© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2559-2567 doi:10.1242/dev.104588
REVIEW
Heterogeneity and plasticity of epidermal stem cells
ABSTRACT
The epidermis is an integral part of our largest organ, the skin, and
protects us against the hostile environment. It is a highly dynamic
tissue that, during normal steady-state conditions, undergoes
constant turnover. Multiple stem cell populations residing in
autonomously maintained compartments facilitate this task. In this
Review, we discuss stem cell behaviour during normal tissue
homeostasis, regeneration and disease within the pilosebaceous
unit, an integral structure of the epidermis that is responsible for hair
growth and lubrication of the epithelium. We provide an up-to-date
view of the pilosebaceous unit, encompassing the heterogeneity
and plasticity of multiple discrete stem cell populations that are
strongly influenced by external cues to maintain their identity and
function.
KEY WORDS: Stem cells, Epidermis, Regeneration, Tissue
homeostasis
Introduction
The skin is the largest organ of the body and consists of multiple
layers with distinct developmental germ layer origins. The
epidermis forms the outermost, water-impermeable layer that
protects against the hostile environment and retains bodily fluids
inside. It is composed of epithelial cells (both keratinocytes and
Merkel cells) in addition to small populations of Langerhans
cells, gamma delta (γ∂) T-cells and melanocytes. The dermis
underneath the epidermis forms the basis for complex interactions
with fibroblasts, endothelial, neuronal, muscle and immune cells
and provides a three-dimensional framework that supports the
maintenance of the epidermis.
The main component of the epidermis is the interfollicular
epidermis (IFE), which forms the protective barrier against the
outside environment. The IFE is a stratified epithelium in which
proliferating cells are anchored to the basement membrane closest
to the dermis. As cells lose affinity for the basement membrane,
they initiate terminal differentiation and, following extensive
remodelling of intracellular proteins, intercellular junctions, lipid
extrusion and nuclear fragmentation, the cells eventually become
highly cross-linked scales that are exfoliated from the surface of
the skin. The pilosebaceous unit (PSU) is a prominent structure
associated with the IFE (Fig. 1). It has important functions within
the tissue that are mediated via its components: infundibulum,
isthmus, sebaceous glands and the hair follicle. The infundibulum
1
BRIC - Biotech Research and Innovation Centre, University of Copenhagen, Ole
2
Maaløes Vej 5, Copenhagen N DK-2200, Denmark. Wellcome Trust & Medical
Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge
CB2 1QR, UK.
*Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly
attributed.
forms the upper part of the PSU between the IFE and the isthmus.
The isthmus forms the mid-region starting at the top of the bulge
and ending at the infundibulum, where it segregates hair follicle
and interfollicular differentiation markers and creates a funnel for
the hair shaft (Fig. 1). The upper region of the isthmus adjacent to
the infundibulum and the sebaceous gland is defined as the
junctional zone (JZ) (Jensen et al., 2009). The sebaceous gland
produces sebum, an antiseptic oily substance that lubricates the
hair and the surface of the skin. Similar to the IFE, proliferating
cells in the sebaceous gland anchored to the basement membrane
support turnover of differentiated cells as they burst and release
their lipid content. The lower, permanent PSU can be divided into
two parts: the bulge and the hair germ. Hair follicle stem cells
reside in the bulge, whereas the hair germ is located directly
above the dermal papilla and forms the germinal centre for hair
follicle growth (Fig. 1) (Silver and Chase, 1977).
In mice, PSU size varies depending on the auxiliary functions:
large sensory vibrissae (whiskers) originate from a large PSU,
whereas the smaller zig-zag hairs that make up the heatinsulating coat tend to have a correspondingly small PSU.
Despite these variations, the overall composition of the PSU is
the same (Sundberg and Hogan, 1994). The lower, permanent
part of the PSU supports successive rounds of hair growth,
which continues throughout life in three distinct phases: anagen,
catagen and telogen. Depending on the mouse strain and body
site, the initial phase of PSU morphogenesis ceases at postnatal
day (P) 14-18, from which point hair cycling is initiated by
regression of the hair structure, known as the catagen phase.
During this process, the bottom of the hair follicle undergoes
rapid apoptosis. After a short resting phase of 3-4 days known as
the telogen phase, hair growth is reinitiated in anagen phase and
the cycle continues. Hair cycling through the three phases
continues throughout life; however, it is only the first postnatal
cycle that is uniformly synchronised in the mouse. This ends
with another catagen phase and a long telogen phase at ∼P42-49,
which lasts 2-3 weeks (Alonso and Fuchs, 2006). Subsequent
hair cycles of individual follicles are then asynchronous and are
instructed by local signals (Plikus et al., 2008). The cyclic
nature of this process is governed by an intricate interplay
between dermal cells and epidermal keratinocytes in the lower
PSU (Fig. 1) (Plikus et al., 2008; Driskell et al., 2009; Greco
et al., 2009).
