Cell Stem Cell
Article
A Distinctive DNA Damage Response in Human
Hematopoietic Stem Cells Reveals an
Apoptosis-Independent Role for p53 in Self-Renewal
Michael Milyavsky,1,2,7 Olga I. Gan,1,2,7 Magan Trottier,2,5,7 Martin Komosa,5 Ofer Tabach,6 Faiyaz Notta,1,2
Eric Lechman,1,2 Karin G. Hermans,1,2 Kolja Eppert,1,2 Zhanna Konovalova,1,2 Olga Ornatsky,3 Eytan Domany,6
M. Stephen Meyn,2,4,5 and John E. Dick1,2,*
1Department of Stem Cell and Developmental Biology, Campbell Family Cancer Research Institute, Ontario Cancer Institute, Toronto General
Research Institute, University Health Network
2Department of Molecular Genetics
3Institute for Biomaterials and Biomedical Engineering
4Department of Pediatrics
University of Toronto, Toronto, ON M5G 1L7, Canada
5Program in Genetics and Genome Biology, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
6Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot 76100, Israel
7These authors contributed equally to this work
*Correspondence: jdick@uhnres.utoronto.ca
DOI 10.1016/j.stem.2010.05.016
SUMMARY
Highly regenerative tissues such as blood must
possess effective DNA damage responses (DDR)
that balance long-term regeneration with protection
from leukemogenesis. Hematopoietic stem cells
(HSCs) sustain life-long blood production, yet their
response to DNA damage remains largely unex-
plored. We report that human HSCs exhibit delayed
DNA double-strand break rejoining, persistent
gH2AX foci, and enhanced p53- and ASPP1-depen-
dent apoptosis after g-radiation compared to pro-
genitors. p53 inactivation or Bcl-2 overexpression
reduced radiation-induced apoptosis and preserved
in vivo repopulating HSC function. Despite similar
protection from irradiation-induced apoptosis, only
Bcl-2-overexpressing HSCs showed higher self-
renewal capacity, establishing that intact p53 posi-
tively regulates self-renewal independently from
apoptosis. The reduced self-renewal of HSCs with
inactivated p53 was associated with increased
spontaneous gH2AX foci in secondary transplants
of HSCs. Our data reveal distinct physiological roles
of p53 that together ensure optimal HSC function:
apoptosis regulation and prevention of gH2AX foci
accumulation upon HSC self-renewal.
INTRODUCTION
Corruption of genetic information by endogenous and exoge-
nous sources of DNA damage threatens the health of an
organism. A complex network of DNA damage responses
(DDR) has evolved to sense and respond to DNA damage. In
multicellular organisms, activation of DDR results in two primary
186 Cell Stem Cell 7, 186–197, August 6, 2010 ª2010 Elsevier Inc.
outcomes: repair of DNA damage and genomic restoration or,
if damaged DNA cannot be sufficiently repaired, execution of
cell death or senescence programs (Vousden and Lane, 2007).
In highly regenerative tissues like blood, short-lived but highly
proliferative committed progenitors (colony-forming cells, CFCs)
generate large numbers of differentiated cells. In turn, CFCs are
derived from more primitive multipotential progenitors (MPPs)
with limited self-renewal potential (Majeti et al., 2007). The entire
hematopoietic system is ultimately generated from HSCs that
have both the greatest longevity and the highest potential for
self-renewal (Benveniste et al., 2003; Rossi et al., 2008). A single
HSC can sustain hematopoiesis for the lifetime of a mouse,
pointing to its potency (Osawa et al., 1996). Because of geno-
toxic stresses, the bone marrow (BM) is likely to harbor progen-
itor cells with acquired mutations (Ji et al., 1995). However, their
short lifespan precludes the accumulation of multiple mutations
required for malignant transformation in a single progenitor cell
clone. By contrast, the long lifespan of HSCs provides time to
potentially accumulate enough DNA damage-induced mutations
to initiate a leukemogenic process (Dick, 2008). Despite a high
rate of hematopoietic regeneration, leukemia is a relatively rare
disease; it is likely that that strong evolutionary pressure exists
for HSCs to develop distinct DDR strategies that emphasize
genetic integrity over survival in the face of genotoxicity. Indeed,
of all tissues, the hematopoietic system is the most radiosensi-
tive, and it is DNA damage to HSCs, not progenitors, that ulti-
mately limits the regeneration of hematopoiesis (Dainiak, 2002;
Mettler and Voelz, 2002; Nijnik et al., 2007; Rossi et al., 2007).
