-3? has been down-regulated remain in the stem cell com-
partment and maintain p63 during serial cultivation. The identifi-
cation of p63 as a keratinocyte stem cell marker will be of practical
importance for the clinical application of epithelial cultures in cell
therapy as well as for studies on epithelial tumorigenesis.
(1–3). These stem cells generate transient amplifying (TA) cells
that terminally differentiate after a discrete number of cell
divisions (4). In vivo, keratinocyte stem cells are usually slow-
cycling and retain labeled DNA precursors, whereas TA cells
divide rapidly and dilute their label quickly (4–8).
Human keratinocyte stem and TA cells when isolated in
culture give rise to holoclones and paraclones, respectively
(9–11). The great proliferative potential of holoclones (9–12),
the capacity of a single holoclone to generate a mature epithe-
victims by means of grafts of autologous cultured keratinocytes
(14–16), provide compelling evidence that keratinocyte ‘‘stem-
ness’’ can be preserved in culture. The proliferative compart-
ment of squamous epithelia also contains a third type of cell, the
meroclone (9), which is considered a ‘‘young’’ TA cell endowed
with a greater proliferative capacity than the paraclone (11).
Some polypeptides are more abundant in putative epidermal
stem cells than in TA cells (17, 18), but no polypeptide confined
exclusively to the stem cells has yet been identified.
The p63 transcription factor belongs to a family that includes
two structurally related proteins, p53 and p73 (19). Whereas p53
plays a well-established role in tumor suppression, p63 and p73
play unique roles in morphogenesis (20–23). In particular,
p63?/?mice have major defects in their limb and craniofacial
development, as well as a striking absence of stratified epithelia
(20, 21). This phenotype could be explained by either inability of
the p63?/?ectoderm to develop into epithelial lineages (20), or
by lack of stem cell character necessary to sustain epithelial
morphogenesis and renewal (21).
population of keratinocyte stem cells in defined locations
governs the renewal of mammalian stratified epithelia
Here we investigate the expression of p63 in epithelial stem
and TA cells and show that p63 is a specific marker of human
corneal and epidermal stem cells.
Materials and Methods
Cell Culture. 3T3-J2 cells (a gift from Howard Green, Harvard
Medical School, Boston) were cultivated as described (16).
Donors provided informed consent for biopsy. Permission was
obtained for specimens taken from organ donors. Epidermal
as described (16). Limbal/corneal keratinocytes were obtained
from ocular biopsies taken from organ donors and cultivated as
described (11). For serial propagation, cells were passaged at the
stage of subconfluence, until they reached senescence (11).
(100–1000) from each biopsy and from each cell passage were
plated onto 3T3-feeder layers and cultured as above. Colonies
were fixed 9–12 days later, stained with Rhodamine B, and
scored under a dissecting microscope. CFE values are expressed
as the ratio of the number colonies to the number of inoculated
cells. The number of aborted colonies was calculated as de-
scribed (9, 11).
The number of cell generations was calculated by using the
following formula: x ? 3.322 log N/No, where N is the total
number of cells obtained at each passage and No is the number
of clonogenic cells. Clonogenic cells were calculated from CFE
data, which were determined separately in parallel dishes at the
time of cell passage.
inoculated onto multiwell plates containing a feeder layer of 3T3
cells. After 7 days of cultivation, clones were identified under an
inverted microscope and photographed. Each clone was trans-
ferred to three dishes. One dish (1/4 of the clone) was fixed 9–12
days later and stained with Rhodamine B for the classification of
clonal type (9, 11). The clonal type was determined by the
percentage of terminal colonies (scored as in ref. 9) formed by
the progeny of the founding cell. When 0–5% of colonies were
terminal the clone was scored as holoclone. When more than
95% of the colonies were terminal the clone was classified as
paraclone. When more than 5%, but less than 95% of the
colonies were terminal, the clone was classified as meroclone (9,
11). The second dish was used for serial cultivation and evalu-
ation of the number of cell generations. Cells plated in the third
Abbreviations: TA, transient amplifying; PCNA, proliferating cell nuclear antigen; PVDF,
†G.P., E.D., and O.G. contributed equally to this work.
