SERIES ON STEM CELL BIOLOGY
PERSPECTIVE
Tissue-specific designs of stem cell hierarchies
Jane E. Visvader and Hans Clevers
Recent work in the field of stem cell biology suggests that there is no single design for an adult tissue stem cell hierarchy, and that
different tissues employ distinct strategies to meet their self-renewal and repair requirements. Stem cells may be multipotent or
unipotent, and can exist in quiescent or actively dividing states. ‘Professional’ stem cells may also co-exist with facultative stem
cells, which are more specialized daughter cells that revert to a stem cell state under specific tissue damage conditions. Here, we
discuss stem cell strategies as seen in three solid mammalian tissues: the intestine, mammary gland and skeletal muscle.
For decades, the haematopoietic system has
been the poster child of the adult stem cell
field, and has laid the foundation for our cur-
rent understanding of self-renewal hierarchies.
The haematopoietic system represents one of
the few mammalian tissues in which stem cells
drive a process of continuous production of
relatively short-lived, differentiated cells under
physiological conditions. Other examples
include the epidermis and its appendages, the
intestinal tract epithelia, and the testis. Most
tissues, such as liver, pancreas or muscle, dis-
play relatively little or no proliferative activity
in the steady state, but can activate their stem
cells following tissue damage.
Classic cell separation and transplantation
assays have led to the definition of long-term
haematopoietic stem cells, and clonogenic
assays have described the potency of indi-
vidual cells1,2. Together, these approaches have
led to the current view of the haematopoietic
stem cell system as a hardwired hierarchy.
Rare quiescent (dormant) stem cells occupy
the apex of this hierarchy, and asymmetric
division of these cells recreates new stem cells
(‘self-renewal’) that give rise to more actively
dividing progenitor cells, which can be either
Jane E. Visvader is in the Stem Cells and Cancer
Division, The Walter and Eliza Hall Institute
of Medical Research, Parkville, Victoria 3052,
Australia, and the Department of Medical Biology,
The University of Melbourne, Parkville, Victoria
3010, Australia. Hans Clevers is at the Hubrecht
Institute-KNAW (Royal Netherlands Academy of Arts
and Sciences), 3584 CT Utrecht, The Netherlands,
and the University Medical Center Utrecht, Cancer
Genomics Netherlands, 3584 CG Utrecht, The
Netherlands.
e-mail: visvader@wehi.edu.au; h.clevers@hubrecht.eu
Published online: 21 March 2016
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 
long- or short-lived. As cells flow through this
system unidirectionally, their differentiation
repertoire becomes progressively more lim-
ited through a series of ordered, irreversible
fate decisions to eventually generate the full
complement of blood cell types. It has been
tacitly assumed that all adult stem cell hierar-
chies adhere to this basic design, yet evidence
indicates that multiple additional designs exist.
Although stem cells are generally multipo-
tent, this feature is not mandatory. If a tissue
essentially consists of only a single cell type,
its stem cells are by definition unipotent.
Examples are the epidermis, in which basal
cells generate only keratinocytes; muscle, in
which satellite cells function as unipotent
stem cells; and the testis, with spermatocytes
as the only cellular output. By contrast, in the
bilayered epithelium that constitutes the mam-
mary ductal tree and prostate, unipotent stem/
progenitor cells contribute to maintenance of
the two layers separately 3–5.
Quiescence is a common feature of diverse
tissue stem cells, including hematopoietic and
neural stem cells. The satellite cell of striated
muscle is probably the most illustrative exam-
ple of a quiescent resident tissue stem cell,
waiting to be called into action. However, qui-
escence is not a requisite feature of stem cells,
as exemplified by the constantly dividing stem
cells of the intestinal crypt and the squamous
oesophageal epithelium6,7. Of note, intestinal
crypts also harbour a non-dividing ‘reserve’
stem cell type, called the +4 cell (discussed
in further detail in the next section). Another
example is the hair follicle bulge, which com-
prises non-dividing stem cells in the resting
phase8. Thus, quiescent and dividing cells with
© 2016 Macmillan Publishers Limited. All rights reserved
stem cell potential might coexist within the
same hierarchy. Yet, this setting does not imply
that one of these cells occupies a position that is
‘higher up’ in the hierarchy relative to the other.
In some tissues, stem cells may simply transit
between quiescent and proliferative states.