Patients with inherited DDR defects and mice that model these
diseases frequently exhibit marked genomic instability, progres-
sive BM failure resulting from impairment of HSC function, and
a high incidence of hematopoietic malignancies (Niedernhofer,
2008). Collectively, these data argue that DDR is essential for
maintaining normal HSC function and for preventing leukemo-
genesis.
In humans, where genotoxic stress is widely employed clini-
cally in chemo- and radiotherapy, BM injury is thought to be
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DNA Damage Response in Hematopoietic Stem Cells
caused primarily by induction of apoptosis (Gudkov and Komar-
ova, 2003). Both p53 and Bcl-2 proteins are well-characterized
regulators of apoptosis. The p53 tumor suppressor is a widely
expressed transcription factor that, in addition to apoptosis
induction, plays critical roles in several DDR pathways, including
activation of cell cycle checkpoints, suppression of homologous
recombination, and others (Bertrand et al., 2004; Vousden and
Lane, 2007). As with other DDR genes, germline mutations in
p53 increase the incidence of hematological malignancy (Nie-
dernhofer, 2008), and somatic aberrations of p53 and Bcl-2 are
frequent in human leukemias and lymphomas (Prokocimer and
Rotter, 1994; Yip and Reed, 2008). Neutralization of Bcl-2 by
the direct transcriptional target of p53, such as Puma, is respon-
sible for apoptosis in many cell types (Michalak et al., 2008) and
overexpression of Bcl-2 alone counteracts many apoptotic
stimuli (Domen et al., 1998; Domen and Weissman, 2003;
Strasser et al., 1994). The recent identification of additional
components of the p53 pathway may help explain the mecha-
nism that underlies cell-specific variations in sensitivity to p53-
mediated apoptosis (Vousden and Lane, 2007). In vitro experi-
ments suggest that p53-interacting proteins, such as
apoptosis-stimulating proteins of p53 (ASPPs) family and others,
fine tune cellular fate toward apoptosis or survival by altering the
transcriptional activity of p53, although their physiological signif-
icance in cells that comprise the hematopoietic hierarchy is
unknown (Samuels-Lev et al., 2001).
The high susceptibility of hematopoietic cells to genotoxic
stress, such as radiation, prompted numerous studies in murine
models that established that BM progenitors vary substantially in
their radiosensitivity and functional recovery after irradiation
(Down et al., 1995; Meng et al., 2003; van Bekkum, 1991; Wage-
maker, 1995; Wang et al., 2006). However, modern advances in
flow cytometric analysis and functional assays of early hemato-
poietic development limit the impact of these pioneering studies.
In some cases (Down et al., 1995; van Bekkum, 1991; Wage-
maker, 1995), the precise cell surface phenotype of population
subsets was not assessed, whereas in others putative stem
cell populations are now known to comprise both progenitors
and HSCs (Meng et al., 2003; Wang et al., 2006).
It has also been difficult to establish the function of p53 in
HSCs compared to other hematopoietic cells via mouse models.
BM progenitors from p53 knockout as well as Bcl-2 transgenic
mice exhibit increased resistance to radiation-induced apo-
ptosis in short-term survival assays (Lotem and Sachs, 1993;
Michalak et al., 2008; Wu et al., 2005). However, the rapid trans-
formation of p53 null BM cells complicates analysis of p53 func-
tion in normal nonmalignant long-term HSCs, also precluding
examination of self-renewal by retransplantation (Dumble et al.,
2007; TeKippe et al., 2003). When attempts were made to
examine the function of putatively nonmalignant HSCs from
p53-deficient mice, conflicting results were obtained; HSC repo-
pulation was either increased, similar, or even decreased (Chen
et al., 2008; Liu et al., 2009; Marusyk et al., 2010). Moreover, with
increased p53 dosage, HSCs exhibit reduced competitive repo-
pulation potential (Dumble et al., 2007).
Finally, species-specific differences in irradiation suscepti-
bility (van Bekkum, 1991), oncogene function, cytokine require-
ments, telomere/telomerase biology, and the inbred nature of
mouse strains complicate direct extrapolation of the knowledge
obtained in rodents to humans (Rangarajan et al., 2004).
