Stems Cells and the Pathways
to Aging and Cancer
Derrick J. Rossi,1,* Catriona H.M. Jamieson,2and Irving L. Weissman3
1Immune Disease Institute, Harvard Stem Cell Institute, and the Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
2Moores Cancer Center, Division of Hematology/Oncology and Department of Medicine, University of California, San Diego, CA 92093, USA
3Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Cancer Center, and Department of Pathology,
Stanford University School of Medicine, Stanford, CA 94305-5323, USA
The aging of tissue-specific stem cell and progenitor cell compartments is believed to be central to
the decline of tissue and organ integrity and function in the elderly. Here, we examineevidence link-
ing stem cell dysfunction to the pathophysiological conditions accompanying aging, focusing on
the mechanisms underlying stem cell decline and their contribution to disease pathogenesis.
Most tissues and organs contain minor populations of primitive
stem cells and progenitor cells. These cells are integral first in
the developing fetus for the generation of tissues and organs
and later in the adult for ongoing tissue maintenance and regen-
eration after injury. Differences in developmental potential and
lineal relationships between stem and progenitor subsets estab-
lish a hierarchical structure for primitive stem cell compart-
ments. Stem cells reside at the apex of the hierarchy and give
rise to multipotent progenitors, which in turn give rise to progen-
itors with more restricted lineage potential. Although best exem-
plified in the hematopoietic system (see the Review by S.H.
Orkin and L.I. Zon, page 631 of this issue), the existence of hier-
archical relationships between stem and progenitor cells is
emerging as a common feature of other tissue-specific stem
cell compartments as well (Bryder et al., 2006). All stem cells
are capable both of self-renewing to give rise to daughter
stem cells and of differentiating into a variety of mature cell
types. These are the defining properties of stem cell biology
thatensure thattissues can befunctionally sustained throughout
the lifetime of the organism, while avoiding the onset of hypopla-
sia and atrophy.
Although certainly one of the most recognizable characteris-
tics of human biology, aging remains one of the least under-
stood. This is largely attributable to the fact that aging is both
gradual and inherently complex, with almost all aspects of phys-
iology and phenotype undergoing steady modification with ad-
vancingage. The complexity of the aging process does not allow
ing diverse systems, methodologies, and model organisms have
begun to build a consensus regarding the central physiological
characteristics of aging. Indeed, such studies have shown that
the process of aging is invariably accompanied by a diminished
capacity to adequately maintain tissue homeostasis or to repair
tissues after injury. When homeostatic control diminishes to the
point at which tissue/organ integrity and function are no longer
sufficiently maintained, physiologic decline ensues, and aging
is manifested. Consistent with this, many of the pathophysiolog-
ical conditions afflicting the elderly, such as anemia, sarcopenia
(loss of muscle mass), and osteoporosis, suggest an imbalance
between cell loss and renewal. The fact that homeostatic main-
tenance and regenerative potential of tissues wane with age has
implicated stem cell decline as a central player in the aging pro-
cess. However, the degree to which aging is attributable to stem
cell dysfunction or instead reflects a more systemic degenera-
tion of tissues and organs will likely differ substantially between
different tissues and their resident stem cells. Nonetheless,
mounting evidence points to stem cells as an important contrib-
uting factor to at least some of the pathophysiological attributes
of aging in a number of different tissues. It therefore seems likely
that a central focus of future research on mammalian aging will
involve a careful evaluation of the contribution and mechanisms
of stem cell dysfunction on a tissue-by-tissue basis.
That advancing age is accompanied by an increased inci-
dence of cancer is incontrovertible. As cancers arise only after
the acquisition of multiple mutagenic events, long-lived cells
are the only cells capable of serving as such reservoirs. Stem
cells represent ideal cellular targets for the accumulation of pre-
cancerous damage as the central properties of stem cells—self-
renewal and differentiation—enable mutations acquired in the
stem cell compartment to be propagated to both self-renewing
progeny and downstream progenitors over the lifetime of the or-
ganism. The danger posed by mutagenic accumulation in stem
cells is held in check by the activity of tumor suppressor proteins
that censor potentially malignant clones by eliciting apoptosis
or permanent growth arrest. However, as the homeostatic de-
mands of tissues require ongoing stem and progenitor cell activ-
ity, tumor suppressor pathways may inadvertently lead to stem
cell attrition and in such a manner contribute to aging.
In this Review we focus on several aspects of stem cell
biology, aging, and disease pathogenesis, beginning with an
overview of the changing roles that stem cells play during ontog-
eny. We then discuss studies indicating that stem cell aging con-
emphasis on mechanisms known to drive cellular aging, and
examine how such mechanisms impact stem cell biology. We
Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc. 681
conclude with a discussion of the connection between the
increased incidence of cancer with advanced age and how, at
least in the hematopoietic system, the clonal progression to can-
cer operates through the stem cell compartment.
Stem Cell Ontogeny: Age Matters
The question of when physiological aging begins in the context
of stem cell biology is germane to this discussion and requires
a consideration of stem cell ontogeny. Stem cells are vital to all
stages oflife, yetthespecific rolesthatstemcellsplayduringdif-
ferent stages of ontogeny change considerably. During early
embryogenesis, pluripotent stem cells differentiate to give rise
to the three germ layers that establish the body plan (see Review
by C.E. Murry and G. Keller, page 661 of this issue). As develop-
ment proceeds, distinct subsets of stem cells emerge to orches-
trate the construction of tissues and organs—processes that are
often incomplete at birth and carry over into postnatal life, as is
the case with mammalian reproductive tissues. Once tissues
sue maintenance, which persists throughout adult life. A funda-
mental assumption that underlies stem cell models of aging is
that at some point during development, all tissue-specific stem
cells derive from pre-existing stem cells by self-renewal, and
not by de novo generation from more primitive progenitors (Fig-
ure 1). In this view, stem cells in both young and old adults alike
are ultimately derived only from stem cells of fetal origin through
their ability to sustain long-term self-renewal, and thus the ‘‘ag-
finitive specification of that lineage and subsequent to any addi-
tional input from more primitive progenitors.
Coincident with the changing roles that stem cells play during
distinct stages of ontogeny, their basic properties change too.
This is probably best exemplified in the hematopoietic system
where hematopoietic stem cells (HSCs) of fetal origin differ
from those of the adult by a number of criteria such as surface
phenotype (Morrison et al., 1995; Osawa et al., 1996; Yoder
et al., 1997), cell-cycle status (Bowie et al., 2006), and the ana-
tomical location where stem cell activity resides (see Review
by S.H. Orkin and L.I. Zon). Different ontological stages are
also marked by characteristic changes in HSC developmental
potential. For example, fetal murine HSCs have the capacity to
give rise to distinct repertoires of T cell (Ikuta et al., 1990) and
B cell (Kantor and Herzenberg, 1993) subsets, which is lost in
adulthood. More gradual transitions are also at play with fetal
HSCs having a greater capacity to give rise to lymphoid lineage
cells than stem cells from young adult mice (Morrison et al.,
1995), which in turn possess greater lymphoid lineage potential
than do HSCs from old mice (Kim et al., 2003; Rossi et al.,
2005; Sudo et al., 2000). The timing of these developmental
changes suggests the existence of molecular switches that are
tightly regulated during ontogeny. Evidence of this is found
upon the examination of the global gene expression profiles of
stem cells from fetal, young, and old mice, which indicate
profound differences in the transcriptome that are reflective of
changing developmental potentials at different stages of ontog-
eny (Ivanova et al., 2002; Phillips et al., 2000; Rossi et al., 2005).