From the haematopoietic system, it has been
deduced that the flow of cells through stem
cell hierarchies is unidirectional such that all
arrows in the hierarchy point away from the
stem cells. Accumulating evidence in solid
tissues (particularly following damage) indi-
cates that fate decisions within hierarchies are
reversible and that even fully differentiated
cells can dedifferentiate and re-acquire stem
cell properties, thus acting as facultative stem
cells. Examples are secretory progenitor cells in
the crypt and various differentiated cell types in
stomach, lung and kidney (reviewed in ref. 9).
In this Perspective, we discuss stem cells in
selected adult tissues that exhibit markedly dif-
ferent strategies: the intestine, mammary gland
and skeletal muscle. These tissues highlight the
diversity and complexity that can evolve within
distinct stem cell compartments.
Heterogeneity within tissue-specific stem
cell compartments
Here we describe features of stem cells resident
within the intestine, mammary gland and skel-
etal muscle, heterogeneity within their stem cell
compartments, and models of the emerging
cellular hierarchies.
The small intestinal crypt–villus unit. The
most rapidly self-renewing tissue in mammals is
probably the intestinal epithelium, which is char-
acterized by continuous proliferation of stem
1
PERSPECTIVE SERIES ON STEM CELL BIOLOGY

a Enteroendocrine cell Paneth cells, were proposed to serve as stem

Villus Enterocyte Migration
Goblet cell
Crypt Secretory
progenitor
+4 LRC
TA cell
Paneth cell
CBC
b Alveolar luminal cell
Alveolar bud
Luminal stem cell
Ductal luminal cell
Stem cell Myoepithelial cell
c Satellite cell Basal lamina
Plasmalemma
Myonuclei Myofibre
Figure 1 Schematic structures of the small intestine, mammary epithelial tree and skeletal muscle, and
the location of their stem cells. (a) The epithelial surface of the small intestine is folded into villi and
crypts. The crypt base columnar (CBC) stem cells, located at the base of the crypt, are interdigitated
with Paneth cells, an important component of the niche. Intestinal stem cells give rise to transit-
amplifying (TA) cells (progenitor cells) that migrate upwards and yield differentiated cells (enterocytes,
goblet cells and enteroendocrine cells). LRC, label-retaining cell. (b) The mammary epithelial tree
is a bilayered structure that comprises primary, secondary and tertiary branches, emanating from a
common point attached to the nipple. Alveolar buds arise during oestrus cycling. The basal layer of
the epithelium harbours bipotent stem cells, but the stem cell niche is yet to be defined. At least 30%
of luminal cells in the adult gland correspond to luminal progenitor cells that are relatively long-lived.
(c) In uninjured muscle, the satellite cell (muscle stem cell) resides between the plasmalemma of the
muscle fibre and the basal lamina (basement membrane). A mature muscle fibre contains hundreds to
thousands of differentiated nuclei. In response to injury, satellite cells proliferate, differentiate to form
multinucleated cells, and then fuse to regenerate myofibres. A small subset of satellite cells self-renew
and return to quiescence after repair.
cells at the base of the crypt. Dividing daugh- (goblet cells, Paneth cells or enteroendocrine
ter cells differentiate into either the abundant cells) (Fig.  1). Originally, slender crypt base
absorptive enterocytes or into secretory cells columnar (CBC) cells, wedged between the
cells10. This concept was incorporated into the
subsequent stem cell zone model of Bjerknes
and Cheng11. Much later, the G-protein-coupled
receptor Lgr5 was identified as a specific marker
of CBCs, and these cells were confirmed by
lineage tracing as bona fide actively dividing,
yet long-lived, crypt stem cells that are able to
generate all differentiated lineages of the small
intestinal epithelium12.
In an intestinal crypt, a clone generated from
one of the fifteen stem cells replaces all other
stem cells over time, as evidenced by the propa-
gation of spontaneous somatic mutations13,14.
Quantitative/multi-colour lineage tracing has
revealed how clonal dynamics progresses. The
15 equipotent Lgr5+ stem cells each divide
symmetrically every 24  hours to yield Lgr5+
daughter cells that stochastically compete for
crypt niche space. At the single cell level, all
are equal, yet those at the base of the crypt are
‘more equal’ than the others, as their position
gives them the highest chance of being retained
as stem cells15–18.