Together, little is known about regulators of DNA damage and
apoptosis within human HSCs and the effects of p53 and Bcl-2
alterations on long-term HSC function.
The development of tools to purify and assay human HSCs
(Majeti et al., 2007; McKenzie et al., 2006), combined with the
ability to genetically manipulate HSCs (Mazurier et al., 2004),
provides the basis for a genetic approach to gain mechanistic
insight into the responses of human HSCs to DNA damage.
Our experiments establish that HSCs delay DSB rejoining,
exhibit persistent DDR activation, and are more sensitive to the
cytotoxic effects of g-radiation compared to committed progen-
itors. Irradiated human HSCs and progenitors undergo apo-
ptosis via p53/ASPP1 and Bcl-2. Upon serial transplantation,
HSCs with disabled p53 accumulated DNA damage and
exhibited decreased self-renewal capacity compared to Bcl-2-
overexpressing cells, pointing to distinct functions of p53 in
apoptosis and maintenance of self-renewal.
RESULTS
Delayed Double-Strand Break Rejoining and Sustained
DDR Foci in Primitive Hematopoietic Cells
The human hematopoietic system is hierarchically organized
where cells at various stages of differentiation can be distin-
guished by cell surface phenotype: HSCs are highly enriched
in mature lineage-negative (Lin!) CD34+CD38! fractions, and
even more so in the Lin!CD34+CD38!CD90+CD45RA! fraction,
whereas Lin!CD34+CD38!CD90!CD45RA! population con-
tains fewer HSCs and many MPPs. More mature progenitor/
precursor cells, including CFCs, are Lin!CD34+CD38+ (Majeti
et al., 2007). We designate these fractions as ‘‘HSCs’’,
‘‘MPPs’’, and ‘‘Progenitors’’, respectively.
To investigate the response of these phenotypic subpopula-
tions to genotoxic stress, purified fractions of cord blood (CB)
cells were irradiated ex vivo. The most biologically significant
DNA lesion induced by ionizing radiation (IR) is a double-strand
break (DSB). To investigate DSB repair, we used the neutral
comet assay to detect DSB rejoining in cells exposed to 15 Gy
g-IR. We found that the Progenitor fraction rejoined 19.4% and
36.4% of breaks by 30 and 60 min post IR, respectively (Fig-
ure 1A), a rate that is slower than what we and others observe
in human fibroblasts (Figure S1 available online; Stenerlow
et al., 2003). The initial response of the HSC/MPP population
was even more striking: no detectable rejoining was seen in
the first 30 min (<1%) and significantly less repair than in the
Progenitor population at 1 hr post IR (29.9% versus 36.4%)
(Figure 1A).
The delay in initiating DSB rejoining suggested that the DDR of
the quiescent HSC/MPP population differs from more differenti-
ated hematopoietic cells and human fibroblasts. To investigate
this possibility, we examined another facet of the damage
response: the formation of subnuclear foci of phosphorylated
H2AX (gH2AX) at and around DSB sites. One hour after 3 Gy
IR, no detectable differences in the induction of gH2AX foci
were observed between HSC/MPP and Progenitor fractions;
both populations displayed approximately 26 induced gH2AX
foci/cell (Figures 1B and 1C). However, 12 hr after IR, signifi-
cantly more gH2AX foci remained in HSC/MPP compared to
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Figure 1. IR-Induced Cellular Response in Hematopoietic Fractions In Vitro

Lin-CB cells were sorted into CD34+CD38!/lowCD45RA! (HSC- and MPP-containing fraction) and CD34+CD38+ (Progenitors).
(A) DSB rejoining measured after 15 Gy IR via the neutral comet assay in HSC/MPP (blue bars) and Progenitor (red bars) fractions. Percent of initial olive tail
moment (OTM) was calculated by dividing OTM at each time point by OTM immediately after IR. 150–350 nuclei were scored for each time point (bars represent
means ± SE, *p < 0.005).
(B) Number of gH2AX foci per cell in HSC/MPP and Progenitor subpopulations 1 and 12 hr after damage. 80–120 nuclei were scored for each time point (bars
represent means ± SE, *p < 0.001).
