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Colloquium
Stem cells of the skin epithelium
Laura Alonso and Elaine Fuchs*
Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10021
Tissue stem cells form the cellular base for organ homeostasis and
repair. Stem cells have the unusual ability to renew themselves
over the lifetime of the organ while producing daughter cells that
differentiate into one or multiple lineages. Difficult to identify and
characterize in any tissue, these cells are nonetheless hotly pursued
because they hold the potential promise of therapeutic reprogram-
ming to grow human tissue in vitro, for the treatment of human
disease. The mammalian skin epithelium exhibits remarkable turn-
over, punctuated by periods of even more rapid production after
injury due to burn or wounding. The stem cells responsible for
supplying this tissue with cellular substrate are not yet easily
distinguishable from neighboring cells. However, in recent years a
significant body of work has begun to characterize the skin
epithelial stem cells, both in tissue culture and in mouse and human
skin. Some epithelial cells cultured from skin exhibit prodigious
proliferative potential; in fact, for >20 years now, cultured human
skin has been used as a source of new skin to engraft onto
damaged areas of burn patients, representing one of the first
therapeutic uses of stem cells. Cell fate choices, including both
self-renewal and differentiation, are crucial biological features of
stem cells that are still poorly understood. Skin epithelial stem cells
represent a ripe target for research into the fundamental mecha-
nisms underlying these important processes.
The skin is the first line of defense to protect the body from
dehydration, injury, and infection. To meet these needs, the
skin has evolved an elaborate differentiation process that results
in a tough, water-impermeable outer covering that is constantly
renewable. Mammalian skin consists of both dermal and epi-
dermal components; this discussion will be restricted to the
epidermal cells, referred to as keratinocytes. The mammalian
epidermis is a stratified tissue, anchored to a basement mem-
brane (Fig. Lay. The layer of cells directly contacting the
basement membrane, termed the basal layer, contains prolifer-
ating cells. Like all keratinocytes, cells of the basal layer possess
a network of 10-nm keratin intermediate filaments (IFs), but
they are otherwise relatively undifferentiated. As the population
of basal cells expands because of division, some cells detach from
the basement membrane and begin to move outward toward the
skin surface. The first change to occur is a strengthening of the
IF network to increase the tensile strength of each cell. Cells
achieve this by synthesizing large numbers of new sets of keratins
which assemble into IFs that aggregate into more resilient
bundles or cables of IFs. IF cables anchor to cell-cell junctions
called desmosomes, thus distributing force not over individual
cells but over the entire tissue (reviewed in ref. 14. As the
suprabasal cells, now connected by desmosomes, move in tan-
dem toward the skin surface, they deposit and enzymatically
cross-link proteins beneath the plasma membrane to form the
corniced envelope. These cells also make lamellar granules filled
with lipids, which are extruded onto the cornified envelope
scaffold, providing a water-impermeable seal that prevents the
unregulated escape of fluids (2~. After production of all mate-
rials is complete, the cells cease transcriptional and metabolic
activity and undergo a programmed cell death that shares some
similarities with apoptosis (3~. The cells (squames) that are
11830-11835 1 PNAS 1 September30, 2003 1 vol. 100 1 suppl. 1
sloughed from the skin surface consist largely of dead protein-
aceous sacs of IF cables; these cell remnants are continually
replaced by inner cells moving outward.
In mouse skin, as measured by autoradiography, the entire
differentiation process from basal layer to squame takes 10-14
days (4~. Human epidermis turns over more slowly; however, the
proliferative reserve of human skin epithelial stem cells, which
supply sufficient progeny to maintain 1-2 m2 of skin for decades,
must be enormous.
An early observation in the field of skin biology was that
epidermal keratinocytes could be grown in culture. As opposed
to many other cell types that require transformation to be
cultured effectively, epithelial cells taken directly from the skin
can be passaged for many generations when cultured in the
presence of a fibroblast feeder layer (54. When grown in the
presence of an epidermal growth factor (EGF) receptor ligand
such as EGF or transforming growth factor ax (TGFa), human
keratinocytes can be expanded by a factor of 10~6 (6~. A careful
analysis of the growth potential of human skin keratinocytes
revealed three different types of cells based on the size of the
clones they are capable of generating in a single plating (7~.