Multiple studies have described an alterna-
tive crypt stem cell, able to retain DNA label
and located just above the Lgr5+ stem cell niche,
at the +4 position. Lineage tracing based on
Bmi1, mTert, Lrig1 or Hopx promoter-driven
expression19–23 implied a role in intestinal
homeostasis for this quiescent cell. The rela-
tionship of +4 cells with Lgr5+ stem cells has
been the subject of intense debate.
Using a diphtheria toxin receptor (DTR)
knock-in mouse model to selectively deplete
Lgr5+ cells, it was noted that their loss is tol-
erated, at least in the short term24. Cells that
express Bmi1 expand following ablation of
Lgr5+ cells to compensate for the loss of the
actively cycling stem cell pool. Lgr5+ cells
rapidly reappear in  vivo, implying that the
Bmi1+ cell population converts into actively
cycling Lgr5+ stem cells24. Removal of Lgr5+
cells by radiation similarly showed that Bmi1-
expressing cells can replace lost Lgr5+ stem
cells25. Lineage tracing using the Hopx model
further revealed that a quiescent stem cell
pool was mobilized following irradiation of
the small intestine23. Re-evaluation of the four
+4 cell markers revealed broad expression of
their mRNAs in the crypt, typically with the
highest levels in Lgr5+ stem cells26–29. These
findings suggest that lineage tracing may have
originated from double-positive Lgr5/Hopx or
Lgr5/Bmi1-expressing stem cells. On the basis
of the same Lgr5 DTR allele, the importance

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SERIES ON STEM CELL BIOLOGY
of Lgr5-expressing progeny of CBCs was
documented during regeneration following
radiation-induced damage30. Depletion of
Lgr5+ stem cells strongly affects the regenera-
tive response, demonstrating that the reserve
stem cell population cannot cope with the
increased demand for stem cell activity under
these conditions.
The intestine provides a striking example
of cellular adaptability that has evolved within
the differentiation hierarchy under conditions
of stress or damage. Secretory precursor cells
located just above the stem cell zone express
the Notch ligand Dll131,32. These Dll1+ cells
produce small, short-lived clones that con-
tain various combinations of Paneth, goblet,
enteroendocrine and tuft cells. However, when
the stem cell pool is ablated by irradiation, the
same Dll1+ cells regenerate entire crypt–villus
units, including enterocytes, suggesting that
secretory progenitors are able to dedifferenti-
ate to multipotent Lgr5+ stem cells following
damage in vivo33.
Several recent studies have demonstrated the
existence of non-dividing Lgr5+ cells. These
rare cells differ from cycling Lgr5+ stem cells
and reside above the Paneth cells around the +4
position. On the basis of gene expression stud-
ies, these cells seem to represent early secretory
daughter cells that directly derive from Lgr5+
stem cells and have retained Lgr5 mRNA,
co-expressed with the canonical +4 mark-
ers26,28,34,35. Could these cells be the +4 reserve
stem cells? Winton and colleagues developed
a method for the specific genetic labelling of
quiescent (DNA/chromatin-label-retaining)
cells. Their elegant approach allows the study
of the identity and function of these rarely
dividing cells during homeostasis and follow-
ing damage36. Two quiescent populations have
been identified36,37: the long-lived Paneth cells,
located at the crypt base where they form an
important constituent of the niche; and a more
heterogeneous population predominantly
localized around the +4 position. A subset of
the heterogeneous population co-expressed
Lgr5 and +4 cell markers, whereas most cells
expressed Paneth cell and enteroendocrine cell
markers, suggesting that this population con-
sists of secretory progenitor cells. Remarkably,
expression levels of proposed +4 markers such
as Bmi1, mTert, Lrig1 and Hopx were similar
in the label-retaining cells and Lgr5+ stem cells.
When lineage tracing was induced in these
label-retaining cells, they were found to be rela-
tively short-lived and did not contribute to the
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 
stem cell pool during homeostasis. Induction
of cytotoxic damage with doxorubicin, how-
ever, prompted quiescent cells to give rise to
proliferative, multipotent stem cells36.
Based on these collective studies, the fol-
lowing picture emerges (Fig.  2): actively
cycling Lgr5+ stem cells are responsible for the
daily generation of all cell lineages. They do so
during the entire lifetime of large mammals,
undergoing thousands of divisions. Among
their immediate progeny are non-dividing
secretory precursors, located just above the
stem cells around the +4 position. Under
homeostatic conditions, these non-dividing
cells exist transiently owing to their terminal
differentiation. Yet, these precursors can read-
ily revert back to a Lgr5+ stem cell phenotype
following stem cell loss — so a ‘professional’,
non-committed quiescent stem cell pool may
not exist. As individual quiescent precursor
cells are relatively short-lived, they should
not be considered stem cells in the strictest
sense. However, new secretory progenitors
are continually generated by the cycling Lgr5+
stem cells, and a pool of these cells is therefore
always available to be called into action fol-
lowing tissue damage. Each secretory progeni-
tor can therefore be considered a ‘facultative
stem cell’.