Underlying mechanisms likely include the utilization and depen-
dence on different transcriptional networks, which play funda-
mental roles in distinguishing embryonic, fetal, and adult stem
cells. For example, whereas the transcription factor Oct4 is
essential for the establishment of embryonic stem cells (Nichols
et al., 1998), it is entirely dispensable for self-renewal and main-
tenance of adult stem cells (Lengner et al., 2007). Similarly, the
transcription factor Sox17 is required for the maintenance and
function of fetal murine HSCs but not for adult HSCs (Kim
et al., 2007). In contrast, other transcription factors such as
Gfi-1 (Hock et al., 2004a; Zeng et al., 2004) and Etv6 (Hock
et al., 2004b) are more important for self-renewal and mainte-
nance of adult stem cells but are less important during fetal
The regulatory mechanisms underlying the transition of stem
cells into old age are less well defined. Nonetheless, important
insights have been gained from global gene expression studies
of stem cells purified from young and old mice. Such studies
have implicated the involvement of higher-order chromatin
dynamics and epigenetic regulation in stem cell aging, as sug-
gested by coordinated age regulation of lineage specification
genes (Rossi et al., 2005), chromosomal regions (Chambers
stem cell aging. For example, overexpression of the Polycomb
group (PcG) protein Ezh2 can extend the replicative capacity
of mouse HSCs during serial transplantation (Kamminga et al.,
2006). Another PcG protein, Bmi-1, is required for maintaining
Figure 1. Models of Stem Cell Aging
Stem cell compartments established during fetal development are maintained
throughout adult life by self-renewal and not by de novo generation from more
primitiveprogenitors. Inonemodel ofstemcell aging (top), functionally distinct
clones emerge within the stem cell pool due to differences in the state of their
genome and epigenome (such clones may or may not already exist within the
fetal stem cell pool). During aging, certain clones expand and come to pre-
dominate the pool, whereas other clones fail to thrive. The success or failure
of different clones can be related to differences in genetic and epigenetic
makeup and clonal fitness to respond and flourish within the aging microenvi-
ronment. In a second model (bottom), all stem cells in the stem cell pool are
imbued with more or less similar functional capacity, which gradually and co-
ordinately changes over time. Changes in functional potential of stem cells in
both models may be cell autonomous or derive from instructive cues from the
stem cell niche or aging systemic environment. It should be noted, however,
that these models are not mutually exclusive, and both may act in concert or
at different stages of ontogeny.
682 Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc.
self-renewal of adult HSCs (Iwama et al., 2004; Park et al., 2003),
neural stem cells (NSCs) (Molofsky et al., 2003), and leukemic
stem cells (LSCs) (Lessard and Sauvageau, 2003). Bmi-1 has
also been implicated in aging through its capacity to repress
the p16Ink4a-p19Arflocus (Jacobs et al., 1999), which encodes
two tumor suppressors: p16INK4A, an inhibitor of Cdk4/6, and
p19/ARF that regulates p53 stability through inactivation of the
ubiquitin ligase Mdm2. By repressing the p16Ink4a-p19Arflocus,
Bmi-1 averts growth arrest or apoptosis of stem cells (Molofsky
et al., 2005; Oguro et al., 2006). Intriguingly, Bmi-1-mediated
suppression of the p16Ink4a-p19Arflocus is dependent upon the
through which reduced expression of Ezh2 in aged HSCs (Rossi
et al., 2005) may lead to induction of p16Ink4a(Janzen et al.,
2006).Together thesestudiesdemonstrate thatthemechanisms
regulating stem cell activity change during ontogeny and sug-
gest that the utilization of different transcriptional networks
driven by epigenetic regulation of chromatin accessibility is cen-
tral to these changes.
Hematopoietic Stem Cell Aging
Advancing age is accompanied by a number of pathophysiolog-
ical changes in the hematopoietic system, whose etiology sug-
itor cell involvement. The most clinically significant of these
changesare the decreased competence ofthe adaptive immune
system (Linton and Dorshkind, 2004), the increased incidence of
myeloid diseases including leukemias (Lichtman and Rowe,
2004), and the onset of anemia in the elderly (Beghe et al.,
2004).Theability ofhuman HSCsto giverise to primitiveprogen-
itors declines substantially during the ontological transitioning
from fetal liver, to cord blood, to adult bone marrow (Lansdorp
et al., 1993), suggesting a progressive loss of stem cell activity
withage. Consistent withthis,arecent meta-analysis conducted
using the National Marrow Donor Program (NMDP) registry as-
sessed multiple donor traits (including donor age, cytomegalovi-
rus serologic status, ABO blood group compatibility, race, and
sex) on various parameters of bone marrow transplant success
in 6978 transplant recipients (Kollman et al., 2001). This study
revealed that advanced donor age was significantly associated
with overall decreased disease-free survival. These studies sug-
gest that the proliferative and regenerative capacity of human
HSCs diminishes with age, and that diminished stem cell activity
is largely cell intrinsic. These conclusions have been corrobo-
rated and expanded upon in studies of mice.
Models of aging often presuppose that loss of homeostatic
control and regenerative potential of tissues is driven by diminu-
tion of stem cell reserves, yet extensive evidence indicates that
HSC numbers increase substantially with advancing age in com-
mon strains of laboratory mice (de Haan et al., 1997; Morrison
et al., 1996; Rossi et al., 2005; Sudo et al., 2000). The expansion
aged donors exhibit a greater capacity, than young controls, to
self-renewto giveriseto phenocopies ofthemselves upontrans-
plantation in young recipients (Pearce et al., 2006; Rossi et al.,
2005). Studies have shown that murine HSCs have the ability
to reconstitute successive recipients through serial transplanta-
tion, indicating that the replicative capacity of HSCs far exceeds
HSCs from old mice were assayed for bioactivity, numerous
functional deficiencies came to light, including altered homing
and mobilization properties (Liang et al., 2005; Xing et al.,
2006; see Review by D.J. Laird et al., page 612 of this issue), di-
minished competitive repopulating ability (Kim et al., 2003; Mor-
rison et al., 1996; Rossi et al., 2005; Sudo et al., 2000), and
a skewing of lineage potential from lymphopoiesis toward mye-
2005; Sudo et al., 2000). The latter property correlates with the
reduced frequency of lymphoid progenitors in old mice (Miller
and Allman, 2003; Rossi et al., 2005), whereas myeloid progen-
itors are maintained or even expanded in old mice (Rossi et al.,
2005). Importantly, the differential capacity of HSCs from old
mice to give rise to lymphoid and myeloid progenitors was found
to be a transplantable, cell-autonomous property of HSC aging
underwritten by the suppression of numerous genes involved
in specifying lymphoid fate and the upregulation of genes in-
volved in myeloid specification (Rossi et al., 2005). Cumulatively,
these studies suggest that two clinically important pathophysio-
logical features of the aged hematopoietic system, namely,
immune system decline and the development of myelogenous
disease, may have their origins in age-dependent functional and
molecular changes intrinsically arising in stem cells (Figure 2).
A growing body of evidence suggests that the stem cell pool
may be comprised of a number of clonal lineages possessing
heritable differences in functional capacity (reviewed in Muller-
Sieburg and Sieburg, 2006). As aging proceeds, certain clones
may out-compete others while still remaining under the homeo-
static regulatory mechanisms imposed by the systemic environ-
ment or the stem cell niche microenvironment (see Review by
S.J. Morrison and A.C. Spradling, page 598of this issue). For ex-
ample, the myeloid lineage bias of aged HSCs may result from
clonal expansion of stem cell clones with a myeloid bias during
eage potential in the aged hematopoietic system implies a
certain fitness of myeloid-biased clones to thrive under the
environment (Figure 1). In an alternative model, the developmen-
tal potential of most or all of the cells in the stem cell population
could gradually and coordinately change over time perhaps in
a cell-autonomous fashion or in response to instructive cues
from the aging environment. In this latter model, stem cell aging
may be viewed as an ongoing developmental process with fetal
development and old age as the start and end points, respec-
tively (Figure 1). Within this framework it can be argued that
changes in lineage potential characteristic of HSC aging may
simply reflect the developmental demands of distinct stages of
ment of immunological memory in the adaptive immune system,
the myeloidbiasof aged stemcellsmay reflect theneed tomain-
tion in the elderly.