Holoclones, which have the greatest proliferative potential,
contain cells that almost all (95%) go on to form proliferative
colonies on passaging. Meroclones have intermediate prolifer-
ative potential, and paraclones abort and differentiate after very
few passages. Holoclone cells can transition to meroclone and
paraclone cells, but the reverse transition was not observed in
this study. It is tempting to speculate that holoclone-generating
cells in vitro might be stem cells in viva.
Where Are the Skin Epithelial Stem Cells Located?
On initial histological evaluation of mammalian skin, there is no
obvious morphologically distinct region, or niche, of the basal
layer where stem cells might be located. It has been known from
the 1970s that the epidermis is organized into columns of
maturing cell layers ~10 cells wide (see Fig. 1B) (8~. It was
initially hypothesized that the entire basal layer consisted of stem
cells, then later that the Langerhans cells were stem cells.
Radiation dose-survival studies suggested that stem cells might
comprise 2-7% of basal layer cells (reviewed in ref. 84. One
method of retrospectively demonstrating the presence of stem
cells in epidermal cultures is to label the population of cells and
then use them to reconstitute epidermal tissue in vivo. Thus,
when retrovirally tagged murine epidermal cultures expressing
the I3-galactosidase reporter gene were grafted onto a mouse, the
reconstituted skin exhibited clonal columns of p-galactosidase-
expressing epidermal cells in the host animal (Fig. 1B) (9~. The
size of the columns over the 12-week study period suggested that
This paper results from the Arthur M. Sackier Coiloquium of the National Academy of
Sciences, "Regenerative Meclicine," held October 18-22, 2002, at the Arnold anc] Mabel
Beckman Center of the National Acaclemies of Science and Engineering in Irvine, CA.
Abbreviation: ERC, iabei-retaining ceil.
*To whom correspondence should be acidressed. E-mail: fuchsib@?rockefeller.ecJu.
2003 by The National Acaclemy of Sciences of the USA
www. p n as. org /cg i /doi /10.1 073/ pn as. 1734203100
OCR for page 15
Stratum
corneum
Epithelium
Basement
membrane
Pious
layer
Differentiating progeny of
one epidermal stem ceil
Enidermal
stem cell
Transiently
Amplifying
Cells
Fig. 1. (A) Diagrammatic representation of skin epithelial histology. Cells~of the basal layer attach to an underlying basement membrane. Basal cells are
mitotically active, but they lose this potential when they detach from the basement membrane and embark on the outward trek toward the skin surface. As basal
cells enter the spinous layer, they strengthen their cytoskeletal and intercellular connections, gaining resilience to mechanical stress. Once this task is completed,
the cells enter the granular layer, where they produce the epidermal barrier. The barrier precursors consist of two major components: (/D glutamine- and
Iysine-rich cornified envelope precursor proteins, which are synthesized and deposited beneath the plasma membrane, and (i/) lamellar granules, which are filled
with lipid bilayers. As the cells enter the final phases of terminal differentiation, a flux of calcium activates the enzyme transglutaminase, which biochemically
cross-l inks the corn if fed envelope protei ns through s-(~-g I uta myl) Iysi ne isopeptide bonds a nd wh ich activates the extrusion of the I i pid hi layers onto th is scaffold.
Cell death ensues, leaving dead, flattened squames at the skin surface, the end-process of terminal differentiation. These squames of the stratum corneum
eventually slough from the skin surface, to be replenished continually by inner layer cells moving outward. (B) Diagram of the epidermal proliferative unit. A
putative slow-cycling epidermal stem cell occasionally divides, giving rise to a stem cell daughter and a transiently amplifying daughter. The transiently
amplifying cell divides two to four times, and these progeny then leave the basal layer and execute a program of terminal differentiation. This model is based on
retroviral transduction of a ,6-galactosidase gene into cultured keratinocytes, which were then used in engraftments onto nude mice to trace stem cell
lineages (9).
as many as 10-12% of murine basal layer cells might be "stem
cells" capable of generating a single maturing column of cells.