The mammary epithelium. In the steady state,
the adult mammary epithelium undergoes low
to moderate turnover in response to hormo-
nally driven cycling. By contrast, the epithe-
lium is subject to enormous bursts of activity
during ductal morphogenesis in puberty and
alveolar expansion in pregnancy. Early studies
demonstrated that any segment of the mam-
mary epithelial tree could successfully engraft
following transplantation into the cleared
mammary fat pads of recipient mice38–40, thus
indicating that mammary repopulating cells
are widely distributed throughout the bilay-
ered ductal tree (Fig. 1). Flow cytometric and
transplantation studies identified discrete
mammary epithelial cell subpopulations and
provided functional evidence that mammary
stem cells (MaSCs) within the basal subset can
reconstitute a fully functional epithelial tree41,42.
The adult mammary gland seems to harbour
multiple stem cell populations, including slow-
cycling43 or putative quiescent MaSCs44,45, as
well as a molecularly distinct MaSC subset
that transiently expands during pregnancy46.
A bona fide quiescent MaSC subset, however,
has yet to be isolated.
© 2016 Macmillan Publishers Limited. All rights reserved
PERSPECTIVE
The ability of a single basal stem cell to
repopulate an entire ductal tree following
implantation41,42 has suggested that bipotent
cells reside at the apex of an epithelial hier-
archy. The identification of freshly sorted
basal cells with bipotent capacity in clonal
colony-forming assays lends further support
to the existence of bipotent stem/progenitor
cells41,47. In addition to these cells, at least two
types of committed luminal progenitor cells
(ductal and alveolar) have been prospectively
isolated48. These unipotent epithelial cells are
presumed to drive ductal morphogenesis and
alveologenesis during pregnancy. It is note-
worthy that luminal-restricted cells remain
faithful in the different assays and do not har-
bour bipotent activity in vivo41,42. Nevertheless,
it has become apparent that basal-restricted
cells can exhibit a more restricted differentia-
tion potential under homeostatic conditions
than in transplantation assays4,49.
Findings from lineage tracing studies track-
ing mammary stem/progenitor cells have
fuelled considerable debate about the existence
of bipotent MaSCs. However, it is important
to note that the two models (unipotent versus
bipotent MaSCs) are not mutually exclusive.
Both bipotent 50,51 and unipotent MaSCs have
been tracked using ‘generic’ basal-restricted
gene promoters such as keratin 5 (K5) or K14,
and smooth muscle actin4,50–52, depending on
the precise model explored. Lineage tracing of
more defined subsets marked by Procr (ref. 53)
or Lgr5 (ref.  50) also provided unequivocal
evidence for the existence of bipotent MaSCs
in adult tissue. Procr is a Wnt target in the
mammary gland, where it marks cycling stem
cells that persist across multiple pregnancies53.
Moreover, tracing of other Wnt-responsive cells
has indicated the presence of bipotent MaSCs
within the basal compartment that contribute to
alveologenesis49. The advent of high-resolution
3D imaging technology combined with a multi-
colour reporter read-out has enabled close scru-
tiny of clones from the inner and outer ductal
surfaces across extensive areas of the epithelial
tree. Using this technology, myoepithelial-
only or bi-lineage clones of the same colour
were visualized, but there was no evidence of
luminal-only clones50. Collectively, bipotent (or
multipotent) MaSCs have now been traced in
diverse models49–51,53. An alternative approach
reliant on the barcoding of cells to track their
in vivo differentiation potential demonstrated
that basal cells readily generate bi-lineage cells
within primary ductal outgrowths54.