Itisalsoworthnotingthatalthoughpediatric leukemias tendto
be lymphoid in origin, the leukemias that manifest in old age
often originate in myeloid compartments, suggesting that the
malignant capacity of hematopoietic progenitors changes in
Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc. 683
a manner mirroring the change in lineage potential of HSCs dur-
ing aging (Kim et al., 2003; Liang et al., 2005; Rossi et al., 2005;
Sudo et al., 2000). This raises the possibility that differences in
disease spectra arising at different points during ontogeny may
be influenced by, or directly result from, changes in the develop-
mental potential of stem cells at a given stage. In support of this,
a recent study demonstrated that ectopic expression of the
BCR-ABL oncogene (which causes chronic myeloid leukemia
rent development of a myeloproliferative disease (MPD) and B
cell leukemia, whereas expression of the BCR-ABL oncogene
in marrow cells of old mice gave rise to MPD with little or no lym-
phoid involvement (Signer et al., 2007). Moreover, the observa-
tion that stem cell aging is coupled to elevated expression of
numerous genes, such as Aml1, Pml, and Eto, involved in the
pathogenesis of myeloid leukemia (Rossi et al., 2005) suggests
the possibility that the upregulation of such proto-oncogenes
may act synergistically with the myeloid bias of aged stem cells
to predispose to myelogenous disease and leukemia (Figure 2).
Whether or not the upregulation of such proto-oncogenes in
stem cells with age increases their susceptibility to the types of
cytogenetic rearrangements and translocations that promote
the development of leukemia remains to be determined.
Aging in Other Stem Cell Compartments
Until recently, functional evaluation of stem cells from nonhema-
topoietic tissues was limited by a paucity of appropriate assays
and by the purity of the stem cells in question. Recent technical
advances in assay systems, along with the prospective isolation
of a wide variety of somatically derived stem cells, are now
permitting the types of functional evaluation that have long
suggests that diverse tissue-specific stem cell reserves decline
with advancing age, and that such a decline bears important
pathophysiological consequences for aging of tissues and or-
gans. For example, graying of hair, one of the most recognizable
aspects of human aging, appears to result from an age-depen-
dent loss of melanocyte stem cells (MSCs) from the subcutane-
ous hair follicle bulge region (Nishimura et al., 2005) and may be
exacerbated by telomere dysfunction as suggested by the pre-
mature graying of telomerase-deficient mice (Rudolph et al.,
upon telomere shortening but do not appear to be diminished
(Flores et al., 2005). Conversely, overexpression of the catalytic
component of telomerase drives quiescent epidermal stem cells
into cycle in a manner that is independent of telomere length
(Flores et al., 2005; Sarin et al., 2005). These studies indicate
that age-dependent loss of function of two distinct stem cell
compartments in the hair follicle may be under the control of
both canonical and noncanonical telomerase pathways.
Studies of the murine gastrointestinal tract have shown that
cells from old mice at or near the position of the stem cells within
the crypts of Lieberkuhn are more susceptible to apoptosis un-
der stress (Martin et al., 1998a) and exhibit reduced regenerative
potential despite an age-dependent increase in the number of
clonogenic crypt cells (Martin et al., 1998b). Although signaling
pathways that control self-renewal, such as the Wnt pathway,
are deregulated in the majority of colon cancers, the mecha-
nisms promoting normal crypt stem cell self-renewal have
been less well defined. Until recently, there was a relative dearth
of information regarding the phenotype and function of normal
intestinal stem cells. Recently, small intestine and colon stem
cells were identified based on expression of a Wnt target gene,
Lgr5 (leucine-rich-repeat-containing G protein-coupled receptor
5) (Barker et al., 2007), that was isolated from a panel of Wnt
target genes because of its crypt-restricted expression. This
discovery provides the impetus for investigating the molecular
mechanisms driving normal intestinal stem cell aging and the
sequential events required for the development of colon cancer.
In the central nervous system, the generation of new neurons
continues throughout life in at least two regions of the brain, the
subventricular zone (SVZ) of the lateral ventricles and the sub-
granular zone (SGZ) of the dentate gyrus of the hippocampus
(Eriksson et al., 1998; Palmer et al., 1997; see Review by C.
Zhao et al., page 645 of this issue). Ongoing neurogenesis is me-
diated by the activity of NSCs and is believed to be important for
such as memory and learning. The decline in both sensory and
cognitive functions in the elderly thus implicates age-associated
Figure 2. Properties of Aging HSCs
A model of hematopoietic stem cell (HSC) aging
under steady-state and stress conditions is pre-
sented with the properties of aged stem cells pro-
iologies. In the steady state, the functional and
molecular properties of aged HSCs result in
askewing of lineage potential away from lymphoid
toward myeloid fates, which we propose may un-
derlie the immuno-decline and prevalence of mye-
eral lines of evidence suggest that these changes
may be mediated by epigenetic regulatory mech-
anisms. Under conditions of stress and regenera-
tion, stem cells exhibit a number of functional def-
icits that are believed to contribute to the
diminished regenerative capacity of the aged he-
matopoietic system, and which evidence sug-
gests may result from age-dependent accrual of
684 Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc.
NSC decline as an important contributory factor. In support of
this, NSC numbers and proliferative potential are reduced in
the SVZ with age (Maslov et al., 2004; Molofsky et al., 2006), cor-
relating with the diminished neurogenesis observed in the olfac-
tory bulb of old mice (Molofsky et al., 2006). Similarly, the dimin-
ished proliferative potential of granule cell progenitors in the
dentate gyrus of aged rats may contribute to the decline in hip-
pocampal neurogenesis that occurs with aging (Kuhn et al.,
ity (Drapeau et al., 2003). The diminishing function of NSCs with
age has been linked with increased genomic instability (Bailey
et al., 2004) and induction of p16Ink4aexpression (Molofsky
et al., 2006). As in the hematopoietic system, the spectrum of tu-
mor types in the brain changes with age, with neuroblastomas
andmedulloblastomas predominating inpediatric cases, andtu-
mors of glial origin tending to predominate in aged individuals.
Given that the cellular origin of brain tumors may arise directly
from the progeny of CD133+stem cells (Uchida et al., 2000), it
will be important to determine if changes in the developmental
potential of NSCs during ontogeny underlie the predisposition
to the changing spectrum of brain tumors presented during dif-
ferent stages of life (Figure 1).