Another method of identifying tissue stem cells makes use
of their slow cycling nature. In a pulse-chase experiment, all
dividing cells of a tissue incorporate nucleotide analogs such as
bromodeoxyuridine (BrdUrd) or tritiated [3Hithymidine into
newly synthesized DNAs. When the label is chased, only those
cells that divide rarely and still reside within the tissue over time
will retain their label. In oral epithelium, so-called label-
retaining cells, or LRCs, are located in discrete regions of tongue
and palatal papillae (10~; in murine ear epidermis, LRCs reside
in the basal layer, near the periphery of differentiating cell
columns (11~. Therefore, a model of skin epithelial maintenance
emerged in the 1980s in which the periodic division of slow-
cycling stem cells in the basal layer gives rise to transiently
amplifying cells that populate most of the basal layer, dividing
two or three times and then moving upward while differentiating
into mature skin cells (Fig. 1B).
In the 1990s, researchers using t3H]thymidine to evaluate label
retention in murine haired epidermis discovered that the ma-
jority of LRCs in the skin reside in the "bulge" region of the hair
follicle, with only a small fraction of LRCs in the basal layer of
interfollicular epidermis (12, 13~. The hair follicle is an epider-
mal appendage that consists of an upper, permanent portion,
and a lower, cycling portion that produces the hair (Fig. 2;
reviewed in refs. 14 and 15~. The outer root sheath (ORS) is
contiguous with and biochemically similar to the basal layer of
the epidermis. The inner layers of the hair follicle include three
concentric layers of inner root sheath (IRS) and three concentric
layers of hair-producing cells. At the base of the hair follicle is
the germinative matrix, which contains rapidly proliferating
"matrix" cells that differentiate to populate all of the layers of
the IRS and the hair shaft itself. The hair follicle bulge, which
Alonso and Fuchs
contains the LRCs, resides within the ORS in a small niche just
below the sebaceous gland, at or near the site of insertion of the
arrector pill muscle. At first glance, the bulge is an interesting
place for stem cells to live; it is down below the surface of the
skin, protected by a column of cells in the upper portion of the
hair follicle, as well as by the heavily keratinized hair shaft itself.
Across the basement membrane, it is surrounded by a supportive
dermal pocket that is richly vascularized and innervated.
Two studies confirmed the relevance of the label retention of
bulge keratinocytes by dissecting rat (16) and human (17) hair
follicles and evaluating different regions of the follicles for
clonogenicity in vitro. In rat whisker follicles, of ~740 total
colony-forming cells per follicle, 95% were found in the bulge,
and the remaining 5% were found in the matrix region. In human
scalp follicles, the highest clonogenicity was found in a region
directly below the bulge; these cells were found to have tremen-
dous proliferative capacity, with theoretical output of as many as
1.7 x 1038 progeny from a single cell (17~. In a creative
confirmation that LRCs have stem cell properties, LRCs cul-
tured from epidermis after a long chase were found to be more
clonogenic than pulse-labeled cells from a similarly aged animal
(13~. Thus, the most clonogenic cells and the cells with highest
label-retaining capacity in the mammalian haired epidermis
occur in the hair follicle, in or near the bulge region.
Hair cycling, the repeated regeneration of hair follicles to
produce new hair shafts over the lifetime of the organism, is a
useful model by which to study stem cell properties. According
to the "bulge activation hypothesis," bulge stem cells are stim-
ulated to divide and produce a new germinative hair matrix only
after receiving signals from specialized hair follicle mesenchymal
cells (12~. In support of this theory, bulge cells have been shown
to divide before regrowth of the follicle (18~.
PNAS | September 30, 2003 I vol. 100 | suppl. ~ | 11831
OCR for page 16
Basal layer
Outer root sheath -
Ir~r`~r rams Huh
Hair shaft -'
Dermaloaoilla //' ,' i.
' ~ // ,' A
me'
~ ~ .
Wit :/
1 ~l'
:~ Inf'~nrlih''1'tm
~-~2
I ~ A A)
1 - 1\
Label retaining
keratinocytes
in the bulge
l
Hair follicle matrix
Functional Characteristics of Skin Epithelial Stem Cells
Locating the putative epidermal stem cells represents a major
advance in the field, allowing scientists to move forward with
respect to the biochemical and functional characteristics of this
important population of cells. Other stem cell fields, such as the
hematopoietic system, are replete with cell surface markers that
identify nearly every cell type, starting with stem cells and
extending through the most differentiated forms of the progeny
types. Specific markers of epidermal stem cells, however, are not
yet known. Although these cells can be identified either in vivo
by label retention or in vitro by clonogenicity, neither method of
identification presently allows easy isolation of stem cells for
analysis. Therefore, there is a strong need for specific epidermal
stem cell markers.