3
PERSPECTIVE SERIES ON STEM CELL BIOLOGY

a Small intestine How do we reconcile the discordant data

generated by different lineage tracing studies
Absorptive
progenitor Enterocyte using similar gene promoters? Part of the
disparity reflects whether or not the driver/
Cycling
Goblet cell reporter line recapitulates the full spectrum of
Lgr5+ secretory endogenous gene and cre expression, the sen-
CBC progenitor sitivity of mammary epithelium to tamoxifen
Non-cycling Paneth cell
secretory treatment, and the degree of labelling achieved.
precursor cell In cases where the labelling of a heterogene-
(+4 LRC)
Enteroendocrine cell ous compartment is inefficient, the fate of the
majority of cells within that population comes
b Mammary gland into question. Technicalities aside, key issues
Ductal progenitor
that remain unanswered are the frequency of
bipotent MaSCs in the adult gland and the rela-
tive contribution of bipotent versus unipotent
Ductal luminal cell MaSCs to morphogenesis and homeostasis.
Based on the small Procr+ population53, the
Lum-SC/progenitor presence of bipotent clones in the adenoviral
Alveolar progenitor K14-driven model51 and an estimated sub-
MaSC Aveolar cell
set of 5% MaSCs in the basal compartment
Myo-SC/progenitor (by single cell transplants)41, bipotent MaSCs
Myoepithelial cell seem to constitute 5–10% of basal cells in the
adult mouse. Accruing data are consistent
c Skeletal muscle
with a model in which bipotent MaSCs help
coordinate ductal morphogenesis through
the generation of unipotent stem/progenitor
Satellite cell (Pax7+) Myoblast Myocyte Myotube
cells, which serve as the ‘workhorses’ for epi-
thelial expansion (Fig. 2). In parallel, bipotent
Figure 2 Schematic models of stem cell hierarchies in the small intestine, mammary gland and
MaSCs may initiate expansion in pregnancy
skeletal muscle. (a) The cycling Lgr5+ CBC cell resides at the top of the hierarchy and yields absorptive (given their increased numbers)46, but unipo-
and secretory progenitors. An intermediate secretory precursor cell that is non-cycling and located tent alveolar progenitor cells necessarily drive
around the +4 position lies downstream of the CBC and generates cycling secretory progenitor alveologenesis. Nonetheless, bipotent MaSCs
cells, which produce goblet, Paneth and enteroendocrine cells. Absorptive progenitors give rise to
enterocytes. (b) In the mammary gland, the stem cell compartment is heterogeneous but has yet to seem to orchestrate the remodelling of the
be properly delineated. Bipotent mammary stem cells (MaSCs) give rise to unipotent stem cells or mammary gland during involution and con-
long-lived progenitors for the basal/myoepithelial and luminal lineages (labelled as lum- or myo-SC/ tribute to ductal homeostasis during ageing 50.
progenitor). Multipotent is equally applicable to bipotent MaSCs, as two distinct luminal subtypes
It is not clear whether these stem cells compete
exist. The precise number of stem and progenitor cells has yet to be determined. Distinct luminal
progenitor cells restricted to a ductal or alveolar fate reside in the virgin mammary gland; these are to colonize the ductal domains through neutral
relatively long-lived progenitors and may derive from a common luminal stem cell that lies upstream, drift dynamics as in the intestine18, but very few
although such a cell may not exist. (c) Small, unipotent muscle stem cells (quiescent satellite cells) MaSCs are involved in repopulating individual
produce myoblasts that differentiate into myocytes. These, in turn, generate fully differentiated,
ducts. The precise niche of these cells remains
multinucleated myotubes during muscle regeneration. The progression of satellite cells through the
myogenic program is regulated by a cascade of myogenic regulatory transcription factors. Only stem obscure, but hormone-receptor-positive lumi-
cells are capable of self-renewal, as indicated by rounded arrows. nal cells pose reasonable candidates.

Unipotent stem or long-lived progenitor
cells reside in both the luminal4,50,51,55,56 and
basal epithelial compartments4,49,52,57. In some
cases, these populations have been shown to
remain stable over entire reproductive cycles,
suggesting that ‘unipotent stem cell’ is a more
apt term. Although these cells play a key role
in independently sustaining each lineage, the
emerging picture is quite complex. Even for the
luminal lineage, the different reporter models
are capturing seemingly distinct cellular sub-
sets with different developmental potential.
For example, Elf5 marks luminal-restricted
progenitor cells that dramatically expand in
puberty, but are largely replaced over a 20-week
chase50, whereas Notch-1, Notch-3, K8 and
K18 target longer lived cells, but these differ
markedly in their labelling kinetics and their
output of oestrogen receptor-positive (ER+)
versus ER– progeny 4,51,55,56. The mammary cell
fate switches apparent at different points of the
morphogenetic cycle represent an additional
layer of complexity 49. To resolve these issues, a
more stochastic approach that is independent
of cell-specific promoters is required to map
the fate of cells in vivo.