(C) Representative immunofluorescence staining for gH2AX (red) and DNA (blue) in HSC/MPP- and Progenitor-enriched fractions 1 and 3 hr after 3 Gy IR.
(D) Assessment of cell death in sorted primitive HSC, MPP, and Progenitor fractions 18 hr post IR by double staining with Annexin and 7-AAD (n = 3).
(E) Survival of clonogenic progenitors from HSC, MPP, and progenitor fractions calculated by dividing number of colonies scored in the irradiated plate by the
number of colonies from the same fraction counted in the nonirradiated plate (n = 3, bars represent means ± standard deviation, *p < 0.05 between progenitors
and other fractions). See also Figure S1.

Progenitor fraction (7.1 versus 2.7 foci/nucleus, respectively),
and the number of gH2AX foci/nucleus remained above the unir-
radiated baseline in 82% of HSCs/MPPs compared to only 44%
Progenitor cells (Figures 1B and 1C). Taken together, these data
indicate that DDR foci persist longer in primitive cells compared
to progenitors.
Increased Apoptosis Induction in Primitive
Hematopoietic Cells
In some cells, the DDR machinery responds to IR-induced DNA
damage by triggering apoptosis (Vousden and Lane, 2007). Both
early (Annexin+, 7AAD!) and late (Annexin+, 7AAD+) stages of
apoptosis were detected 18 hr after 3 Gy IR for all fractions
(Figure 1D). HSC and MPP fractions were investigated sepa-
rately. Both populations showed a similar proportion of apo-
ptotic cells (59% and 55%, respectively) that was significantly
higher than irradiated Progenitor cells (35%). With loss of clono-
genic potential as a biological readout of cell death, irradiated
HSC and MPP fractions exhibited decreased clonogenic survival
compared to the Progenitor fraction (Figure 1E), correlating
closely with their higher apoptotic rate.
To examine the possible effect of the microenvironment on
IR-induced apoptosis in HSC/MPP and Progenitor fractions,
we performed in vivo IR experiments. NOD/SCID mice were
transplanted with Lin! human CB cells to obtain human hemato-
poietic engraftment. Upon IR (3 Gy) of the engrafted recip-
ients (5–10 weeks posttransplantation), the apoptosis rate
of Lin!CD34+CD38! and Lin!CD34+CD38+ human cells was

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Table 1. Differential Induction of Apoptosis in Response to In
Vivo Irradiation in Stem and Progenitor Cell Subpopulations
Annexin V + Cells (%)
Lin!CD34+CD38! Lin!CD34+ CD38+
Fraction Fraction
(Mean ± SE) (Mean ± SE)
Nonirradiated 6.9 ± 1.8 7.6 ± 4.2
marrow (n = 3)
Irradiated 33.3 ± 9.7 17.9 ± 6.0
marrow (n = 3)
Fold increase in 4.8 2.3
apoptosis in
response to radiation
Human hematopoietic cells were purified from NOD/SCID mice trans-
planted with CB cells 5–10 weeks before. The cohort of the recipients
was irradiated with 3 Gy and sacrificed 1.5 hr later. In every experiment
BM cells were obtained from 2–3 nonirradiated mice and from 4–6 irradi-
ated recipients. Lin! human cells were purified and incubated in
cytokine-supplemented medium for the additional 18 hr followed by
CD34, CD38, and Annexin staining for flow cytometry analysis. Mean
percentage ± standard error (SE) of Annexin V+ SYTOX Blue! cells
in each fraction is shown. n indicates the number of independent
experiments.
assessed (Table 1). IR-induced apoptosis of human hema-
topoietic cells in the BM microenvironment was 2-fold higher
in Lin!CD34+CD38! cells compared to Lin!CD34+CD38+
Progenitors.
Collectively, our in vitro and in vivo irradiation data indicated
that the HSC/MPP fraction possessed higher susceptibility to
IR-induced apoptosis than Progenitor cells.