The distinct components of the epidermis are maintained
throughout life by resident stem cells. In humans, the first
seminal experiments suggesting the existence of epidermal stem
cells came from in vitro cultures, in which epithelial cells from
small skin biopsies were serially propagated and shown to form
stratified squamous epithelium with more advanced keratinisation
of upper cell layers (Rheinwald and Green, 1975). Stem cell
behaviour was proven by the successful engraftment to, and longterm maintenance of, cultured keratinocytes in burns victims
(Gallico et al., 1984). In general, a high degree of cellular
heterogeneity defined by marker expression, cell division rate and
2559
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Troels Schepeler1, Mahalia E. Page2 and Kim B. Jensen1,2,*
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Development (2014) 141, 2559-2567 doi:10.1242/dev.104588
governed by a number of equipotent stem cell populations with
discrete functions during homeostasis. In this Review, we will
discuss the basis for this model and its functional relevance.
Fig. 1. Hair follicle stem cell compartments. Different stem cell populations
are shown in the mouse resting (telogen) adult hair follicle. Each stem cell
compartment is defined by distinct protein expression and gene promoter
activity (see key); cells with multiple colours express multiple markers.
ultrastructure, has been observed both within the basal layer of the
human IFE (Jones et al., 1995; Li et al., 1998; Jensen et al., 1999)
and in the PSU (Cotsarelis et al., 1990; Rochat et al., 1994; Lyle
et al., 1998; Ohyama et al., 2006). These observations led to the
proposal that stem cells exist within distinct niches and that these
cells can give rise to progeny with limited proliferative potential,
also known as transit amplifying cells. Similar observations
have been made for the mouse epidermis, which will be the focus
of this Review.
The prevailing model for epidermal maintenance places
multipotent stem cells at the apex of a cellular hierarchy. This is
based on a combination of cell culture, lineage-tracing and
transplantation studies (Jaks et al., 2008; Snippert et al., 2010;
Blanpain et al., 2004; Claudinot et al., 2005; Jensen et al., 2008).
However, it is not clear whether transplantation studies provide a
true reflection of multipotency during steady-state homeostasis and,
furthermore, the exact location of the multipotent stem cells remains
unclear. Recent data from live-imaging studies and long-term fatemapping experiments have demonstrated regionally restricted
contributions from multiple distinct stem cell niches in the PSU
during homeostasis (Ghazizadeh and Taichman, 2001; Morris et al.,
2004; Levy et al., 2005; Jaks et al., 2008; Brownell et al., 2011; Page
et al., 2013). Furthermore, transplantation and injury studies
demonstrate that such regional restriction of discrete stem cell
populations breaks down after tissue damage, as stem cells have
been observed to regenerate all structures of the epidermis under
such conditions (Levy et al., 2005, 2007; Nowak et al., 2008; Jensen
et al., 2009; Brownell et al., 2011; Page et al., 2013). This forms the
basis for an updated model of tissue maintenance, which is
2560
The epidermis forms as a flat single-layered epithelium from the
surface ectoderm. The appearance of PSUs proceeds in waves
depending on the associated hair type, starting with whisker follicles,
then awl/auchene follicles and lastly zig-zag hairs. Although the size
of the PSU varies between the different hair types, they all follow
essentially the same morphological transitions (reviewed by SchmidtUllrich and Paus, 2005). Focal elevation in Wnt signalling initiates
PSU formation and the growing structure subsequently extends into
the underlying mesenchyme (Gat et al., 1998; St-Jacques et al., 1998;
Huelsken et al., 2001). Analysis of the developing PSU demonstrates
co-expression of the future adult stem cell markers Sox9, Lgr6 and
Lrig1 (Nowak et al., 2008; Jensen et al., 2009; Snippert et al., 2010;
Frances and Niemann, 2012). As the PSU extends further into the
dermis, expression of these stem cell markers segregates into distinct
domains. These include a quiescent region that is positive for future
bulge stem cell markers, such as Sox9, Nfatc1 and Tcf3, as well as a
distinct Lrig1-expressing region above the prospective bulge from
which sebaceous glands subsequently emerge (Fig. 2) (Nowak et al.,
2008; Jensen et al., 2009; Frances and Niemann, 2012). Other stem
cell markers such as Plet1 (recognised by antibody MTS24)
and CD34 are not expressed until after sebaceous gland formation
and the first completed hair cycle, respectively (Watt and Jensen,
2009; Frances and Niemann, 2012). The outcome from these early
developmental events is a patterned PSU with defined compartments
demarcated by markers of the future stem cell niches.
Extensive cellular heterogeneity exists within the mature PSU and
this has been the topic of a number of excellent recent reviews (Arwert
et al., 2012; Rompolas and Greco, 2014; Solanas and Benitah, 2013).