The skeletal muscle. Muscle satellite cells are
a largely dormant population, reflecting the
low turnover of this tissue in the absence of
injury (Fig. 1). The first definitive demonstra-
tion that satellite cells possess stem cell activity
derived from engraftment of a single myofibre
with its satellite cells into radiation-ablated
muscles58. Further studies showed that the
satellite cell pool comprises a small subset of
unipotent muscle stem cells (MuSCs), which
demonstrate long-term self-renewal ability
in serial engraftment assays at the single cell
level and over multiple generations58–60. Under

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SERIES ON STEM CELL BIOLOGY
homeostatic conditions, MuSCs are mitotically
quiescent and contribute minimally to myofi-
bre maintenance (reviewed elsewhere61,62).
However, when muscle tissue is exposed to
growth stimuli or damage, these cells rapidly
re-enter the cell cycle and either undergo
asymmetric divisions to initiate myogenesis
or symmetric cell divisions to reconstitute
the stem cell pool. In myogenesis, MuSCs
give rise to committed satellite cells, so-called
myogenic progenitors, that differentiate into
myocytes and form new fibres through fusion
with each other or to damaged fibres (Fig. 2).
Interestingly, multiple muscle fibre types exist
that differ in size and contractile properties.
In addition, their intrinsic transcriptional
networks vary in different anatomical loca-
tions62. The proportion of satellite cells can
differ according to muscle type, with apparent
heterogeneity among satellite cells residing in
slow versus fast muscles62. Notably, although
MuSCs are unipotent within skeletal muscle,
they have the capacity to generate brown adi-
pocytes during embryogenesis or when chal-
lenged with cold exposure63–65.
Pax7 is a defining marker of satellite
cells61,62, and conditional ablation of Pax7+
cells revealed that these cells are essential
for early postnatal growth. Satellite cell pre-
cursors first emerge in late embryogenesis
and their numbers decline during postna-
tal growth, implying that only a subset of
embryonic Pax7+ cells form the self-renewing
MuSC pool in the adult 66. In adult muscle,
Pax7+ cells are essential for tissue regenera-
tion in response to acute injury 67–69 and for
replenishment of the MuSC pool, but are
dispensable for general homeostasis of mus-
cle fibres67 and increase in muscle mass70. As
Pax7+ satellite cells are exclusively recruited
during tissue repair, other types of cells prob-
ably do not contribute substantially to the
regenerated muscle fibres71. Notably, Pax7 is
not a specific marker of MuSCs, as activated
satellite cells also express this gene. Curiously,
Pax7+ MuSCs asymmetrically segregate their
template DNA strand during muscle regen-
eration59, although this mechanism does not
seem to hold for other tissue stem cells.
Despite the relative simplicity of the mus-
cle differentiation hierarchy, the satellite cell
compartment is functionally heterogeneous
based on gene expression signatures, self-
renewal capacity, myogenic commitment and
cell cycle status59,61,62,72,73. Pax7high satellite cells
exhibit slower cell cycle entry kinetics and have
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 
PERSPECTIVE
Self-renewal
Cell cycle
entry
Quiescent stem cell Transit stem cell Active stem cell
Stem cells Progenitors Mature cells
Figure 3 A general model depicting the hierarchical organization of tissue-specific stem and progenitor
cells. Although common principles apply to stem cell compartments in different tissues, there is
no single design. In the configuration depicted here, a multipotent or bipotent stem cell exists in
a quiescent state and can be activated to enter the cell cycle by tissue damage or a physiological
stimulus. The discovery of intermediate stem cell populations (for example, in blood and striated
muscle) implies that a continuum of stem cell states might exist to confer a higher degree of
flexibility. The activated stem cell self-renews or returns to a quiescent state when tissue repair or the
physiological process has been completed. The balance between stem cell quiescence versus self-
renewal and differentiation is central to homeostasis and averting cell transformation. Progenitors (such
as those in the small intestine) can be co-opted under stress or injury to dedifferentiate into stem-like
cells to facilitate tissue repair.
a lower metabolic state than Pax7low cells59. Intriguingly, satellite cells can spend time out
Myf5 also distinguishes different populations of their niche while moving between fibres dur-
of satellite cells, with subsets of committed ing postnatal growth or injury.