Extrinsic Regulation of Stem Cell Aging
Stem cells from a variety of tissues reside in close proximity to
specialized support cells that extrinsically regulate stem cell
self-renewal, differentiation, and, as emerging evidence indi-
cates, aging. For instance, in the fruit fly Drosophila, aging of
both male and female germline stem cells (GSCs) is regulated,
at least in part, by a niche-dependent decline resulting from
either diminished signaling via bone morphogenetic proteins
(BMPs) in the ovary (Pan et al., 2007) or diminished signaling
by a ligand called Unpaired in the testis (Boyle et al., 2007; see
Review by S.J. Morrison and A.C. Spradling). In both cases,
reduced cadherin-mediated adhesion of GSCs to the niche
and weakening of direct contacts between stem cells and niche
cells with age correlated with the decline in GSC numbers and
function. The systemic milieu has also been shown to regulate
stem cell decline during aging as demonstrated by experiments
in which exposure of muscle satellite cells of old mice to a young
systemic environment through heterochronic parabiosis and the
establishment of a surgically conjoined circulatory system was
from old mice, in part, through the restoration of Delta-Notch
signaling (Conboy et al.,2005). The impaired ability of aged mus-
cle satellite cells to adequately respond to injury has been con-
nected to declining numbers of these cells (Shefer et al., 2006)
and the tendency of satellite cells to convert from their normal
myogenic fate to a fibrotic fate with age due to elevated Wnt sig-
naling originating from the serum of old mice (Brack et al., 2007).
Activated Wnt signaling also appears to suppress stem/progen-
itor cell numbers in Klotho-deficient mice (Liu et al., 2007), which
develop degenerative phenotypes linked to impaired vitamin-D
homeostasis. Studies aimed at elucidating the function of Wnt
signaling in HSC biology underscore the cell type and context-
specific effects of Wnts in development. Differences in model
systems have resulted in some studies indicating that Wnt
signaling, through the canonical pathway, enhances stem cell
self-renewal capacity (Reya et al., 2003; Willert et al., 2003),
whereas others suggest alternative effects (Cobas et al., 2004).
It has, however, become apparent that too much Wnt signaling
through constitutive activation of b-catenin can severely blunt
etteret al.,2006;Scheller etal.,2006). Soalthough Wntsignaling
may be beneficial to stem cells in certain physiological settings,
imbalances in Wnt, the activation of noncanonical pathways by
Wnt, or ontological differences in the way that Wnt signals are
received may suppress stem cell activity in other settings such
Although much of the functional decline that accompanies
HSC aging is cell intrinsic, the influence of the stem cell niche
on HSC biology isnotin question, andthusitwould besurprising
if HSC aging were not extrinsically modulated to some degree.
Along these lines it is noteworthy that caloric restriction (CR),
the only intervention known to extend longevity across species,
significantly improved HSC function in aged mice, even after
transplantation into non-CR recipients (Chen et al., 2003). CR
has also been reported to suppress myeloid leukemic develop-
ment in an irradiation-induced mouse model of leukemia, coinci-
dent with a decline in the numbers of primitive hematopoietic
stem/progenitor cells, the presumed targets of disease develop-
ment in this model (Yoshida et al., 2006). These provocative find-
ings suggest that stem cell numbers, aging, and diseases of
stem cell origin may be modulated by pathways believed to be
involved in mediating the antiaging effects of CR across species,
such as the insulin-like growth factor (Igf1) pathway (Kenyon,
2005) or the Sir2 family of deacetylases (Chen and Guarente,
DNA Damage and Its Contribution to Stem Cell Aging
One mechanism believed to be central to the aging of cells and
tissues is cumulative damage to cellular macromolecules such
as proteins, lipids, RNA, and DNA. Of these, none has been
more intimately linked to aging than DNA damage (reviewed in
Lombard et al., 2005). This is likely due to the fact that in contrast
to other cellular polymers such as proteins and RNA, DNA is
neither appreciably turned over nor recycled. Furthermore, the
sheer magnitude of genomic insult that cells must endure is
believed to be staggering, with some estimates suggesting
drolysis alone (Lindahl, 1993). At thecellular level,genomic dam-
age is imparted by a variety of sources including reactive oxygen
species (ROS), the natural by-products of oxidative metabolism,
which are considered a major source of damage contributing
to aging. It is clear, however, that in addition to ROS, a much
broader range of extrinsic and intrinsic sources, such as UV irra-
diation, alkylating agents, telomere attrition, and DNA replication
the biological impact of such damage, cells have evolved a num-
ber of pathways that recognize, respond to, and repair different
types of lesions (reviewed in Hoeijmakers, 2001). Due to the
imperfect nature of these repair systems, however, a certain
degree of DNA damage evades repair and accumulates with
age (Hamilton et al., 2001), although the rates at which damage
accrues in different cell types appear to be tissue specific (Dolle
Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc. 685
et al., 1997). Compelling genetic evidence that genomic damage
contributes to the pathophysiology of aging has emerged from
the study of human segmental progeria syndromes (often re-
ferred to as premature aging disorders) such as Werner syn-
drome, Cockayne syndrome, Trichothiodystrophy, Bloom syn-
drome, and Ataxia telangiectasia. These diseases have all
been found to have deficits in pathways that mediate DNA-dam-
age responses or DNA-damage repair (reviewed in Martin,
2005). This concept has been strengthened in studies of mouse
strains harboring DNA-damage repair, response, and mainte-
nance deficiencies, which exhibit a spectrum of degenerative
phenotypes reminiscent of ‘‘accelerated’’ aging (Lombard et al.,
2005). It should be noted, however, that there is no clear consen-
sus as to whether human progeria syndromes, or the murine
strains that model them, accurately reflect aging at an acceler-
atedpaceorsimply exhibitpathologiesthat share certainpheno-
types characteristic of aged individuals.
Stem cells are subject to a similar array of insults as somatic
like somatic cells, however, lesions arising in the stem cell com-
partment—except those resulting in growth arrest, or cell
death—can be propagated both to daughter stem cells and to
downstream lineages through the processes of self-renewal
and differentiation. In such a manner, the impact of damage
accrued in individual stem cells can be potentiated with possible
ramifications for all levels of the developmental hierarchy. Al-
though certain types of genomic lesions can modulate cellular
behavior directly, the consequence of DNA damage on the phys-
response. Depending upon the nature and extent of the damage,
cytostatic, cytotoxic, or mutagenic lesions arising in stem cells
have the potential to drive cells to senescence, apoptosis, or
transformation, respectively (Figure 3). If diminution of the func-
tional stem cell reserves surpasses levels of self-renewal and
mature cell production, then diminished homeostatic control
and reduced regenerative potential—the physiological hallmarks
of aging—would be predicted to ensue. Alternatively, if unre-
paired or imprecisely repaired DNA damage proves sufficiently
cur. From the point of view of clonal selection, stem cells persist-
cess even further if lesions imparting a growth or survival advan-
tage are acquired. However, as in other cell types, potent tumor
suppressor pathways are active in stem cells to ensure that cells
harboring potentially dangerous lesions are removed from the
stem cell pool. In the context of stem cells and progenitor cells
that must proliferate throughout life,the action of tumor suppres-
sor pathways also provides an avenue through which stem cell
activity may be prematurely truncated during aging (Figure 4).
Figure 3. DNA Damage in the Stem Cell
Heritable DNA damage accrued in stem cells
elicits different responses depending upon the na-
ture and extent of the lesions. Examples of each
type of lesion are shown, although the cellular re-
sponse and outcomes are not mutually exclusive.
Mutagenic lesions in stem cells can lead to trans-
formation of stem cells or their progeny upon ac-
quisition of a full repertoire of oncogenic lesions.
Cytostatic and cytotoxic lesions lead to stem cell
senescence or apoptosis, respectively, which
over time could lead to diminution of the stem
cell pool. Other types of damage can lead to de-
regulation of the mechanisms governing stem
cell biology and to dysfunction. Cumulatively, cy-
tostatic, cytotoxic, and deregulatory lesions can
lead to diminished homeostatic control and re-
duced regenerative potential, the physiological
hallmarks of aging.
Figure 4. Stem Cell Aging and Tumor Suppressor Pathways
Cellular and genomic damage resulting from endogenous and exogenous
sources activate tumor suppressor pathways including those mediated by
the tumor suppressor proteins p53 and Rb to ensure that potentially danger-
ous lesions do not lead to malignancy. As aging advances and damage ac-
crues, the activity of such tumor suppressors increases and in so doing has
the potential to negatively modulate stem cell function through the induction
of apoptosis or senescence.