Several different populations of cells have been shown to maintain the
PSU, and are characterised by the expression of a variety of different
markers (see Fig. 1). These include: Lgr5, CD34 and Krt15, which
mark the hair follicle bulge stem cells (Liu et al., 2003; Trempus et al.,
2003; Jaks et al., 2008); Blimp1 (Prdm1), which marks the sebaceous
gland stem cells (Horsley et al., 2006); Gli1 and Lgr6, which mark
stem cells in the lower isthmus (Snippert et al., 2010; Brownell et al.,
2011); and Lrig1 and Plet1, which mark stem cells in the upper
isthmus/JZ (Nijhof et al., 2006; Jensen et al., 2009; Raymond et al.,
2010). It is not yet clear whether these different patterns of gene
expression reflect specific functions; however, the functional
significance of cellular heterogeneity is beginning to emerge from
the differences observed in cell behaviour. Several lines of
investigation now support a model whereby the epidermis is
regionally compartmentalised into functional units that are
maintained autonomously (Ghazizadeh and Taichman, 2001; Levy
et al., 2005; Page et al., 2013). Here, the lower PSU supports hair
follicle growth, while the sebaceous gland, infundibulum and IFE are
replenished as independent, yet connected, compartments. This
concept opposes the idea that a single population of multipotent stem
cells is responsible for epidermal maintenance (Taylor et al., 2000;
Lavker et al., 2003, Snippert et al., 2010). In order to understand how
long-term homeostasis of the epidermis is maintained, it is necessary
to assess distinct cell behaviours within each PSU compartment, as
well as their specific requirements for cellular replenishment.
The PSU and tissue compartmentalisation
The development of improved strategies for transgenesis has
enabled the generation of numerous mouse strains that allow
DEVELOPMENT
The emergence of cellular heterogeneity within the PSU
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Development (2014) 141, 2559-2567 doi:10.1242/dev.104588
temporal and spatial labelling of specific subpopulations of cells in
the PSU (reviewed by Kretzschmar and Watt, 2012; Blanpain and
Simons, 2013; Alcolea and Jones, 2014). These sophisticated
approaches have made it possible to gain new insights into the longterm maintenance of the epithelium and to characterise cellular
heterogeneity during steady-state homeostasis, allowing a
comprehensive view of how each individual compartment within
the PSU is both maintained and replenished.
The hair follicle
The best-characterised stem cell population in the PSU resides in the
bulge region (bulge stem cells; Fig. 1). These cells have been the focus
of numerous investigations because of their prominent location, highly
quiescent nature (Cotsarelis et al., 1990), extensive clonogenic
capacity in vitro (Oshima et al., 2001) and their expression of a set
of distinct markers (reviewed by Fuchs and Horsley, 2008). Bulge stem
cells were initially believed to contribute to the entire epithelium during
steady-state homeostasis (Taylor et al., 2000; Lavker et al., 2003).
However, fate-mapping experiments have since established that these
cells are restricted to lower hair follicle lineages during steady-state
homeostasis (Morris et al., 2004; Jaks et al., 2008; Youssef et al., 2010;
Page et al., 2013). It has been suggested that hair follicle stem cells
contribute to the sebaceous gland (Petersson et al., 2011); however, the
minimal Krt15 promoter used to control Cre expression in these
experiments has been associated with ectopic expression in the IFE
and the sebaceous gland, providing a false impression of the hair
follicle stem cell contribution (Lapouge et al., 2011). The molecular
characterisation of bulge stem cells has provided new insights into
their biological properties. Importantly, although the overall hair
follicle stem cell niche appears homogenous, an underlying cellular
heterogeneity exists, including circadian oscillation within the stem
cell pool that is important for long-term tissue homeostasis (Janich
et al., 2011). Furthermore, the localisation of stem cells within the
niche dictates their involvement in hair cycling (Rompolas et al.,
2013).
The IFE and infundibulum
Despite forming a continuous epithelial sheet, the infundibulum is
maintained independently from the IFE (Levy et al., 2005; Nowak
et al., 2008; Page et al., 2013). Fate-mapping studies in combination
with mathematical modelling have proposed various models for
tissue maintenance of the IFE. These suggest that IFE maintenance
relies either on a combination of largely quiescent stem cells
together with proliferating committed progenitors (Mascre et al.,
2012) or on a single population of progenitors (Clayton et al., 2007;
Doupe et al., 2010; Lim et al., 2013). Importantly, the models were
based on four different Cre models and the differing outcomes
might reflect the behaviour of distinct populations within the
epithelium. Moreover, different epidermal regions – ear, palm and
tail epidermis – were studied. The possibility remains that the
distinct patterning of the tail epithelium enables the formation of
distinct stem cell niches that would not exist in other body parts and
that a two-tier system of quiescent stem cells and committed
progenitors only operates in restricted areas (Gomez et al., 2013).
Quantitative studies are still lacking for lineage-tracing experiments
within the infundibulum. The available data, however, demonstrate
that proliferating Lrig1-expressing stem cells located in the upper part
of the JZ rapidly replenish the infundibulum (Page et al., 2013;
Veniaminova et al., 2013). Interestingly, clones arising in the JZ
gradually fill up the infundibulum without contributing to the IFE,
suggesting that this compartment is maintained as an autonomous unit
(Page et al., 2013). The labelling dynamics imply that transit
amplifying cells are rapidly replaced from within the JZ. The
requirement for cellular replacement is very likely a reflection of the
mechanical stress exerted by movements of the hair, as it is channelled
through the infundibulum. Increased shedding of the differentiated
barrier within the infundibulum due to this stress is evident by the
expression of pro-inflammatory markers in Lrig1-expressing cells.
These include β-defensins, which are endogenous antimicrobial
peptides, and the monocyte attractants Cxcl12, Ccl2 and Ccl7 (Nagao
et al., 2012; Page et al., 2013).
It has been proposed that cells from the PSU participate in the
maintenance of the IFE (Morris et al., 2004; Snippert et al., 2010).