¶To whom reprint requests should be addressed at: Laboratory of Tissue Engineering IDI,
Istituto Dermopatico dell’Immacolata, Via dei Castelli Romani, 83/85 00040 Pomezia
(Roma), Italy. E-mail: firstname.lastname@example.org.
The publication costs of this article were defrayed in part by page charge payment. This
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§1734 solely to indicate this fact.
March 13, 2001 ?
vol. 98 ?
dish were cultivated for 4–5 days, then used to prepare cell
extracts destined to Western analysis.
Immunohistochemistry and Western Analysis. Ocular biopsies were
obtained from organ donors. Sheets of cultured epithelium were
detached from the vessels with Dispase II (24). Specimens were
fixed in paraformaldehyde (4% in PBS) 30 min at room tem-
perature and embedded in paraffin. For immunohistochemistry
(performed as described in ref. 11), sections were stained with
a p63-specific mAb (21, 25), a Keratin-3-specific mAb (AE5, a
gift from Tung-Tien Sun, New York University Medical Center,
New York) (26), and a proliferating cell nuclear antigen
(PCNA)-specific mAb (27) (Santa Cruz Biotechnology).
For immunoblots, mass or clonal cultures were extracted on
ice with RIPA buffer (0.15 mM NaCl/0.05 mM Tris?HCl, pH
7.5/1% Triton X-100/1% sodium deoxycholate/0.1% SDS) (12).
Equal amounts of samples were electrophoresed on 7.5%–12,5%
SDS-polyacrylamide gels and transferred to poly(vinylidene
difluoride) (PVDF) filters (Immobilon-P, Millipore). Immuno-
reactions were carried out as described (12), using polyclonal
antibodies to 14-3-3? and to pan-14-3-3 (Pan) (a gift from
Alastair Aitken, University of Edinburgh, Edinburgh), and mAb
to p63 (21, 25) and to PCNA (27). Immobilon-bound antibodies
were detected by chemiluminescence with ECL (Amersham
Expression of p63 by Human Limbal/Corneal Stem and TA Cells.
Identification of epithelial stem cells may rely on label-retaining
experiments in vivo (4–8) or on clonal analysis of multiplying
keratinocytes in vitro (9–13). In corneal epithelium, the two
criteria identify the same cells. Slow-cycling corneal cells and
human corneal holoclones are confined to the basal layer of the
the conjunctiva (4, 7, 11). The corneal epithelium is formed
exclusively by rapidly dividing TA cells that migrate millimeters
away from their precursor limbal stem cells (4, 26).
Fig. 1A shows immunohistochemical analysis of sections of the
human ocular surface spanning the peripheral cornea (P) and
the limbus (L). Nuclear p63 was abundantly expressed only in the
basal layer of limbal epithelium (L, arrows). Within the limbus,
patches of cells expressing very low levels of p63 (L, asterisks)
were interspersed with the more numerous p63?cells. The TA
cells covering the corneal surface (P) do not express detectable
levels of p63 (P). Very low levels of the protein were observed
in occasional basal cells of the peripheral cornea, adjacent to the
limbal epithelium (P, arrowheads).
To investigate the expression of p63 in isolated stem and TA
cells, single keratinocytes were isolated from subconfluent pri-
mary limbal cultures. After 7 days of cultivation each clone was
transferred to three dishes. One dish was used for the classifi-
cation of the clonal type (9, 11). The second dishes was used for
evaluation of the number of cell generations. Cells plated in the
third dish were cultivated for 4–5 days, then used to prepare cell
extracts destined to Western analysis.
were classified as TA cells, whereas stem cells (holoclones)
represented 15.1% of total clones. Limbal holoclones were
p63-specific mAb. Eight different inclusions from four different biopsies were analyzed. The transition from limbus to peripheral cornea was identified by the
(arrows) expresses p63, and that patches of p63?cells (asterisks) stagger p63?cells. (B) Clonal analysis of subconfluent primary limbal cultures obtained from
two different donors (LC3 and LC5). Cell extracts were prepared from cultures generated by holoclones (H), paraclones (P), and meroclones (M). Equal amounts
to pan-14-3-3 (Pan). Identical results were obtained with two other limbal strains (LC1 and LC25). (C) Densitometric analysis of the blots shown in B.