686 Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc.
Evidence that DNA damage contributes significantly to stem
cell decline has principally been generated in the context of the
hematopoietic system using mice bearing mutations in media-
tors of diverse DNA-repair pathways. For example, reduced
plantation experiments using mice with mutations in FancD1/
Brca2 (Navarro et al., 2006), Msh2 (Reese et al., 2003), and
Lig4 (Nijnik et al., 2007). That DNA damage impacts stem cell
function during aging was demonstrated in a series of experi-
ments evaluating HSC reserves and functional capacity in young
and old mice deficient in several different DNA-repair pathways
(Rossi et al., 2007a, 2007c). Surprisingly, whereas HSC reserves
were preserved during aging in these strains, stem cell function
was severely attenuated when quiescent stem cells were forced
into the cell cycle under conditions of stress or regeneration. The
observation that stem cells from older mutant mice performed
poorly compared to those from young mutant mice suggested
that DNA damage may be accumulating in quiescent stem cells
during aging and contributing to the diminished ability of old
stem cells to regenerate tissues after injury (Figure 2). Consistent
with this, purified quiescent HSCs from old wild-type mice—
known to be deficient in regenerative capacity—were found to
accrue considerable DNA damage with age as shown by the
accumulation of gH2AX foci, a marker of DNA damage (Rossi
et al., 2007a). These findings help to explain why mice bearing
mutations in diverse DNA-repair/genomic maintenance path-
ways exhibit relatively normal hematopoiesis under steady-state
conditions yet readily reveal stem cell functional impairment
upon challenge (Carreau et al., 1999; Haneline et al., 1999; Nav-
arro et al., 2006; Reese et al., 2003; Rossi et al., 2007a; Samper
et al., 2002). It is unlikely that DNA damage underlies all aspects
of stem cell aging, however, as the myeloid/lymphoid lineage-
skewing characteristic of advanced aging in repair-competent
HSCs (Kim et al., 2003; Liang et al., 2005; Rossi et al., 2005;
Sudo et al., 2000) was not recapitulated during aging of DNA-
repair-deficient mouse strains in which stem cell lineage poten-
tial has been evaluated (Rossi et al., 2007a) (Figure 2).
Although it seems clear that DNA damage contributes to the
diminished capacity of stem cells to adequately respond to
stress, the contribution of damage in stem cells to the general
steady-state decline of aged tissues and organisms is less clear.
Stochastic destabilization of transcription resulting from global
DNA damage has been implicated in the age-dependent decline
of certain postmitotic somatic cells (Bahar et al., 2006), yet this
mechanism does not appear to be active in other cell types
including stem cells (Warren et al., 2007). An alternative possibil-
ity is suggested by a recent study in which the DNA-damage
response protein ATR (ataxia telangiectasia and Rad3 related)
was conditionally ablated in adult mice to generate tissues that
were mosaic for ATR deficiency (Ruzankina et al., 2007). These
mice undergo rapid massive attrition in multiple tissues with
high cell turnover yet were rescued by the emergence of stem
cells and progenitor cells that had evaded ATR ablation resulting
in restoration of tissue homeostasis to near normal levels. Within
a few months, however, the mice developed several age-related
phenotypes concomitant with homeostatic imbalances and also
showed reduced stem/progenitor cell numbers in certain com-
partments including hair follicular bulge stem cells and bone
marrow LSK (lineage?Sca1+ckit+) cells (a population containing
hematopoietic stem and progenitor cells). These results suggest
that excessive replicative stress of ATR-competent stem/pro-
genitor cells in response to a massive homeostatic catastrophe
may lead to their premature exhaustion. On the other hand, the
fact that HSCs can be serially transplanted through multiple suc-
cessive recipients and still retain functional capacity even after
prodigious activity does not support the idea that such a mecha-
nism could exhaust stem cell functional reserves in animals over
the course of one lifetime (Harrison, 1979). Moreover, given that
HSC reserves do not diminish but rather quite substantially in-
crease with advanced age in common laboratory strains of
mice (Morrison et al., 1996; Rossi et al., 2005; Sudo et al.,
2000), it seems likely that the loss of LSK cells in aged ATR
mosaic mice may bea result of lasting consequences of ATRde-
ficiency in niche cells (Ruzankina et al., 2007). Consistent with
this, telomerase deficiency has been shown to lead to a lasting
decline in the ability of the bone marrow microenvironment to
support HSC engraftment and normal lineage potential (Ju
et al., 2007). These studies highlight the importance of extrinsic
signaling pathways and the aging of the environment for stem
Stem Cell Aging and Reactive Oxygen Species
Prolonged exposure to ROS has long been postulated to con-
tribute to aging. Indeed, this is the essential axiom of the oxida-
tive stress or free radical hypothesis of aging posited by Harman
in 1956 (Harman, 1956). Some lines of evidence support Har-
man’s hypothesis, including studies demonstrating that oxida-
tive lesions accumulate with age and the finding that increased
longevity associated with caloric restriction is associated with
reduced oxidative damage (Sohal and Weindruch, 1996).
Furthermore, genetic manipulation of pathways that control cel-
lular responses to oxidative stress have been shown to extend
longevity in flies (Orr and Sohal, 1994) and in rodents (Holzen-
berger et al., 2003; Migliaccio et al., 1999). The argument behind
accumulation in the mitochondrial genome and progressive
mitochondrial dysfunction and is supported by genetic studies
using mice bearing a proofreading-deficient mitochondrial DNA
(mtDNA) polymerase, which exhibit a spectrum of degenerative
phenotypes reminiscent of aging (Kujoth et al., 2005; Trifunovic
et al., 2004). Recent studies have, however, cast doubt on this
paradigm with the suggestion that point mutation accrual in
mtDNA may not determine the rate of aging in wild-type mice
(Vermulst et al., 2007). Moreover, a recent meta-analysis of
data from 68 randomized trials showed no evidence that antiox-
idant dietary supplements had any beneficial effects on mortality
but rather demonstrated, quite startlingly, that several common
antioxidant dietary supplements actually increased the risk of
theless pointed to myriad deleterious consequences of ROS in
cells such as limiting the replicative capacity of certain cell types
and the induction of permanent growth arrest. Importantly,
a number of recent studies have provided clear evidence of
a role for ROS in the preservation of stem cell function. In an
elegant series of studies, the DNA-damage response protein
ATM (ataxia telangiectasia-mutated) was shown to be vital for
Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc. 687
regulating ROS levels and their effects on stem cells. HSCs from
ATM-deficient mice had elevated intracellular levels of ROS with
a concomitant decrease in functional capacity that could be re-
stored upon treatment with the antioxidant N-acetyl-L-cysteine
(NAC) (Ito et al., 2004). Loss of HSC activity in ATM-deficient
mice in response to ROS is dependent upon activation of p38
MAP kinase (Ito et al., 2006) and induction of p16Ink4a(Ito et al.,
2004). In another study, HSCs from mice lacking Foxo transcrip-
tion factors (Foxo1/Foxo3/Foxo4 triple mutants) showed en-
hanced short-term yet diminished long-term repopulation activ-
ity associated with increased cycling, apoptosis, and reduced
stem and progenitor cell reserves (Tothova et al., 2007). Strik-
ingly, Foxo-deficient HSCs showed elevated intracellular ROS
levels that could be reduced upon treatment with the antioxidant
NAC, which additionally restored HSC pool size, cell-cycle pro-
file, and apoptosis to normal levels. In a related study, Foxo3a
deficiency alone was sufficient to diminish HSC function, elevate
intracellular ROS, disrupt stem cell quiescence, and decrease
the size of the HSC compartment during aging (Miyamoto
et al., 2007). Cumulatively, these studies identify intracellular
management of ROS levels—by ATM, p38 MAP kinase, and
p16Ink4aas well as by Foxo transcription factors—as an impor-
and function during aging.