These conclusions arise from fate-mapping experiments using both
Lgr6 and Krt15 promoters to drive inducible Cre recombinases.
However, it is clear that the ectopic expression activity observed
from both of these promoters within the IFE complicates such
studies (Lapouge et al., 2011; Snippert et al., 2010; Page et al., 2013;
Liao and Nguyen, 2014). The observation that the PSU is
maintained independently of the IFE (Levy et al., 2005; Nowak
et al., 2008; Brownell et al., 2011; Page et al., 2013) makes it
exceedingly difficult to reconcile these apparently contradictory
observations, and further investigations are needed to resolve this
outstanding question.
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Fig. 2. Emergence of distinct
stem cell populations during
morphogenesis of the
pilosebaceous unit. During
development, pilosebaceous formation
is initiated from an early epidermal
structure (the placode) that develops
into a fully formed pilosebaceous unit
(PSU) through a series of steps
involving complex interactions with
existing dermal cells. Initially, various
stem cell markers are co-expressed
within the same region of the
developing PSU, but at later stages
marker expression is associated with
segregation of cells into distinct
domains. Cells with multiple colours
express multiple markers.
The sebaceous gland
The sebaceous gland plays a very prominent role in lubricating
and waterproofing the epidermis. The gland requires constant
replenishment to remain functional throughout life, but the identity
of sebaceous gland stem cells remains enigmatic. Early data
suggested that Blimp1 specifically marked a population of
sebaceous gland stem cells (Horsley et al., 2006). However, it
was subsequently shown that Blimp1 is more widely expressed in
the epidermis, including in differentiated cells of the sebaceous
gland, and it has therefore been rejected as a specific marker of
sebaceous gland stem cells (Magnusdottir et al., 2007; Cottle et al.,
2013). Fate mapping from a minimal Krt15 promoter and from the
Lgr6 promoter driving Cre expression have led to the conclusion
that stem cells residing within the bulge and the lower isthmus
replenish the sebaceous gland (Petersson et al., 2011; Snippert
et al., 2010). However, the ectopic expression observed from both
the Lgr6 and Krt15 promoters in the sebaceous gland reinforces the
fact that additional studies are required to resolve the issue of
whether cells from the bulge and isthmus contribute to sebaceous
gland maintenance. Recent lineage-tracing data using Lrig1, which
marks basal cells in both the JZ and the sebaceous gland, strongly
support the contention that basal cells within the sebaceous gland
form an autonomous source for cellular replenishment and that the
sebaceous gland is maintained independently of all other
compartments (Page et al., 2013).
Merkel cells
Merkel cells link the epithelium to the nervous system and provide
sensory functions. They reside either in enervated regions of the
whisker PSU or in touch domes next to PSUs within the IFE
(Maricich et al., 2009; Doucet et al., 2013). It was initially believed
that this cell type was derived from neural crest, based on its
function and expression of neuronal markers; however, conditional
knockout, transplantation and lineage-tracing studies have
demonstrated an epidermal origin (van Keymeulen et al., 2009;
Morrison et al., 2009; Woo et al., 2010). Merkel cells have a slow
turnover, which has made analysis exceedingly difficult, but elegant
studies involving fate mapping with incorporation of nucleotide
analogs have demonstrated that epidermal keratinocytes are
responsible for their replacement (van Keymeulen et al., 2009).
Whether a specialised subset of cells within the epidermal keratinocyte
compartment is responsible for Merkel cell replacement has not yet
been addressed.
Tissue compartmentalisation
The structural composition achieved via epithelial compartmentalisation
provides an attractive model for tissue replenishment, since stem cells
only need to cater for local cell replacement needs. This design allows
compartments within the tissue to undergo renewal at different rates
without compromising tissue integrity. This is illustrated by the
spatial separation of the highly proliferative JZ and the quiescent
bulge region. Interestingly, a tissue compartmentalisation model has
recently been proposed for homeostasis of other epithelial tissues
such as the prostate, mammary gland and tongue (Van Keymeulen
et al., 2011; Choi et al., 2012; Ousset et al., 2012; Tanaka et al., 2013).
Such compartmentalisation implies that tissues generally build up
seemingly invisible borders between structural elements and that this
is relevant for maintaining long-term tissue homeostasis.
The exact molecular mechanisms for maintaining the structural
integrity of the individual elements remain unresolved. Insights from
elegant developmental studies illustrate that combinations of
mechanical forces and cell sorting can form the basis for establishing
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Development (2014) 141, 2559-2567 doi:10.1242/dev.104588
and maintaining tissue compartmentalisation. Boundaries between
different compartments are readily established but their subsequent
maintenance requires restricted movement within the tissue. In the
developing Drosophila wing disc, this is achieved by a zone of
non-proliferating cells that forms a boundary between distinct
compartments (O’Brochta and Bryant, 1985). Cell sorting has also
been associated with the differential expression of adhesion molecules
to generate differential adhesion or repulsion between juxtaposing cell
populations. Alternatively, the differential expression of secreted
signalling molecules can establish distinct cellular identities that
preclude disruption of the compartment boundaries (reviewed by
Dahmann et al., 2011).