satellite cells (Myf5+) and sublaminar MuSCs
(Myf5–). As anticipated, Myf5– satellite cells Transitioning between quiescence and
possess higher self-renewal and regenerative activation
capacity than Myf5+ cells in transplantation Many tissue stem cells exist in a reversible
assays72. Part of the observed heterogeneity state of quiescence or dormancy (G0), which
might also reflect transitioning between dif- presumably evolved to protect the genomic
ferent states. Indeed, satellite cells transit from integrity of long-lived cells (Fig. 3). Quiescent
the G0 (resting) cell cycle phase into a poised cells are activated in response to injury or a
state (Galert) that enables rapid activation of physiological stimulus and then return to
deeply quiescent stem cells in response to quiescence once homeostasis has been re-
injury 73. These cells are primed for cell cycle established. MuSCs and HSCs are among
entry, and exhibit a dramatic increase in cell the best-characterized quiescent cells, distin-
size, mitochondrial metabolism and mTORC1 guished largely by their low RNA content and
signalling. HSCs primed by muscle injury in label-retaining ability 77. Quiescent stem cells
the contralateral muscle also show changes typically exist in a low metabolic and cycling
indicative of a poised state with increased state, with downregulation of the molecular
functional potential73. machinery required for cell cycle progression,
Spatially, satellite cells are positioned DNA replication and mitochondrial func-
between the plasma membrane and basal lam- tion77. Moreover, dormant cells demonstrate
ina of the muscle fibre (Fig. 1). Interestingly, higher repopulating potential than cycling
the muscle fibre is a major component of the stem cells in transplantation assays, suggesting
satellite stem cell niche: following contact with that intrinsic mechanisms link the quiescent
a freshly isolated single muscle fibre, satellite state to regenerative potential78. Finally, loss
cells can return to their quiescent state74,75. This of quiescence can lead to impaired organ or
process is regulated in part through Notch sig- tissue function, with the haematopoietic sys-
nalling, which coordinates homing of MuSCs tem providing a poignant example. Persistent
to their niche and adhesion to the basement activation of HSCs from their dormant state in
membrane and underlying muscle fibre76. response to physiological stress results in stem
Conversely, removal of the plasmalemma cell exhaustion, accumulation of DNA damage
drives quiescent satellite cells into cycle74. and functional decline79.
5
© 2016 Macmillan Publishers Limited. All rights reserved
PERSPECTIVE
Conclusions
It is becoming increasingly apparent that
different stem cell types with tissue renewal
capacity can reside within the same tissue.
Both transplantation and lineage tracing
assays have proved to be indispensable in dis-
secting stem cell compartments. The small
intestine comprises multipotent stem cells
that are actively cycling to replenish all cells
of the crypt–villus unit. Adding an apparent
layer of complexity, both bipotent and uni-
potent epithelial stem cells seem to reside in
the mammary epithelial tree. However, their
cycling status and precise roles during ontog-
eny remain to be clarified. By contrast, skeletal
muscle harbours deeply quiescent, unipotent
stem cells that are poised for activation fol-
lowing injury — yet even these cells exhibit
functional heterogeneity. Although the ‘qui-
escent’ precursor cell that lies downstream
of the intestinal stem cell possesses remark-
able adaptability following tissue damage,
the degree of flexibility within the mammary
epithelial and muscle hierarchies under physi-
ological conditions remains to be elucidated.
Functional diversity within a stem cell com-
partment is presumably established through
the complex interplay between intrinsic and
extrinsic signals. The integration of single cell
RNA-seq data with functional assays should
help decipher heterogeneity in the stem cell
compartment at the molecular level and iden-
tify signalling networks specific to cellular
subtypes. As these pathways unfold and more
refined tools become available to investigate
stem cell populations, it should be feasible
to devise improved methods for long-term
expansion of stem cells outside their niche for
regenerative therapy.
ACKNOWLEDGEMENTS
We thank P. Maltezos (WEHI, Melbourne, Australia)
for assistance with figure preparation. J.E.V. is
supported by a NHMRC Australia Fellowship, and
thanks the NHMRC IRIISS, Australian Cancer
Research Foundation and National Breast Cancer
Foundation.
ADDITIONAL INFORMATION
Reprints and permissions information is available
online at www.nature.com/reprints. Correspondence
should be addressed to J.E.V. or H.C.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
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