The maintenance of telomeres (specialized nucleoprotein struc-
tures at the ends of chromosomes) by the enzyme telomerase
represents a specialized form of genomic maintenance and is
possess the ability to maintain telomere length indefinitely either
by overexpressing telomerase or by utilizing an alternative telo-
mere lengthening pathway (ALT) that relies upon exchange of
formed cells, when telomere attrition reaches a critical point, ca-
suppressor proteins p53 or p16Ink4aare activated leading to the
elimination or permanent growth arrest of these cells. Telomere
dysfunction therefore represents a potent tumor suppressor
mechanism meant to limit the proliferative capacity of malignant
cells and to prevent uncontrolled growth. However, activation of
tumor suppressor pathways may be a potential avenue by which
stem cell activity is prematurely blunted with age (Figure 4). This
such as the blood, gut, and skin, with high turnover. Perhaps as
a mechanism to counter this, telomerase activity is primarily
restricted to primitive stem and progenitor cell compartments
(Harrington, 2004). Nonetheless, as telomere shortening still
takes place in human HSCs during aging (Vaziri et al., 1994)
and during serial transplantation of murine HSCs (Allsopp et al.,
2003a, 2003b), telomerase expression in stem cells may slow
down telomere shortening but not stop italtogether. Importantly,
expression of the catalytic component of telomerase was able to
prevent telomere shortening in HSCs during serial transplanta-
suggesting that telomere-length-independent mechanisms may
ultimately limit the replicative capacity of murine HSCs (Allsopp
et al., 2003b). Indeed, direct experimental evidence demonstrat-
ing that telomere shortening attenuates stem cell function or
contributes to organismal aging in mice or humans possessing
functional telomerase activity is limited.
There is evidence, however, that in the absence of functional
telomerase, telomere attrition both diminishes stem cell and
progenitor cell activity and reduces life span. For example, the
clinical presentation of pancytopenia and bone marrow failure
inpatients suffering fromaplasticanemia ordyskeratosis conge-
nita with mutations in the catalytic component (Yamaguchi et al.,
2002)oftelomerase suggestsclearhematopoietic stemandpro-
genitor cell involvement. Mice deficient in telomerase activity
have a reduced life span upon telomere shortening and develop
progressive pathologies in proliferative tissues such as the gut,
blood, and skin that are underwritten by diminished homeostatic
maintenance capability and reduced regenerative potential
(Blasco et al., 1997; Herrera et al., 1999; Lee et al., 1998; Ru-
dolph et al., 1999). When directly assayed, stem and progenitor
cell compartments were particularly affected in these mice with
HSCs exhibiting reduced replicative capacity in serial transplan-
tation assays (Allsopp et al., 2003a) and diminished repopulating
ability (Rossi et al., 2007a; Samper et al., 2002). The proliferative
potential of neural stem and progenitor cells in late generation
telomerase-deficient mice was also diminished both in vitro (Fer-
ron et al., 2004; Wong et al., 2003) and in vivo (Ferron et al.,
2004). The impact of telomere attrition in several stem cell com-
partments appears dependent on the Cdk inhibitor p21, whose
absence partially rescues stem/progenitor cell activity and at
the same time extends the longevity of telomerase-deficient
mice without promoting cancer (Choudhury et al., 2007). Simi-
larly, ablation of the mismatch repair gene Pms2 extends life
span and abrogates the degenerative phenotypes of telome-
rase-deficient mice in part by attenuating p21 induction (Siegl-
Cachedenier et al., 2007). Interestingly, loss of either p21 or
Pms2 appears to rescue proliferation defects but not apoptosis
in cells from telomerase-deficient mice, suggesting that these
molecules may be common to a pathway that signals cell-cycle
arrest in response to dysfunctional telomeres. Incontrast, loss of
p53acts positivelyupon thegerm cellcompartment inyoungtel-
omerase-deficient mice yet exacerbates tumorigenesis later in
life, which is likely due to the fact that p53 deficiency rescues
both the proliferative defect and apoptosis associated with telo-
mere dysfunction (Chin et al., 1999). A different outcome is ob-
served in mice lacking the DNA-damage repair protein ATM on
a telomerase-deficient background. In these animals, telomere
dysfunction is exacerbated within several stem and progenitor
cell compartments and longevity is compromised, yet there is
diminished formation of T cell lymphomas normally associated
with ATM deficiency (Wong et al., 2003). These studies highlight
the complex interplay between signals emanating from dysfunc-
tional telomeres and tumor suppressor pathways that regulate
stem cell biology, aging, and cancer.
Stem Cell Aging and Tumor Suppressor Pathways
Somatic cells respond to potentially deleterious lesions gener-
ated by exogenous and endogenous sources by activation of
the retinoblastoma (Rb) and p53 tumor suppressor pathways,
which act to permanently arrest or kill damaged cells. The role
688 Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc.
that inactivation of these pathways plays in the development of
cancer is unequivocal, yet it is becoming increasingly evident
that activation of these pathways may also contribute signifi-
cantly to aging (Figure 4). Elevated expression of the Rb effector
p16Ink4ahas been demonstrated in numerous tissues with age
(Krishnamurthy et al., 2004; Zindy et al., 1997) and has emerged,
along with senescence-associated b-galactosidase activity (SA-
b-gal) (Dimri et al., 1995), as one of the principal biomarkers
of aging. Several studies have demonstrated not only that
p16Ink4aincreases in several different stem cell compartments
with age but also that this induction has functional conse-
quences. For example, NSCs in the SVZ of the mammalian brain
diminish in number and function with age (Maslov et al., 2004;
Molofsky et al., 2006), concomitant with increasing p16Ink4aex-
pression (Molofsky et al., 2006). Importantly, these phenotypes
were partially mitigated in p16Ink4a-deficient mice, with a corre-
sponding improvement in neurogenesis in the olfactory bulb of
old mice (Molofsky et al., 2006). In a related study, p16Ink4aex-
pression was elevated in HSCs from old mice, whereas age-as-
sociated repopulating deficits and serial-transplant capacity
were improved in aged p16Ink4a-deficient mice (Janzen et al.,
2006). Taken together these studies establish a causal role for
p16Ink4ain the reduced functional capacity of aged HSCs and
NSCs. It is important to note that neither of these studies has
established that p16INK4ainduction leads to cellular senescence
in either of these stem cell compartments. In fact, evidence from
clonal culture experiments indicates that HSCs from aged mice
have a comparable capacity to give rise to progeny as HSCs
from young mice (Morrison et al., 1996; Sudo et al., 2000). More-
over, loss of Bmi-1 and derepression of the p16Ink4a-p19Arflocus
are not sufficient to impart a senescent phenotype to stem cells,
as demonstrated by experiments in which clonally cultured
cle and giving rise to daughter cells as wild-type control cells
(Iwama et al., 2004). These studies suggest that cellular senes-
cence and permanent growth arrest may not be a significant
physiological outcome of normal HSC aging. Consistent with
this, p16Ink4adeficiency in the hematopoietic system appears
to allay stress-associated apoptosis rather than cellular senes-
cence (Janzen et al., 2006). Thus the question of whether or
not stem cell aging is accompanied by increased cellular senes-
cence remains an important unresolved issue.
cell aging is more complex. Loss of p53 predisposes to a spec-
trum of neoplasms (Donehower et al., 1992), whereas mice over-
expressing a short isoform (Maier et al., 2004) or truncated acti-
vated form of p53 (Tyner et al., 2002) exhibit suppression of
tumorigenesis yet develop early degenerative phenotypes remi-
niscent of aging. Conversely, mice with increased but normally
regulated expression of p53 and its positive regulator Arf are
resistant to tumorigenesis, live longer, and have reduced levels
of age-associated damage to proteins, lipids, and DNA (Matheu
et al., 2007). Mice with increased p53 activity resulting from a
hypomorphic allele of Mdm2 are also tumor resistant yet age
can have either proaging or antiaging effects depending on con-
text. In accordance with these results, several studies have indi-
cated that modulation of p53 expression in stem cells may also
differentially impinge upon stem cell behavior and aging. For
example, in p53-deficient mice, there were increased numbers
assays and were more capable of giving rise to phenocopies of
themselves in primary transplant recipients than controls, indi-
cating an elevated self-renewal capacity (TeKippe et al., 2003).