Inactivation of p53 or Bcl-2 Overexpression Blocks
IR-Induced Apoptosis
The roles of p53 and Bcl-2 in IR-induced apoptosis are not
well established in immature human hematopoietic cells. To
determine their role, we constructed lentivirus vectors express-
ing either shRNA targeting p53 (shp53) or a dominant-negative
form of p53 (GSE56) (Ossovskaya et al., 1996) and the human
Bcl-2 cDNA (Figure 2A). Transduced hematopoietic cells
showed considerable downregulation of p53 protein, overex-
pression of inactive p53, or overexpression of Bcl-2, respectively
(Figure 2B). After 3 Gy IR, the expression of several p53 tran-
scriptional target genes, p21, PUMA, and Killer/DR5, was abro-
gated in Lin-CB cells that expressed either the shp53 or GSE56
lentiviruses (Figure 2C), confirming the functional inactivation of
p53 with both vectors. Transduction of Lin-CB cells with the Bcl-
2 lentivirus did not affect the induction of p53 target genes estab-
lishing the specificity of these genetic tools (Figure 2C).
To determine the involvement of p53/Bcl-2 in cell death of
human hematopoietic cells, we measured apoptosis induction
in Lin-CB cells expressing shp53, GSE56, or Bcl-2 at 18–24 hr
after IR. Inactivation of p53 (p53 knockdown [KD]) or overexpres-
sion of Bcl-2 resulted in a significant decrease in the post-IR
apoptotic frequency compared to controls (Figure 3A). Clono-
genic assays revealed that both genetic modifications increased
the number of CFC after IR, but this partial functional rescue
was much less effective than the protection from IR-induced
apoptosis (Figure 3B; Figure S2A). These data provide evidence
that CFCs respond to genotoxic stress via a p53/Bcl-2-depen-
dent apoptotic mechanism, and this mechanism must be
partially responsible for the loss of their clonogenic potential.
To determine whether the differences in DDR between HSCs/
MPPs and Progenitor cells could be due to the differential
expression of molecular regulators of p53-dependent apoptosis,
we carried out global gene expression profiling of both cell frac-
tions, with and without IR, and with and without p53 inactivation
(unpublished data). Because these studies required short-term
culture (4 days), we were not able to employ CD38 in the sorting
strategy because it becomes dissociated from HSC/MPP func-
tion upon culture (Dorrell et al., 2000). As an alternative, we
employed CD90 because it is a useful marker to distinguish
a population enriched for HSCs (CD34+CD90+), whereas CD34+
CD90! cells are Progenitor enriched, but devoid of HSCs (Danet
et al., 2001). One molecular regulator of p53, expression of which
was much higher in CD34+CD90+ was PPP1R13B mRNA,
encoding the apoptosis-stimulating protein of p53 (ASPP1).
Independent validation of ASPP1 expression by real-time PCR
confirmed its higher expression in CD34+CD90+ cells and in
freshly isolated Lin!CD34+CD38! HSC-enriched cells as com-
pared with CD34+CD90! and Lin!CD34+CD38+ Progenitors,
respectively (Figure 3C; Figure S2B). To elucidate the functional
significance of ASPP1 after induction of apoptosis, we knocked
down ASPP1 protein expression (Figure S2C). After transduction
of Lin-CB cells with shASPP1-encoding lentiviruses and IR,
the proportion of IR-induced Annexin+ cells was significantly
reduced only in the CD34+CD90+ primitive fraction and not in
the more mature CD34+CD90! cell population (Figures 3D and
3E), providing evidence that the HSC-specific expression of
ASPP1 is functionally important.
These results reveal that in response to IR, the HSC-enriched
fraction is more sensitive to p53-dependent apoptosis, whereas
more mature cells undergo lower apoptosis induction and they
are also less sensitive to p53 inactivation. Furthermore, the
high ASPP1 expression in HSC-enriched fraction is partially
responsible for the inherent susceptibility of those cells to
IR-induced apoptosis.
Abrogation of p53/Bcl-2-Mediated Apoptosis Allows
Myelopoiesis Recovery after IR
Because our results showed a clear distinction in apoptosis
induction between HSCs/MPPs and Progenitor cells as well as
only partial rescue of clonogenic activity when apoptosis was in-
hibited by disabling p53 or overexpressing Bcl2, it was important
to examine the biological consequences on primitive cells after
abrogation of IR-induced apoptosis. Lin-CB cells were trans-
duced with control, GSE56, or Bcl-2 lentiviruses, irradiated
(3 Gy), and grown in cytokine-dependent cultures with equal
input cell numbers. Continuous production of immature CD34+
cells, as well as functionally defined CFCs in this long-term
culture assay, serves as a functional indicator for the mainte-
nance of primitive cells, including HSCs (Piacibello et al., 2002).