Within the epidermis a number of these mechanisms might work in
concert. The quiescent nature of stem cells within the bulge, with
respect to both cell division and migration (Cotsarelis et al., 1990;
Rompolas et al., 2012), could potentially hinder the downward
migration of progeny of highly proliferative stem cells in the JZ, much
like the boundary zone formed by non-proliferating cells in the
Drosophila wing disc (O’Brochta and Bryant, 1985). In a similar
manner, balanced cellular replenishment between the IFE and the
infundibulum could restrict movement between compartments during
steady-state homeostasis. In this case, the structural arrangements of
the PSU at an angle to the IFE could create mechanical tension at the
junctions between the two compartments and facilitate the formation of
a seemingly invisible border. In a more classical manner, the
differential expression of adherens molecules such as CD34 and
Necl2 (Cadm1) in distinct stem cell populations is likely to affect
compartmentalisation (Trempus et al., 2003; Giangreco et al., 2009).
The importance of Necl2 in regulating stem cell proliferation is evident,
although it is still unknown how loss and gain of Necl2 affect tissue
compartmentalisation (Giangreco et al., 2009). The Eph and ephrin
family provides a classical example of sorting molecules (Batlle et al.,
2002; Solanas et al., 2011); although members are differentially
expressed within the epidermis (Genander et al., 2010), their
involvement in epidermal compartmentalisation is still unclear.
Lastly, the differential expression of growth factor receptors and
ligands, as well as extracellular matrix molecules, might endow specific
properties within different stem cell niches and thereby contribute to
distinct behaviours associated with certain compartments. Bone
morphogenetic protein (BMP) receptor signalling represents one
such example, as it instructs quiescence in bulge stem cells but drives
cellular differentiation within the IFE (Plikus and Chuong, 2008;
Ahmed et al., 2011). The extent to which the mechanisms that govern
tissue compartmentalisation are reliant on differential proliferative
behaviours or the differential expression of signalling and/or adhesion
molecules is likely to be resolved by the combination of long-term fatemapping studies and genetic knockout models. Understanding these
mechanisms will provide important insights into how homeostasis
is maintained.
Pilosebaceous stem cells and tissue regeneration
In addition to their important role in homeostasis, stem cells actively
engage in tissue repair following injury. The regenerative process is
initiated instantly following injury and can be divided into three
partly overlapping phases. Initially, injury causes activation of the
immune system and initiates the first inflammatory phase.
Following the early stimulation of the complement system and
bleeding, activated neutrophils, macrophages and lymphocytes
enter the wound area to remove pathogens and cellular debris. As
the adaptive immune response peaks, keratinocytes enter the
proliferative phase of the re-epithelialisation process to regenerate
the barrier. Lastly, the regenerated zone, including the underlying
DEVELOPMENT
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Development (2014) 141, 2559-2567 doi:10.1242/dev.104588
dermis, undergoes significant remodelling in order to reinstate
tissue contraction and limit scarring. The entire regenerative process
is driven by numerous paracrine and autocrine signalling loops
between inflammatory cells, fibroblasts and epidermal keratinocytes
(reviewed by Gurtner et al., 2008).
In order for stem cell progeny from the PSU to participate in the
regenerative response within the IFE, the existing tissue
compartmentalisation boundaries need to be broken. Once this has
occurred, cells from within the PSU can contribute to both the acute
re-epithelialisation and long-term maintenance of the wounded area.
The extent of tissue injury, and thus the experimental method used,
will very likely influence the regenerative response and the
contribution from the PSU. The most commonly employed
experimental model for skin injury is full-thickness wounding. This
procedure involves taking a biopsy that excises the full thickness of
the skin, thereby exposing the underlying muscle facia. Whereas
superficial wounds are readily healed by relatively simple processes,
full-thickness wounds provide a means to study numerous aspects of
regeneration, including stem cell activation. The size of biopsies taken
also influences the regenerative response: regenerated epithelium
from smaller wounds (<7 mm in diameter) is devoid of hair follicles,
whereas larger wounds (>1 cm in diameter) result in de novo follicle
formation (Ito et al., 2007).
Tissue regeneration: breaking down the boundaries
Fig. 3. Mobilisation of stem cell progeny from the PSU following
wounding. Upon full-thickness wounding of intact skin (A), stem cell progeny
from multiple PSUs along the wound edge migrate toward the centre of the
wound area (white arrows, B) to participate in the regenerative response within
the interfollicular epidermis. Two lineage-marked populations represented by
blue cells and violet cells are depicted. Re-epithelialisation of the wound area
occurs with a larger fraction of blue cell progeny present than violet cell
progeny, which can result from the differential timing of stem cell activation or
different labelling efficiencies, as shown in C. According to a stochastic model
(D), the long-term contribution of stem cell progeny in the wound area is
proportional to their initial representation in the wound epidermis. Since there is
no difference in the fitness between the blue and violet cells, blue cells
outnumber violet cells because of their initial numerical advantage. By
contrast, a selective model (E) assumes that intrinsic fitness differences
between stem cell progeny do exist. Here, violet cells have an advantage
compared with blue cells, so despite being under-represented they eventually
dominate the wound area.