One class of candidate stem cell markers is the integrin family
of transmembrane receptors, whose members are responsible for
the attachment of the basal layer of the epidermis to its
underlying substratum, the basement membrane (reviewed in
ref. 19~. Basement membrane is rich in extracellular matrix
(ECM) (20) proteins, many of which constitute the ligands for
integrin a,B heterodimers. When cultured human keratinocytes
were isolated by fluorescence-activated cell sorting (FACS) on
the basis of their surface integrin {31 levels, cells with the highest
fluorescence displayed a moderately increased colony-forming
efficiency in vitro (21~. In this study, colony-forming efficiency
correlated with the speed of cell adherence to integrin ligands,
including type IV collagen (c~2,(31) and ECM proteins secreted
by keratinocytes (21~. In a different study, human keratinocytes
sorted for the hemidesmosomal integrin old, which partners with
Epidermal layers
Sebaceous gland
Lineages derived from
bulge stem cells
Differentiation pathways
supplied by bulge stem cells
11832 1 www.pnas.org/cgi/doi/10.1073/pnas.1734203100
Fig. 2. Diagram of the hairfollicle and cell lineages supplied
by epidermal stem cells. A compartment of multipotent stem
cells is located in the bulge, which lies in the outer root sheath
(ORS) just below the sebaceous gland. Contiguous with the
basal layer of the epidermis, the ORS forms the external
sheath of the hair follicle. The interior or the inner root
sheath (IRS) forms the channel for the hair; as the hair shaft
nears the skin surface, the IRS degenerates, liberating its
attachments to the hair. The hair shaft and IRS are derived
from the matrix, the transiently amplifying cells of the hair
follicle. The matrix surrounds the dermal papilla, a cluster
of specialized mesenchymal cells in the hair bulb. The multi-
potent stem cells found in the bulge are thought to contrib-
ute to the lineages of the hair follicle, sebaceous gland, and
the epidermis (see red dashed lines). Transiently amplifying
progeny of bulge stem cells in each of these regions differ-
entiates as shown (see green dashed lines).
,B4 to robustly attach to basement membrane component laminin
5, were shown to have higher proliferative potential than those
sorted for the focal adhesion integrin p1, which partners pro-
miscuously with cr2 (type IV collagen), or3 (laminin 5), or5
(fibronectin), and or9 (tenascin) in keratinocytes (22~.
Integrin expression levels can change when cells are trans-
ferred from living skin to culture conditions, potentially intro-
ducing caveats to the extrapolation of in vitro integrin expression
data to in viva stem cells. In this case, however, the colony-
forming efficiency of human keratinocytes obtained directly
from skin also correlated with rapid adhesion to type IV
collagen, a feature characteristic of cells with elevated or2
integrin (23~. Additionally, immunohistochemical analysis of
intact human skin from different regions seems to display
heterogeneity in ,B1 integrin expression levels; patches of in-
creased expression have been postulated to contain stem cells
(234. Early studies suggested a role for 131 integrin signaling in
the prevention of terminal differentiation (20~. Mice condition-
ally lacking integrin ,B1 in skin epithelial cells exhibit severe
defects in basement membrane assembly and organization,
underscoring a role for these integrins not only in attachment to
but also assembly of extracellular matrix (refs. 24 and 25; see also
ref. 26~. Wound healing is impaired in these mice; although I31
null keratinocytes proliferate adequately in vivo after wounding,
they migrate ineffectively, resulting in delayed reepithelializa-
tion (27~. Embryonic stem cells lacking I31 integrin show reduced
ability to differentiate into keratinocytes, a defect that can be
partially rescued by dermal derived growth factors (28~. Whether
the skin stem cell compartment depends on ,{31 integrin has been
Alonso and Fuchs
, . .
OCR for page 17
more difficult to judge given the severity in phenotype of the
f1-null skin.
Recently, two studies investigating the transcriptional profiles
of hematopoietic, neural, and embryonic stem cells have found
integrins to be up-regulated in these stem cells as compared with
their transiently amplifying progeny (29, 30~. Integrin or6 was
present in both lists, and 91 was present in one of the two.