In contrast, stem and progenitor cells in mice expressing a
liferative and repopulating capacity compared to wild-type con-
trols, whereas the same cells from p53+/?mice exhibited
increased activity (Dumble et al., 2007). Although apoptosis of
stem cells and progenitor cells was not addressed in either of
these studies, p53-mediated apoptosis is critically involved in
the physiological regulation of HSC population size and function.
For example, mice carrying a mutant form of the Rad50 DNA-re-
pair protein experience precipitous bone marrow failure due to
hypermorphic signaling through an ATM-Chk2-p53-dependent
apoptotic pathway (Bender et al., 2002; Morales et al., 2005).
Conversely, enforced expression of the antiapoptotic protein
BCL-2 in the hematopoietic compartment of mice expands
HSC numbers and improves repopulating potential (Domen
et al., 2000), in a manner similar to that seen with p53 deficiency
(TeKippe et al., 2003). Cumulatively, these studies illustrate the
tenuous balance between tumor suppression and stem cell
may be delayed without a concomitant increase in cancer inci-
The vast majority of somatic cells comprising tissues and organs
are terminally differentiated and postmitotic, yet stem cells must
retain mitotic potential throughout the lifetime of an animal in
order to be able to respond to homeostatic demands. In accor-
dance with the biological importance of such a duty, stem cells
from a diverse array of tissues seem to have evolved a number
of mechanisms aimed at maintaining genomic integrity beyond
that of somatic cells. For instance, stem cells from a variety of
tissues express high levels of ABC transporter proteins that are
known to have physiological roles in cytoprotection through their
capacity to actively pump genotoxic and xenobiotic compounds
that has been reported for certain tissue-specific stem cells is
somes to more differentiated progeny at mitosis while retaining
the original and possibly error-free chromosome within the
stem cell (reviewed in Lansdorp, 2007). This mechanism does
not, however, appear to be universal to all stem cell compart-
ments (Kiel et al., 2007), and indeed the notion that asymmetric
strand segregation is used by stem cells to limit exposure to rep-
lication errors has recently been challenged (Lansdorp, 2007).
Nonetheless, the fact that many tissue-specific stem cells pri-
marily reside in the quiescent (G0) phase of the cell cycle
throughout their adult lifetime achieves the same end by limiting
passage through the cell cycle and the possibility of attendant
DNA replication errors. Moreover, quiescent stem cells are
also relatively metabolically inactive with concomitantly low gen-
eration of endogenous free radicals and ROS (Tothova et al.,
Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc. 689
The benefits afforded by residing in a quiescent state, how-
ever, appear to come at a cost for stem cells. HSCs from old
mice accumulate considerably more damage-induced gH2AX
foci in their genomes than more actively proliferating progenitors
(Rossi et al., 2007a). DNA damage accrued in proliferating pro-
genitor cells is either repaired or the cells are eliminated by apo-
ptosis as they pass through the cell cycle, whereas quiescent
stem cells appear to only undergo apoptosis when they are
forced into the cell cycle. These results suggest that quiescence
itself might be a critical factor involved in facilitating the accrual
from the fact that the cellular machinery responsible for eliciting
so intimately linked to cell-cycle progression and cell-cycle-de-
pendent checkpoint control mechanisms (reviewed in Zhou
and Elledge, 2000). Consistent with this, cultured cells driven
to quiescence by serum starvation must re-enter the cell cycle
2000) because global genomic DNA repair was suppressed
under experimentally induced quiescence (Bielas and Heddle,
2004). Whether or not the repair properties of serum-starved
cells in culture will extend to the physiology of stem cells is
clearly an important and unresolved question. Nonetheless, the
finding that stem cells specifically accumulate DNA damage
with age suggests a mechanism whereby stem cells themselves
may serve as a reservoir for the acquisition of mutations required
for disease pathogenesis or oncogenic transformation (Rossi
et al., 2007a).
At the Interface of Cancer, Aging, and Stem
Cancer is the leading cause of death for individuals younger than
85 (http://seer.cancer.gov/). However, mortality rates have de-
creased by less than 1.5% per year despite the introduction of
a plethora of new anticancer therapies over the last decade. Be-
cause a multiplicity of molecular events that correspond with
stem cell aging also occur in tumors in the elderly, research to
elucidate molecular aberrationsin stem and progenitor cellsdur-
ing aging may provide insights into cancer formation and point
the waytoward new therapeutictargets. Manycancersincluding
colon, breast, brain, head and neck, pancreatic, and hematopoi-
etic malignancies contain minor populations of tumor-initiating
cells or cancer stem cells (CSCs) (reviewed in Clarke and Fuller,
2006). CSCs share many of the functional properties of normal
stem cells including the potential for unlimited self-renewal.
Inamanner similarto normalstemcells, CSCshavethe potential
to giverisetomostorall ofthecelltypeswithinthetumorandare
ultimately responsible for ongoing tumor maintenance. More-
over, CSCs, like normal stem cells, are believed to express
high levels of many ABC/MDR transporter proteins that mediate
drug efflux, a property that may enable CSCs to evade the
ing to relapse. In fact, the leading cause of death in cancer
patients continues to be the acquired or intrinsic resistance of
tumor cells to therapy. The extent to which CSCs are derived
directly from transformed stem cells or are the result of transfor-
mative events that impart stem cell-like properties onto more
committed progenitors is only just beginning to be elucidated
and is likely to vary among different cancers. However, signifi-
cant deregulation of the mechanisms governing self-renewal,
survival, and cell-fate decisions is implied as these are normally
tightly regulated processes.