Variability in the initial labelling of cells is dependent in part on the
experimental setting, but early studies of wound healing did not take
this into account (Ito et al., 2005). Interestingly, when normalising
fate-mapping data to the initial contribution of PSU-derived cells to
the wound epidermis, different PSU stem cell populations contribute
in a comparable manner to long-term tissue maintenance (Page et al.,
2013). This suggests that cellular identity within the epidermis is very
plastic and that the long-term contribution of PSU-derived stem cells
to the maintenance of the IFE is not determined by the cellular
heritage but appears instead to be a stochastic event (Fig. 3).
It remains to be shown whether PSU-derived cells in the IFE are
indistinguishable from resident IFE cells or if they have intrinsic
properties otherwise associated with their origin in the PSU.
Up until now, it has been argued that bulge stem cells are
multipotent based on their ability to be mobilised and enter the IFE
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DEVELOPMENT
In the case of full-thickness wounds, the regenerative process is
elicited immediately following injury and, within 24 h, the progeny
of stem cells in the JZ are detected in the IFE. The response from
bulge hair follicle stem cells is significantly delayed, suggesting that
this population might have a different role in the repair process
(Page et al., 2013). The fact that PSU-derived cells are detected
within the IFE shortly after injury suggests that normal boundaries
between the PSU and the IFE do not prohibit cell migration and
mixing between compartments following injury (Nowak et al.,
2008; Levy et al., 2007). In contrast to full-thickness wounding,
cells from within the PSU are not required for tissue repair following
incisional wounds in the tail epithelium (Langton et al., 2008), and
lineage-tracing data show that IFE-derived progeny in general are a
major source for regeneration in response to a small biopsy on the
tail (Mascre et al., 2012). It is therefore clear that the severity of
the initial trauma greatly influences the regenerative process and the
cellular response.
Mobilisation of stem cells from the PSU allows them to take part
in the regenerative response (Nowak et al., 2008; Levy et al., 2007).
Not only the timing of stem cell activation but also the subsequent
long-term contribution to the re-epithelialised wound area vary
between different populations of stem cells (reviewed by Plikus
et al., 2012). For example, the majority of progeny that derive from
Krt15-expressing bulge stem cells appear to be rapidly lost
following wound healing (Ito et al., 2005), whereas progeny from
stem cell populations expressing Sox9, Gli1 and Lrig1 located in the
bulge, isthmus and JZ contribute to long-term regeneration (Nowak
et al., 2008; Brownell et al., 2011; Page et al., 2013). One of two
possible models might therefore describe the regenerative response;
a selective model, in which specific stem cell progeny are retained
within the regenerated area at the expense of others; or a stochastic
model, whereby the fraction of retained stem cell progeny within the
regenerated tissue is proportional to their initial contribution
(Fig. 3). Although lineage tracing is indispensable for analysing
the relative contributions of different cell types during regeneration,
one must consider that the end result will inevitably reflect the
fraction of labelled cells mobilised to enter the wounded area.
upon injury or induction of inflammation (Taylor et al., 2000; Braun
et al., 2003; Tumbar et al., 2004). The reverse experiment, in which
IFE cells replace damaged PSU compartments, has been difficult to
perform due to the inability to efficiently eliminate parts of the PSU
(Ito et al., 2005). Elegant lineage-tracing studies using laser ablation
of bulge stem cells have recently demonstrated that cells from the
upper PSU or the IFE can migrate towards the lower PSU and
replace bulge stem cells (Rompolas et al., 2013). Combined with
data from skin reconstitution experiments, this illustrates an
extraordinary plasticity among epidermal stem cells (Jensen et al.,
2009). A model is now emerging whereby tissue-specific stem cells,
irrespective of their ancestry, have the potential to contribute to
essentially all compartments when provided with an appropriate
microenvironment.
Stem cell activation
Epithelial regeneration is driven by multiple signalling pathways,
both paracrine and autocrine, that mediate cell-cell interactions
within the wounded area and guide regeneration through the distinct
phases (Gurtner et al., 2008). Studies of loss- and gain-of-function
mouse models for different signalling networks illustrate the
enormous redundancy in the transforming growth factor (TGF) β
superfamily, the epidermal growth factor (EGF) and fibroblast
growth factor (FGF) receptor family, and in the interleukin and
interferon pathways, as only moderate effects are observed when
these pathways are modulated during the regenerative process
(reviewed by Muller et al., 2012). The hepatocyte growth factor
(HGF) pathway, which consists of one ligand and one receptor
(HGF and c-Met, respectively), is required during wound repair, and
HGF is upregulated very early following trauma (Chmielowiec
et al., 2007). As c-Met is highly expressed by various stem cell
populations, this might suggest a role in stem cell activation
(Chmielowiec et al., 2007). The precise signalling dynamics of the
other pathways remains largely unresolved, and it is therefore
difficult to assess whether they are involved in stem cell activation
and mobilisation or in the subsequent burst of proliferation, or in a
combination of these events.
Stem cell activation and mobilisation during normal hair cycling
may provide insight into the stem cell response during wounding
and regeneration. Hair cycling is initiated by the activation of
subsets of hair follicle stem cells by a combination of Wnt, TGFβ
and FGF stimulation (Gat et al., 1998; Greco et al., 2009; Oshimori
and Fuchs, 2012). Although Wnt promotes hair follicle growth, it is
not required for the normal regenerative process (Ito et al., 2005).