Because stem cells are restricted to the basal layer of either hair
follicle bulge or interfollicular epidermis, molecules instrumen-
tal in cell-substratum adhesion are conceptually interesting stem
cell markers. It is possible that stem cells require strong adher-
ence to the basement membrane to maintain their stem cell
characteristics or their position in the stem cell niche. Despite
the intrigue, most if not all proliferating cells use integrins in
adhesion. Thus, the usefulness of integrins as stem cell markers
is limited by the uncertainty of interpretation of their levels of
expression relative to transit-amplifying stem cell progeny.
The transferrin receptor is another surface marker shown to
differ in its expression between stem cells and proliferating
progeny. In this case, reduced surface expression of the trans-
ferrin receptor has been associated with human keratinocyte
stem cells. Sorting of primary skin cells on the basis of integrin
x6 and transferrin receptor found that LRCs were enriched in
o`6-high, transferrin receptor-low cells, whereas cells actively
dividing were enriched in the c~6-high, transferrin receptor-high
population (314.
Even in the absence of cell surface markers useful for isolation
of stem cells, skin biologists have made advances in understanding
some of the molecules important in conversion from stem cell to
transit-amplifying cell. One such example is the protooncogene
c-myc, a transcriptional regulator of proliferation in a large variety
of cell types, including skin keratinocytes (32, 33~. Interestingly,
overexpression of c-myc in transgenic mouse skin results in what
appears to be depletion of the multipotent skin stem cells of the
bulge, as judged by a reduction in LRCs and impaired wound
healing (34, 35~. Surprisingly, increasing c-myc expression also
seems to cause a cell fate change from hair follicle progenitor cells
to sebum-producing cells, suggesting that c-myc levels may influ-
ence not only the decision of stem cell daughters to become
transit-amplifying cells, but also the decision of which lineage to
adopt.
Another factor associated with stem cells and/or their con-
version to transit-amplifying cells is the transcription factor p63,
a homologue of p53. p63 is known to be expressed in epithelial
stem cells of the corneal limbus in vivo and in the holoclone-
generating skin keratinocytes ~n vitro (364. Mice harboring a
targeted deletion at the p63 gene locus have a profound defect
in epidermal development (37, 38~. In early development, when
the skin epithelium is a single layer, the p63-null skin appears
normal. As the process of stratification proceeds, however,
p63-null skin becomes progressively denuded of epithelium,
leaving only a few remaining cells that express suprabasal and
not basal markers (374.
These studies suggest that p63 and c-myc are both important
regulators of skin keratinocyte function. A major issue still
unresolved for both these factors is the extent to which they
govern stem cell maintenance versus the production of transit-
amplifying progeny.
The Multipotency of Skin Epithelial Stem Cells: What Is the
Relationship Between Interfollicular and Bulge Stem Cells?
Substantial evidence supports the idea that stem cells in the
interfollicular epidermis are less potent than bulge stem cells,
leading to speculation that they are progeny, perhaps unipotent
progeny, of multipotent bulge cells. In contrast to bulge cells,
interfollicular stem cells do not have a clearly defined niche.
There are more slow-cycling stem cells in the bulge than in the
interfollicular epidermis (12), and the longevity of label reten-
Alonso and Fuchs
tion in bulge cells is longer than that in interfollicular cells (39,
404. Cells cultured from the bulge also have a higher clonogenic
potential than interfollicular cells (17~. Curiously, LRCs of the
bulge require repeated treatment with 12-0-tetradecanoylphor-
bol-13-acetate (PMA) to induce proliferation, whereas interfol-
licular LRCs are more easily induced to divide (394. Finally,
superficial burns that destroy the interfollicular epidermis but
leave intact the hair follicles do not require skin grafting,
whereas deeper burns in which the hair follicles are destroyed
cannot regrow epithelium except from the edges (414.