The myeloid differentiation potential in control and experi-
mental cultures beyond 4 weeks of incubation was similar,
although in the first 2 weeks, Bcl-2-overexpressing cells
exhibited EPO-independent differentiation (Figure S3). Overall,
unirradiated cells achieved greater than 1000-fold expansion
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Figure 2. Inactivation of p53 and Overexpression of Bcl-2 in Lin-CB

(A) Diagrams of the lentiviral vectors used for inactivation of p53 (H1-shp53-GIP and MA-GSE56) and overexpression of Bcl-2. Control vectors are: H1-shRFP for
shRNA interference, MA for GSE56 and Bcl-2 overexpression.
(B) Left: western blot analysis of p53 protein levels in TEX cell line after infection with shp53 and IR with 3 Gy. Right: western blot analysis of p53 and Bcl-2 protein
levels in CB cells infected with either control (MA), GSE56-, or Bcl-2-expressing lentiviruses. Accumulation of inactive p53 protein is visible as a result of GSE56
overexpression.
(C) Analysis of p53 mRNA and p53 target genes 4 days after shp53 lentiviral transduction of Lin-CB cells. Relative expression levels of the indicated genes were
determined by Q-PCR. Profiling of p21, PUMA, and Killer/DR5 by Q-PCR in Lin-CB-expressing shRFP, shp53, MA, GSE56, and Bcl-2 (nonirradiated [NT], white
bars; 3 hr post IR [IR], black bars). Representative experiments are separated by vertical lines. Gene-specific expression levels were normalized to the levels of the
GAPDH housekeeping control for the same cDNA sample.

over the course of 10 weeks, with GSE56- and Bcl-2-infected
cultures showing slightly higher cell numbers at later time points
compared to control (Figure 4A). In all groups, nonirradiated cells
exhibited a slight expansion of CD34+ cells over 3 weeks (Fig-
ure 4B). By contrast, irradiated control cells failed to expand,
whereas both inactivation of p53 and overexpression of Bcl-2
permitted sustainable cell production. Upon IR, the control
group showed a sharp decrease in the number of CD34+ cells
and CFCs (Figures 4B and 4C) in agreement with the absence
of overall cell production. However, IR of cells with inactivated
p53 or overexpressed Bcl-2 resulted in a modest initial decrease
in the CD34+ fraction followed by robust recovery up to input
levels (Figure 4B) and steady expansion of CFCs (Figure 4C).
However, p53 inactivation or Bcl-2 overexpression did not
completely restore the expansion potential of hematopoietic
cells after IR: as a result, the total and CFC cell production was
about 10-fold lower relative to the nonirradiated group (Figures
4A–4C). Nevertheless, these long-term in vitro cultures reveal
that blocking IR-induced apoptosis by p53 inactivation or Bcl-
2 overexpression effectively protects primitive hematopoietic
cells that are essential for the recovery of long-term myeloid
cell production after IR.
Prevention of IR-Induced p53-Mediated Apoptosis
Protects HSCs
Human HSCs can be assayed by reconstitution of hematopoi-
esis in vivo utilizing the NOD/SCID repopulation assay (Dick,
2008). Lin-CB cells were transduced with control, p53KD-, and
Bcl-2-expressing viruses, sorted for EGFP+ cells, exposed to
IR or not, and transplanted intrafemorally into NOD/SCID mice
(Figure 5A). Recipients of nonirradiated cells from all three exper-
imental groups were highly engrafted in the injected bone with
human cells (Figure 5B), demonstrating similar multilineage
engraftment provided by transduced HSCs (Figure S4). The
intrafemoral injection method provides a unique ability to study
one of the central functions of HSCs, namely their migration to
other hematopoietic territories. Despite the similar level of the
engraftment in the injected bone, engraftment in the noninjected
bones was significantly higher in recipients of cells overexpress-
ing Bcl-2 or p53KD cells (Figure 5B). The improved repopulation

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Figure 3. p53, Bcl-2, and ASPP1 Regulates Apoptosis and Survival of Lin-CB Cells after IR
(A) Apoptosis of EGFP+ population was analyzed by annexin staining and flow cytometry 18–24 hr after 3 Gy IR. Results obtained separately for shRFP and MA
(designated Ctrl thereafter), as well as for shp53 and GSE56 (designated p53KD [knockdown]) showed similar trend and therefore were combined for mean calcu-
lations (n = 2–8).