Members of the TGFβ superfamily have widespread effects on all
PSU stem cell populations (Plikus and Chuong, 2008; Oshimori and
Fuchs, 2012; Lin and Yang, 2013), and there is evidence to suggest
that the inhibitory effects of BMP signalling on wound repair
involve misregulation of epidermal stem cells (Lewis et al., 2014).
Signalling via basic FGFs is important for PSU maintenance;
however, the pronounced inflammatory effects that ensue following
loss of the FGF receptors prohibits any meaningful conclusions as to
their specific effects on stem cell mobilisation during regeneration
(Meyer et al., 2012; Yang et al., 2010). Analysis of signalling
networks that instruct stem cells outside the hair follicle bulge will
provide additional insight into the mechanisms of tissue repair and
tissue compartmentalisation.
Oncogenic perturbation of stem cell behaviour
Mechanistic insight into stem cell regulation can be gained from
targeting specific cancer-associated gain- or loss-of-function
mutations to individual stem cell populations. Members of the Ras
2564
Development (2014) 141, 2559-2567 doi:10.1242/dev.104588
family of small GTPases are activated downstream of most growth
factor receptor pathways and are found to be mutated in a number
of different cancers (Schubbert et al., 2007). The expression of
constitutively active Ras via its endogenous promoter in cells of
interest in vivo therefore provides a proxy for constitutively active
growth factor receptor activation (Tuveson et al., 2004). Expression of
a constitutively active form of Kras in bulge stem cells and the Lrig1positive stem cell compartment causes increased proliferation
specifically within the targeted compartments (White et al., 2011;
Page et al., 2013). Studies have also shown that, in certain cases, mice
can develop spontaneous papillomas following expression of
constitutively active Kras in bulge stem cells. Fate mapping in this
case suggests that progeny can traverse the normal boundaries
between the PSU compartments and contribute to the sebaceous gland
and the infundibulum (Lapouge et al., 2011). Upon mobilisation into
the IFE, Lrig1-expressing cells also have the capacity to form
papillomas, but only following injury (Page et al., 2013).
Basal cell carcinoma (BCC; see Box 1), which is the most common
type of cancer, arises from mutations in the hedgehog signalling
pathway (reviewed by Kasper et al., 2012). BCC formation can be
efficiently modelled in vivo upon loss of patched (Ptch) or expression
of a constitutively activated form of smoothened (SmoM2) (Kasper
et al., 2012). Modelling BCC induction reveals that certain stem cell
compartments are competent whereas other are refractory to tumour
formation within their normal niches. Historically, it was believed that
BCCs arise from the lower PSU owing to the morphological
resemblance of the tumour cells to hair follicles and the expression
of hair follicle markers such as Krt17 and Krt19 in tumour
tissues (Markey et al., 1992). However, targeting tumour-associated
mutations to bulge stem cells only causes local tissue hyperplasia,
whereas cells within the IFE readily form invasive carcinomas
(Youssef et al., 2010). Dynamic studies illustrate that SmoM2
promotes the reprogramming of IFE cells to a hair follicle fate
similar to that observed during development (Youssef et al., 2012).
Interestingly, however, it appears that it is not the intrinsic properties of
the cells themselves that protects them against tumour formation but
Box 1. Basal cell carcinoma
The most frequently occurring cancer in Caucasians is basal cell
carcinoma (BCC), which will affect three out of ten people. It is a locally
invasive disease that does not metastasise; however, it is still considered
malignant as it causes significant destruction of the surrounding tissue
and extensive disfigurement. BCC formation is associated with
uncontrolled hedgehog (Hh) signalling, and the identification of
disease-associated mutations has helped delineate the pathway. Hh
signalling is mediated via ligand binding (Sonic, Indian and Desert
hedgehog) to the receptor Ptch. This allows Smo to translocate to the
primary cilium, where it releases Gli transcription factors from the
inhibitor Sufu (Epstein, 2008; Kasper et al., 2012).
Excessive Hh signalling via overexpressed ligands, loss of Ptch,
expression of mutant Smo or overexpression of Gli transcription factors
in mouse models recapitulates the human disease (reviewed by Kasper
et al., 2012). The aetiology of BCC has recently been vigorously
debated. Historically, the cell of origin for BCC has been classified as hair
follicle stem cells owing to the histological similarity of tumours and hair
follicles. Recent lineage tracing does however demonstrate that cells
within the IFE are most competent at BCC formation (Youssef et al.,
2010). Timecourse resolution of the early events following tumour
induction demonstrates that BCC development recapitulates early PSU
morphogenesis in a strictly Wnt-dependent manner (Youssef et al.,
2012). BCCs therefore provide an excellent in vivo example of cellular
reprogramming based on the perturbation of a single pathway.