The multipotency of bulge cells is demonstrated by the fact
that these special cells can give rise to all lineages of skin
epithelia, including interfollicular epidermis (Fig. 1B). Cells
isolated from dissected bulge regions are capable of differenti-
ating into stratified epidermis when grown to confluence in
culture and transplanted onto athymic mice (17~. The label-
retaining property of slow-cycling stem cells has been exploited
to evaluate bulge cell contribution to multiple epidermal lin-
eages. Cells that retain label after an 8-week chase, which reside
exclusively in the bulge by the technique used in this study,
contribute to all of the layers of the hair follicle (40~. Pulse
labeling with two different nucleotides, timed precisely (based
on cell cycle length) to label cells of the infundibulum region of
the hair follicle, demonstrated an efflux of hair follicle cells out
into interfollicular epidermis in both normal and wounded states
(404. In another assay, the multipotency of bulge stem cells was
demonstrated by transplantation of I3-galactosidase-expressing
transgenic whisker bulge cells into the bulge region of follicles
from an unlabeled recipient mouse (424. Over time, I3-galacto-
sidase-expressing cells from transplanted bulges populated all of
the epithelial compartments of the resulting chimeric follicles,
including the sebaceous gland and the infundibular region above
the bulge that is thought to be most similar to interfollicular
. .
epic .ermls.
Although the evidence supporting multipotency of the bulge
cells is compelling, the characteristics of stem cells outside this
niche are less certain. Are interfollicular epidermal cells unipo-
tent or multipotent? To what extent, if any, do they differ from
bulge stem cells? The answers to these questions await more
extensive knowledge about the molecular characteristics of bulge
cells. Recently, however, the Wnt signaling pathway has been
linked to the ability of skin epithelial cells to acquire and/or
maintain features of multipotent stem cells (43-454. At the heart
of this pathway is I3-catenin, a multifunctional protein that is
stabilized when cells receive a Wnt signal. h-catenin is required
in some way to activate members of a DNA-binding protein
family referred to as the Lef/Tcf family (reviewed in ref. 464. In
skin, Wnt signals are received by multipotent embryonic skin
epithelial cells before their commitment to form a hair follicle
(47~. When specialized skin mesenchymal cells inhibit a second
signaling pathway, the bone morphogenetic protein (BMP)
pathway, the multipotent epithelial cells express Lefl and be-
come committed to forming a hair follicle (484. Both the Wnt
and BMP signaling pathways appear to be functionally important
to making a hair follicle, as judged by the fact that mice with
disrupted function of Lefl (49-51), Noggin (52), or I3-catenin
(53, 54) are all severely impaired in their ability to form hair
follicles.
Normally, only bulge stem cells are thought to retain multi-
potency in postnatal skin. However, when f-catenin is consti-
tutively stabilized in transgenic mouse skin, the adult interfol-
licular epidermis behaves as embryonic skin, seemingly able to
choose between an epidermal and hair follicle fate (444. Inter-
estingly, when the specialized mesenchymal cells (dermal papilla
cells) of the hair follicle are exposed to Wnt signaling, they too
appear to retain their hair follicle-inducing power (554. There-
fore, Wnt signals may be able to act both on the epithelillm to
PNAS | September 30, 2003 | vol. 100 | suppl. ~ | 11833
OCR for page 18
induce stem cell-like properties and on the mesenchyme to
maintain its stem cell recruiting properties.
The degree of Lefl /Tcf activity may be critical in determining
the outcome of stem cell lineage determination. When Lefl is
overexpressed in the skin and oral epithelium, occasional hairs
and teeth are seen in inappropriate places (43~. When a form of
Lefl that is unable to associate with I3-catenin is overexpressed,
hair follicle cells adopt a sebaceous cell fate in inappropriate
places, and epidermal cysts form in place of some secondary hair
follicles (50, 51~. Similarly, when I3-catenin is conditionally
targeted for removal in skin, epidermal cysts are observed in
place of hair follicles (53~. In the future, it will be interesting to
explore the levels and effects of other proteins that are now
known to influence the status of Lefl/Tcf activity (reviewed in
ref. 56~. In this regard, the recent parallel findings of Kielman et
al. (57) showing that adenomatous polyposis cold (APC) influ-
ences stem cell lineage determination in embryonic stem cells by
controlling the dosage of I3-catenin signaling are fascinating. In
addition, there are now a number of reports that suggest a more
global role for I3-catenin and its partners in stem cells and fate
specification (see refs. 58-604.