(B) Clonogenic potential (CFC) of sorted Lin-CB Ctrl, p53KD-, or Bcl-2-overexpressing cells (n = 5–11).
(C) Relative expression of ASPP1 in CD34+CD90+ and CD34+CD90! cells isolated after 4 days of ex vivo culture.
(D and E) Lin! CB cells were transduced with the indicated shRNAs, sorted 4 days later into CD34+CD90+ and CD34+CD90! fractions followed by 3 Gy IR.
Apoptosis was analyzed by annexin staining and flow cytometry 18–24 hr post-IR.
Bars represent means ± standard deviation, *p < 0.05 versus Ctrl. See also Figure S2.

capacity of p53KD cells was probably due to a 1.7-fold increase
in HSC frequency as determined by limiting dilution analysis
(Table S1).
Irradiated control cells failed to establish grafts in most of the
recipients (17 out of 21) with median engraftment levels <0.1%
(Figure 5C). On the contrary, most recipients of irradiated
p53KD- or Bcl-2-overexpressing cells were engrafted (25 out
of 27 and 15 out of 21, respectively). Median levels of engraft-
ment in these two groups (6.6% and 5.6%, respectively) were
significantly higher in comparison with the irradiated control.
The differentiation of engrafted cells was unaffected by IR in
the p53KD and Bcl-2 groups (Figure S4). Importantly, IR did
not impair the ability of p53KD- or Bcl-2-overexpressing cells
to migrate (Figure 5C).
Examination of engraftment levels does not provide the means
to quantify the number of HSCs contributing to repopulation.
However, applying Poisson statistics to the engraftment data
enabled us to estimate that 3 Gy of IR reduced the HSC
frequency in control and p53KD cells by 154- and 25-fold,
respectively. Interestingly in the control vector-transduced
group, 3 Gy of IR inactivated 99.3% of HSC and 90% of clono-
genic progenitors, whereas p53 inactivation prior to IR resulted
in 96% and 80% inactivation of HSCs and CFCs, respectively.
Therefore, p53 inactivation conferred radioprotection of HSCs
by 5.7-fold, an effect that was much higher than the 1.7-fold
protection of CFCs (Figure 3B; Figure S2A and Table S2).
Based on the long-term in vivo and in vitro results, we
conclude that p53 inactivation or Bcl-2 overexpression offers
significant, but not absolute, radioprotection. We established
that p53/Bcl2-dependent apoptosis regulates human HSCs
under homeostatic and genotoxic stress conditions and that
HSCs are more dependent on this pathway than committed
progenitors.
HSC Self-Renewal Is Regulated by Bcl-2 and p53
Accumulation of DNA damage in several murine genetic models
of DNA repair results in severely diminished HSC function (Nijnik
et al., 2007; Rossi et al., 2007). Our initial observations revealed
that freshly isolated human HSCs exhibited distinct patterns of
DDR as compared to progenitors (Figure 1), so it was important
to determine whether p53 and Bcl-2 modulation would affect
HSC genome integrity after repopulation of NOD/SCID mice.

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Figure 4. Effect of p53 Inactivation or Bcl-2 Overexpression on Long-Term Myelopoiesis In Vitro
Four days after lentiviral transduction, EGFP+ cells were sorted from control (MA), GSE56-, or Bcl-2-overexpressing groups, divided into two groups (NT and IR
with 3 Gy), and placed into long-term cultures (100,000 cells per culture).
(A) Cell expansion.
(B) CD34+ cell number per culture as analyzed by flow cytometry.
(C) CFC dynamics in cultures (n = 3).
See also Figure S3.