DEVELOPMENT
REVIEW
REVIEW
rather the surrounding microenvironment, since mobilisation of bulge
stem cells carrying BCC-promoting mutations into the IFE is sufficient
for rapid tumour induction (Kasper et al., 2011; Wong and Reiter,
2011). Moreover, the observed compartmentalisation is capable of
withstanding the pressure of increased proliferation associated with the
expression of oncogenes (Youssef et al., 2010; White et al., 2011; Page
et al., 2013). This demonstrates that oncogene responses are highly
context dependent and suggests that tissue compartmentalisation could
represent a mechanism for tumour suppression.
A recent report illustrated that tissue compartmentalisation
boundaries within the PSU can be compromised upon perturbation
of the Notch pathway (Veniaminova et al., 2013). Notch signalling is
governed by the expression of receptors and cognate ligands on
neighbouring cells, which forms the basis for lateral signal inhibition,
whereby groups of cells organise themselves within a tissue.
Epidermal Notch signalling is important for cellular identity and
boundary maintenance within the PSU (Pan et al., 2004; Vauclair
et al., 2005; Blanpain et al., 2006; Estrach et al., 2006). Notch also
represents an important tumour suppressor in the epidermis (Nicolas
et al., 2003; Agrawal et al., 2011). In the absence of Notch signalling
there is significant inflammatory infiltrate, which could partly explain
its tumour suppressor function (Demehri et al., 2012). Inhibition of
Notch signalling via expression of dominant-negative GFP-tagged
mastermind-like 1 allows stem cell progeny within the upper PSU to
migrate into the IFE: an event that is usually prohibited during normal
homeostasis (Veniaminova et al., 2013). In light of these observations
and the apparent role of inflammation in the mobilisation of stem cell
progeny, it is pertinent that future work considers the role of stem
cell mobilisation in tissue aging, as evidence points to an important
role of inflammation in the loss of stem cell capacity in vivo (Doles
et al., 2012).
Development (2014) 141, 2559-2567 doi:10.1242/dev.104588
compartments within the epidermis. This provides unprecedented
control of cellular turnover in the distinct compartments and might
even serve as a means to guard against carcinogenesis.
The epidermis is our first defence against the environment, and as
such must be exceedingly versatile in its ability to respond to change.
The boundaries that are so tightly maintained during homeostasis are
rapidly lost upon injury, suggesting that the behavioural patterns
observed during normal homeostasis are endowed by the local
microenvironment rather than by intrinsic cellular properties. It is still
too early to provide a conclusive answer as to which mechanisms
sustain normal compartmentalisation. Nevertheless, indications from
cancer models do suggest that individual compartments maintain
distinct microenvironments that control the ability to respond to
particular oncogenic signalling networks.
A better understanding of how the microenvironment controls
stem cell dynamics and tissue compartmentalisation will be
important for identifying the specific factors that instruct cell
identity and behaviour. Within the skin, interactions between the
dermis and epidermis are integral for homeostasis and for the correct
expression of stem cell markers within the PSU (Brownell et al.,
2011; Liao and Nguyen, 2014); however, the complexity of this
interaction is only starting to be unravelled. Recent evidence reveals
that the dermis is highly complex and contains multiple distinct
lineages (Driskell et al., 2013). Future investigations will shed light
on the reciprocal relationship between populations of cells in the
epidermis and dermis that control stem cell identity and behaviour,
and how tissue compartmentalisation relates to health and disease.
Acknowledgements
We thank members of the K.B.J. laboratory, Cedric Blanpain, Ben Simons and Phil
Jones for stimulating discussions.
Competing interests
Stem cell hierarchy models adapted from the hematopoietic system
have been utilised to explain tissue maintenance in most tissues.
However, such models are not necessarily applicable in a spatially
restricted environment such as the epithelia. Indeed, the observed stem
cell heterogeneity and plasticity, as well as tissue compartmentalisation,
are not exclusive to the epidermis. The compartmentalisation model
was originally proposed within the epithelium of the mammary gland,
where the luminal and basal cell compartments are maintained as
independent entities (van Keymeulen et al., 2011). This model was then
subsequently shown to apply to the epithelium of the prostate as well
(Choi et al., 2012; Ousset et al., 2012). Although it remains to be shown
whether additional tissues are maintained by similar mechanisms, it is
tempting to speculate that parts of the stomach, where multiple
populations of stem cells have been identified, might be maintained via
tissue compartmentalisation (Mills and Shivdasani, 2011; Stange et al.,
2013). In the epidermis, the observed compartmentalisation provides a
functional explanation of the observed stem cell heterogeneity and the
need for cellular replacement (Page et al., 2013), and thus it is
conceivable that other tissues might show the same requirements.
Conclusions
Over the years, a number of models have been proposed to explain
epidermal maintenance. These range from the existence of a single
common multipotent stem cell population to no apparent stem cells at
all. However, recent evidence provides a more comprehensive view
that encompasses different functional compartments of the epidermis.
Here, multiple discrete stem cell populations with restricted lineage
potential under homeostatic conditions serve to maintain specific
The authors declare no competing financial interests.
Funding
Work in the authors’ laboratories is supported by the Lundbeck Foundation,
Wellcome Trust, Medical Research Council UK, Danish Cancer Society, Danish
Medical Council, Association for International Cancer Research, Manufacturer
Vilhelm Pedersen and Wife Memorial Legacy (this support was granted on
recommendation from the Novo Nordisk Foundation) and the EMBO Young
Investigator Programme.
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