Clinical Applications of Skin Epithelial Stem Cells: Grafting of
Cultured Keratinocytes
Basic research into stem cell biology is partly oriented toward the
eventual possibility of harvesting stem cells from a patient,
modifying or expanding them, and reimplanting them to treat
disease. Skin keratinocytes have proved useful for this already,
because of their accessibility and ability to be cultured. The most
prominent clinical use of cultured keratinocytes is in creating
confluent epithelial sheets that can be gently removed from the
culture dish and applied to reconstitute the epithelial portion of
burns, chronic wounds, and ulcers. The advantage of this method
is the use of the patient's own skin, which represents the optimal
long-term repopulation strategy. Today, the most commonly
used skin grafting technique employs a different approach, the
use of split-thickness grafts taken from unaffected skin. This
method is effective, but it is limited by the available surface area
of unaffected skin and creates some degree of additional injury.
The use of cultured keratinocytes allows a much greater surface
to be covered and requires a smaller area of unaffected skin from
which to harvest the keratinocytes for culture. At present, the use
of cultured keratinocytes is limited by the length of time needed
to grow the epithelial sheets in vitro, during which time the
patient is susceptible to infection. The epithelial sheets are also
extremely fragile and do not adhere well to some burn surfaces.
Under development are skin substitutes that could function
as dermal equivalents to hold the expanding keratinocytes,
improve adhesiveness to the burn wound, and form a temporary
wound cover to reduce infection rates. If subconfluent keratin-
ocyte cultures could be implanted into the dermal equivalent,
then this graft could be applied very early after the burn injury,
with the epithelial cover maturing while the dermal equivalent
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11834 1 www.pnas.org/cgi/doi/10.1073/pnas.1734203100
functions as a temporary dressing, obviating the need for two
surgeries (61~.
Grafting of cultured skin epithelial stem cells has other
potential applications besides replacing burned skin; in partic-
ular, it is exciting to consider the possibility of using the cultured
keratinocytes as delivery instruments for gene therapy. Two
groups have devised methods to use cultured human keratino-
cytes to correct inborn metabolic skin diseases (62, 63~. Kera-
tinocytes were harvested from patients with recessive dystrophic
epidermolysis bullosa, and the genetic defect was corrected
either by genomic integration of the correct sequence using a
bacteriophage integrase or by transgene expression using a
lentivirus. The repaired keratinocytes were expanded in culture
and grafted onto nude mice to produce healthy epithelia in which
the defect was corrected. Although neither study included
grafting back onto the original human being suffering from skin
disease, these efforts represent a major advance toward the
possibility of manipulating stem cells to treat human disease.
Conclusions
The search for the biochemical regulators of skin epithelial stem
cell self-renewal and production of daughter cells that will
populate one or several lineages of the epidermis is ongoing. The
field has advanced significantly over the past three decades,
especially with respect to the presence and location of discrete
stem cell compartments. Some initial work has implicated the
integrins as cell surface markers that, although not specific, may
be useful to enrich populations of cells for stem cells to allow
characterization. C-myc and p63 have also been identified as
regulators of stem cell fate. In addition, recent years have
experienced a flurry of reports that implicate Wnt signaling,
13-catenin, and Lefl/Tcf transcriptional regulation in stem cell
maintenance and/or lineage determination. Despite these ad-
vances, scientists are not yet able to reliably isolate stem cells
from skin epithelium to exhaustively study their transcriptional
and functional characteristics. Eventually, when techniques exist
to find the answers to these questions, doctors may be able to not
only use skin epithelial stem cells to grow new skin to treat burns,
but also to treat genetic diseases of skin and possibly even
nonskin origin. We don't yet know whether it might be possible
in the future to genetically engineer keratinocytes to inducibly
secrete peptide hormones, such as insulin as a treatment for
diabetes, or growth hormone as a treatment for growth hormone
deficiency (644. Nor do we know whether keratinocyte stem cells
possess sufficient plasticity to be differentiated into nonkerati-
nocyte cell types to correct defects of other tissues. The studies
of the past three decades indicate that the future of skin stem cell
research holds great promise.
We thank our scientific colleagues and past and present Fuchs laboratory
members, who have advanced our understanding of epithelial stem cells.
E.F. is an Investigator of the Howard Hughes Medical Institute. The
research leading to this article was supported by National Institutes of
Health Grant RO1-AR31737.
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Representative terms from entire chapter:
stem cell