Aberrant Self-Renewal: The Emergence
of Leukemic Stem Cells
One of the most instructive diseases for understanding the
correlation between stem cell aging and the acquisition of the
complement of mutations required for malignant transformation
is chronic myeloid leukemia (CML), which is generally a disease
of the elderly. CML was the first malignancy to be associated
with a characteristic chromosomal abnormality and its constitu-
tively active tyrosine kinase fusion product, BCR-ABL. CML was
also the first cancer shown to be derived from a stem cell and to
be treated therapeutically with a drug rationally designed to
inhibit the BCR-ABL oncoprotein (reviewed in Wong and Witte,
2004). The molecular mechanisms driving progression of human
CML from its chronic phase through myeloid blast crisis have
been extensively investigated, but only recently has the role of
stem cell and progenitor cell biology been evaluated (Jamieson
et al., 2004). In chronic phase CML, quantitative PCR analysis
has shown that the BCR-ABL fusion transcript was present at
the highest levels in phenotypic HSCs, whereas blast crisis
CML was marked by amplified BCR-ABL expression in more
committed granulocyte-macrophage progenitors (GMPs) (Ja-
mieson et al., 2004). After the transition to blast crisis, GMPs
were found to have gained the capacity to self-renew and to
transfer blast crisis leukemia to immunodeficient mice, in part,
as a consequence of self-renewal driven by aberrant Wnt/b-cat-
enin signaling (Jamieson et al., 2004). In mouse models, expres-
sion of BCR-ABL was not sufficient to confer the properties of
LSCs on committed progenitors, whereas ectopic expression
of the MOZ-TIF2 oncogene that induces self-renewal was suffi-
cient (Huntly et al., 2004). In another mouse model, ectopic
expression of the MLL-ENL oncogene in HSCs, common mye-
loid progenitors (CMPs), or GMPs led to immortalization of the
GMPs (Cozzio et al., 2003). Interestingly, expression profiling
itors by ectopic expression of the MLL-AF9 oncogene revealed
that the expression signature of the parental progenitors was
more or less retained, whereas the self-renewing leukemic
clones had acquired a gene expression signature more charac-
teristic of stem cells (Krivtsov et al., 2006). These results suggest
that LSCs can emerge from committed hematopoietic progeni-
torcells without widespread reprogramming ofgene expression,
through the activation of only a critical subset of self-renewal
genes. Moreover, these studies reveal that the potential of differ-
ent oncogenic products to impart self-renewal capacity on
leukemic clones is developmental stage specific.
Although the mutational events that drive leukemogenesis
often act in a cell-autonomous fashion, recent studies have re-
vealed an equally important contribution to myeloproliferative
disorder and leukemia pathogenesis through dysregulation of
the microenvironment. This was shown by experiments in which
wild-type bone marrow cells developed into myeloproliferative
disease upon transplantation into environments deficient in
either the retinoic acid receptor gamma (RARg) (Walkley et al.,
2007a) or retinoblastoma protein (Walkley et al., 2007b). In
690 Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc.
both cases, loss of trabecular bone in the RARg or retinoblas-
toma-deficient settings suggests that aberrant extrinsic signals
emanating from the stem cell niche may be important in setting
the stage for disease pathogenesis.
The cancer stem cell model of tumorigenesis predicts that in
order to be clinically effective in the long-term, cancer therapies
must target the small subset of CSCs that are responsible for
maintaining and spreading the tumor. The fact that many path-
ways critical to tumorigenesis are so intimately linked to normal
stem cell self-renewal processes has raised the possibility that
cancer therapies targeting such pathways might also inadver-
tently ablate normal stem cells. One such self-renewal pathway,
mediated by the tumor suppressor protein Pten, is differentially
used by both normal and LSCs (Yilmaz et al., 2006; Zhang
et al., 2006). This mechanistic distinction allowed for targeted
therapy of the Pten pathway—through suppression of mTOR
byrapamycin—leadingto thedepletion ofLSCs without damage
to the normal stem cell pool (Yilmaz et al., 2006).
Cumulatively, these studies underscore the importance of the
cell-intrinsic and -extrinsic mechanisms that straddle the inter-
face of normal and leukemic stem cell regulation. Elucidating
these mechanisms will inform diagnostic and prognostic strate-
gies as well as the development of new therapies.
Aberrant Fate Determination and Disease
Aberrant cell-fate decisions are a well-documented feature of
aging and are also a hallmark of cancer. Developmental stage-
specific differentiation abnormalities in cancer have been most
highly studied in leukemias with balanced translocations that
result in inappropriate expression of transcription factors that
regulate critical cell-fate decisions. In one-third of cases of
B-progenitor acute lymphoblastic leukemia (ALL), PAX5, an es-
suggesting that deregulated transcription factor expression and
altered cell-fate decisions play a critical role in the pathogenesis
of lymphoid malignancy (reviewed in O’Neil and Look, 2007).
Similar abnormalities in cell-fate decisions are important drivers
polymorphism (SNP) within a highly conserved distal enhancer
myeloid progenitors in complex karyotype acute myeloid leuke-
mia (AML) by blocking binding of the chromatin-remodeling
transcriptional regulator SATB1 (Steidl et al., 2007).
Further evidence that aberrant cell-fate decisions in primitive
progenitors contribute to cancer pathogenesis comes from an
analysis of AML patients harboring t8;21 translocations generat-
ing the AML-1-ETO oncogene, the expression of which leads to
decreased expression of the critical granulocytic differentiation
factor, CEBP/a (Pabst et al., 2001). Although the AML-1-ETOon-
pable of transferring leukemia to immunocompromised mice are
enriched within a CD34+38?/lo subset of blood and bone marrow
cells (Bonnet and Dick, 1997), which represent a primitive multi-
potent hematopoietic progenitor cell subpopulation downstream
of HSCs (Majeti et al., 2007). This suggests that although HSCs
harbor the primary lesion, additional oncogenic events are re-
quired to drive transformation into AML. Consistent with this, ec-
sufficient to induce leukemogenesis unless combined with direct
Figure 5. Model of Leukemic Progression
In this model, HSCs serve as the reservoir for the
accumulation of the geneticand epigenetic events
that eventually lead to blast crisis and leukemia.
Stem cell self-renewal and differentiation enable
heritable mutations acquired in the stem cell com-
partment to be propagated to both self-renewing
progeny and downstream progenitors over the
lifetime of the organism. Stem cells with heritable
lesions act as substrates for additional hits, which
in turn can promote selection of preleukemic
clones if lesions imparting a growth or survival ad-
vantage are acquired. In this model, seven events
are listed as the full complement of events re-
quired for the progression of preleukemic clones
to frank leukemia, but the actual number of events
may vary depending upon the type of cancer and
the nature of the lesions involved as it is possible
that multiple oncogenic properties could be con-
ferred upon a preleukemic clone through the ac-
quisition of a single hit. Although the mutagenic
events accrue in stem cells, the eventual emer-
gence of leukemic clones occurs at the stage of
progenitor cells downstream of HSCs that acquire
the capacity for unlimited self-renewal. In chronic
myeloid leukemia (CML), this can be the granulo-
cyte/macrophage progenitor, whereas in acute
myeloid leukemia, the leukemic clone can emerge
further downstream depending upon the nature of
the oncogenic lesions involved.
Cell 132, 681–696, February 22, 2008 ª2008 Elsevier Inc. 691
mutagenesis using the mutagen ENU (Yuan et al., 2001). To-
gether with the finding that HSCs can accumulate DNA damage
during aging (Rossi et al., 2007a), these results suggest a model
in which the primary oncogenic lesion generating the AML-1-
ETO fusion product occurs in HSCs, which then serve as sub-
strates for the acquisition of additional hits eventually leading
to the transformation of downstream progenitors (Figure 5).
Together, these studies provide evidence that leukemogene-
of seven independent genetic and epigenetic events required for
transition to blast crisis or acute leukemia. In this model, patients
containing n = 1, n = 2, and n = 3 events in addition to leukemic
ure 5). Analysis of LSCs from such patients at the genomic, gene
expression, epigenetic, and proteomic levels should reveal the
full life history of these leukemic clones and shed light on how
to combat their formation and progression.
The number of elderly adults is at a historical high, and concom-
malignant conditions will place a heavy burden on future health
care resources. The need to develop therapeutic strategies to
treat pathophysiological conditions in the elderly is therefore
medically, socially, and economically crucial. Characterization
these goals as such research should be able to identify the
mechanisms underlying stem cell functional decline and inform
strategies for intervention. Of particular importance is the devel-
opment of targeted therapies that will obviate the high mortality
We thank David Lombard, Roi Gazit, Luigi Warren, Nina Korsisaari and David
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