Expression of p63 by human corneal stem and TA cells. (A) Corneal biopsies spanning from the peripheral cornea (P) to the limbus (L) were stained with
Pellegrini et al.
March 13, 2001 ?
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serially propagated and each holoclone produced approximately
100 generations before senescence, accounting for the entire
proliferative capacity of the parental mass culture. In contrast,
paraclones underwent only 15–20 cell doublings before mitotic
possessed high levels of p63 (Fig. 1B, H). In sharp contrast, p63
was undetectable in cultures generated by paraclones (Fig. 1B,
P). Although meroclones possess a significant proliferative ca-
pacity (an average of approximately 40 cell doublings; ref. 11),
p63 was barely detectable in meroclones (Fig. 1B, M). Cultures
generated by all clonal types synthesized almost identical
amounts of a control protein (pan-14-3-3) (Fig. 1B), suggesting
that differences of p63 content between different clonal types
were not due to general alterations of protein synthesis.
The relative content of p63 in clones was quantified by
densitometric analysis of the blots and is shown in Fig. 1C.
Limbal holoclones contained 8–10-fold and ?200-fold more p63
than meroclones and paraclones, respectively.
Expression of p63 by Stem and TA Cells of Epidermal Cultures. In the
epidermis, stem cells and TA cells are not segregated as they are
in the cornea, but are interspersed in the epidermal basal layer
(1, 17). Cultured primary keratinocytes generate cohesive sheets
of stratified epithelium (24). As in natural epidermis (25), p63
was confined to the basal layer of these sheets (Fig. 2A). Cells
with high content of nuclear p63 were located at intervals (Fig.
2A, arrowheads), sometimes in clusters (Fig. 2A, arrows) sepa-
rated by stretches of p63?cells. At higher magnification, it was
quite common to observe cells with abundant p63 (Fig. 2A Inset,
arrow) flanked by cells with little or no p63 (Fig. 2A Inset,
arrowheads and asterisks). Thus, the expression pattern of p63
is reminiscent of the proposed columnar organization of the
p63-specific mAb. p63 was confined to the basal cell layer. Note that p63?cells were located at intervals as single cells (arrowheads) or as patches (arrows), and
were separated by stretches of p63?cells. At higher magnification (Inset), it is possible to observe cells expressing high levels of p63 (arrow) flanked with cells
expressing lower levels of the protein (arrowheads). The latter cells are flanked by p63?cells (asterisks). (B) Clonal analysis of subconfluent primary epidermal
cultures obtained from two different donors (K71 and K100). Cell extracts were prepared from cultures generated by holoclones (H), meroclones (M), and
paraclones (P). Equal amounts of protein were fractionated on 7.5% SDS-polyacrylamide gels, transferred to PVDF filters and immunostained with p63-specific
mAb (p63) and with polyclonal antibodies to 14-3-3? (?). p63 was high in holoclones, nearly absent in meroclones, and undetectable in paraclones; 14-3-3? was
similar in all.
Expression of p63 by human epidermal stem and TA cells. (A) Cultured epidermal sheets prepared from primary keratinocytes were stained with
www.pnas.org?cgi?doi?10.1073?pnas.061032098Pellegrini et al.
epidermis, where each column is thought to be generated by a
single stem cell (28).
We analyzed 120 clones obtained from subconfluent primary
epidermal cultures (9–11). The vast majority (93.3%) of clones
were classified as meroclones and paraclones (TA cells), whereas
holoclones (stem cells) represented 6.6% of total clones. West-
ern analysis showed that cultures generated by holoclones ex-
pressed high levels of p63 (Fig. 2B, H lanes). In sharp contrast,
p63 was undetectable in cultures generated by paraclones (Fig.
2B, P lanes) and barely detectable in cultures generated by
meroclones (Fig. 2B, M lanes). Like limbal holoclones, epider-
mal holoclones possess 8–10-fold and ?200-fold more p63 than
meroclones and paraclones, respectively.
p63 Expression and the Potential for Cell Proliferation. In human
epidermis, hair follicles, and stratified epidermal cultures, p63 is
expressed in the nuclei of cells that are either proliferating or
possess the ability to multiply (25). PCNA is a DNA polymerase
?-associated protein that is synthesized in G1 and S phases of the
cell cycle and is therefore a specific marker of proliferating cells
(27). Staining for p63 and PCNA performed on parallel limbal
sections revealed that cells forming the basal layer of human
PCNA-specific mAb (D–F). Arrows indicate basal cells that expressed p63, but not PCNA. Black asterisks indicate cells that expressed both p63 and PCNA. B and
(LC25) and epidermal (K71) cultures. Cell extracts were prepared from cultures generated by holoclones (H), meroclones (M), and paraclones (P). Equal amounts
of protein were fractionated on 7.5% (G) or on 12.5% (H) SDS-polyacrylamide gels, transferred to PVDF filters and immunostained with p63-specific mAb (p63)
and with PCNA-specific mAb (PCNA).
Expression of p63 and PCNA by keratinocyte stem and TA cells. (A–F) Parallel sections of limbal biopsies were stained with p63-specific mAb (A–C) and
Pellegrini et al.
March 13, 2001 ?
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no. 6 ?
limbus express both p63 (Fig. 3 A–C) and PCNA (D–F). Al-
though most cells expressing PCNA also express p63 (see black
asterisks in A, B, D, and E), it was possible to observe cells
expressing PCNA but not p63 (see red asterisks in B and E).
These latter cells often flanked p63?cells (B and E). Most
frequently, cells expressing high levels of p63 (A–C, arrows) did
not possess PCNA (D–F, arrows). These observations suggest
that p63 is expressed by keratinocytes that possess the ability to
proliferate and not simply by keratinocytes that are duplicating
their DNA, as already shown in human epidermis (25).
in cultured corneal (Fig. 3G) and epidermal (Fig. 3H) stem (H)
and TA (M and P) cells. Western analysis showed that cultures
of p63 and PCNA. In sharp contrast, cultures generated by
paraclones (G and H, P lanes) expressed PCNA, but not p63.
Corneal meroclones (G, M lanes) possessed high levels of
PCNA, but very low levels of p63.
These data confirm that p63 protein is principally restricted to
keratinocyte stem cells. The observation that p63?cells do not
necessarily express PCNA in vivo (Fig. 3 A–F and ref. 25), but
that all cultured p63?clones possess PCNA (Fig. 3 G and H), is
consistent with the notion that epithelial stem cells are slow-
cycling in vivo, but actively proliferating in culture.
Expression of p63 in Cultured Keratinocytes Maintained in the Stem
Cell Compartment. Clonal evolution (i.e., generation of TA cells
from a parental stem cell) is a continuous unidirectional process
occurring during aging, wound healing, and serial cultivation (1,
have completed their clonal evolution and have generated
terminal TA cells (9, 11–13). We have shown that the sigma
isoform of the 14-3-3 family of proteins (31, 32) promotes this
clonal evolution (12). When expression of 14-3-3? was stably
down-regulated in primary human epidermal keratinocytes by
transduction with defective retrovirus carrying a full-length
human 14-3-3? cDNA in an antisense orientation (12), clonal
evolution was prevented and the keratinocytes were maintained
in the stem cell compartment and immortalized (these cell lines
and the procedures to generate them are fully described in ref.
12). We therefore examined expression of p63 in antisense-?-
Clonal analysis performed on control keratinocytes (Fig. 4A,
yellow columns) showed that 95% of clonogenic cells could be
identified as either meroclones (57.5%) or paraclones (37.5%),
whereas stem cells represented 5% of total clonogenic cells. In
contrast, epidermal cultures generated from antisense-?-
transduced cells were formed mainly by stem cell holoclones,
which accounted for about 50% of total clonogenic cells (Fig.
4A, blue columns). As expected, antisense-?-transduced kera-
after bypass of replicative senescence. The frequency of cells
strongly staining for p63 in the transduced keratinocytes shown
in Fig. 4B was compared with that of untransduced cells, such as
those shown in Fig. 2A. The frequencies were 71.2% and 10.7%,
To our knowledge, p63 is the first gene product definitely
distinguishing stem cells from their TA progeny in stratified
squamous epithelia. Identification of p63 as a stem cell marker
is consistent with the phenotype of p63?/?mice (20, 21). It was
known that p63?/?mice lack stratified epithelia and contain
clusters of terminally differentiated keratinocytes on the ex-
posed dermis (21), and that p63 is expressed in the nuclei of
keratinocytes with proliferative potential (25). The finding that
p63 is specifically expressed by stem cells of human epidermis
and limbal epithelium, and not by TA cells (this paper), strongly
suggests that the phenotype of p63?/?mice should be ascribed
to a failure to maintain stem cells (21) rather than to inability of
the p63?/?ectoderm to form epithelial lineages during devel-
Newborns start their life with a high content of keratinocyte
stem cells, but the number declines during aging (1, 9). Further-
more, division of stem cells gives rise to hierarchy of TA cells
with progressively less proliferative potential (4, 8, 9, 11). This
has been clearly shown in corneal epithelium where there is a
well-established centripetal migration of limbus-derived TA
cells, as they progressively decrease their proliferative capacity
(4). We show here that p63 is not expressed by the basal TA cells
of corneal epithelium. We also show that TA keratinocytes,
immediately after their withdrawal from the stem cell compart-
ment (meroclones), already have greatly reduced p63, even
though they possess very appreciable proliferative capacity (9,
11, 17). Furthermore, cultures generated by TA cells express a
cell-proliferation-associated nuclear antigen (PCNA), but not
p63. Taken together, these observations show that possession of
p63 is not simply a property of multiplying cells (ref. 25 and this
paper), but a property of stem cells as they are identified by
Human cornea and epidermis display a slightly different
pattern of p63 localization from that seen in other mucosal
epithelia. For instance, p63 is expressed by all basal and most
suprabasal cells of cervical squamous mucosa (33). More exper-
iments are needed to clarify whether differences in p63 expres-
sion reflect different functions of the protein in maintaining the
proliferative potential of other stratified epithelia. Finally, in the
human hair follicle, p63 is expressed by keratinocytes forming
the outer root sheath as well as by bulb keratinocytes surround-
antisense RNA for 14-3-3?. The expression of 14-3-3? was stably down-
regulated in primary keratinocytes by transduction with defective retrovirus
carrying a full-length human 14-3-3? cDNA in antisense orientation (12).
These cells maintained telomerase activity and became immortal (12). (A)
Clonal analysis was performed on keratinocytes transduced with antisense ?
DNA (K45-AS) or with an empty vector (K45-V). Note that the percentage of
approximately 50% in K45-AS keratinocytes (H, blue column). (B) Cultured
epidermal sheets prepared from antisense-?-transduced keratinocytes were
www.pnas.org?cgi?doi?10.1073?pnas.061032098Pellegrini et al.
ing the surface of the follicular papilla (25). This finding suggests
that keratinocytes endowed with stem cell properties are present
not only in specific regions of the outer epithelial sheath (10, 34),
but also in the hair bulb, as previously postulated (10, 35).
Our findings have major implications for the study of kera-
tinocytes in two different fields. First, cultured keratinocytes
in patients with life-threatening or disabling epithelial defects
(14–16). Permanent regeneration of the epithelium obviously
requires engraftment of stem cells. This engraftment of stem
cells will also be essential for successful gene therapy. Second,
stem cells are thought to be involved in the formation of
malignant tumors (36, 37). In this respect, it is known that most
cells of poorly differentiated squamous cell carcinomas possess
p63 (25). Thus, the definition of this long-sought keratinocyte
stem cell marker will be of crucial importance for proper clinical
application of epithelial cultures in cell therapy as well as for
studies on epithelial tumorigenesis.
This work was supported by Telethon-Italy (Grants A.106 and B-53) and
by Ministero della Sanita `, Italy.
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