After transplantation, HSCs proliferate to initiate hematopoietic
repopulation, and in addition, HSCs must self-renew to ensure
long-term repopulation. Serial transplantation is an effective
assay to measure the capacity of HSCs to self-renew; however,
retransplantation could generate significant replicative or other
forms of stress. We purified human Lin! cells from the BM of
highly engrafted mice and quantified gH2AX foci. We found
that human Lin! cells from primary recipients of control (MA),
p53KD-, and Bcl-2-overexpressing cells harbored approxi-
mately the same mean number and similar distribution of spon-
taneous gH2AX foci as human fibroblasts (Table S3, Figure 6A;
Wilson et al., 2010). However, upon serial transplantation there
was a striking elevation in the number of gH2AX foci per cell
only in human Lin! cells with disabled p53 as compared to the
control or Bcl-2-overexpressing cells that maintain wild-type
p53 (Table S3). Examination of the distribution of foci showed
that human cells with disabled p53 had a high proportion of cells
exhibiting more than 5 gH2AX foci/nucleus and a complete
absence of foci-negative cells compared to the other experi-
mental groups (Figure 6A). By contrast, the Bcl-2-overexpress-
ing cells possessed lower numbers of gH2AX foci/nucleus
even compared to control. These results indicate that the
changes in gH2AX foci numbers are uncoupled from the process
192 Cell Stem Cell 7, 186–197, August 6, 2010 ª2010 Elsevier Inc.
Cell Stem Cell
DNA Damage Response in Hematopoietic Stem Cells
of apoptosis. The increase in gH2AX foci seen solely in p53KD
cells is probably due to a loss of one or more p53 functions
that must be independent of apoptosis, because abrogation of
apoptosis induction by Bcl-2 overexpression did not result in
an elevation in foci numbers.
Our findings that p53 helps to control the accumulation of
spontaneous DNA damage in human HSCs throughout times
of expansion, such as during serial transplantation, raises the
question of whether HSC self-renewal capacity is affected
when p53 is disabled. Equally, the reduced numbers of gH2AX
foci when Bcl-2 is overexpressed brings up a similar query.
To determine whether p53 or Bcl-2 modulation affected HSC
self-renewal, we examined human cell engraftment of secondary
recipients.
In nonirradiated HSCs, only Bcl-2 overexpression resulted in
significantly higher self-renewal ability with median engraftment
levels of 0.15%, 0.58%, and 3.6% for control, p53KD, and Bcl-2
groups, respectively (Figure 6B). The p53KD group showed
a trend to lower engraftment compared to the Bcl-2 group.
When irradiated cells were serially transplanted, we found
a striking difference: HSCs with disabled p53 exhibited signifi-
cantly lower self-renewal capacity in comparison with Bcl-2-
overexpressing cells. Only HSCs from the Bcl-2-overexpressing
Cell Stem Cell
DNA Damage Response in Hematopoietic Stem Cells

Figure 5. Repopulating Potential of Genetically Modified HSCs after IR

(A) Study design. Lin-CB cells were transduced with Ctrl (shRFP or MA), p53KD (shp53 or GSE56), or Bcl-2 lentiviruses, cultured for 4 days, sorted for EGFP+ cells,
divided into two groups (NT and IR with 3 Gy), and injected intrafemorally into preconditioned immunodeficient mice. Seven to ten weeks later, the secondary
transplants were performed and mice were analyzed again 7–10 weeks later.
(B and C) Human engraftment of the nonirradiated (B) and irradiated (C) Lin-CB cells in the injected femurs (RF) and in the noninjected bones (BM) of primary
recipients. Each symbol stands for engraftment level in one recipient. Horizontal lines represent median for each group, *p < 0.05 in comparison with relevant
Ctrl group (n = 6).
See also Figure S4 and Tables S1 and S2.

group were able to consistently re-establish human engraftment
(Figure 6C). This was surprising because p53 inactivation and
Bcl-2 overexpression similarly protected HSCs from radiation
injury resulting in similar levels of engraftment in primary recipi-
ents. Together, these data link the function of these two key
molecular regulators to HSC self-renewal under both homeo-
static and stress conditions.
DISCUSSION
Our results establish that the DDR of human HSCs differs in
multiple ways from more mature hematopoietic populations.
Primitive HSCs/MPPs exhibit delayed DSB rejoining and persis-
tent DDR foci and undergo higher levels of p53/ASPP1-depen-
dent apoptosis compared to Progenitor population in response
to IR. p53 inactivation or Bcl-2 overexpression effectively antag-
onized IR-induced apoptosis and provided profound rescue
of HSC repopulation potential in primary transplanted mice.
However, in serial transplanted mice, HSCs with disabled p53
exhibited persistently high spontaneous gH2AX foci in the
engrafted cells and this correlated with markedly decreased
HSC self-renewal capacity. By contrast, Bcl-2-overexpressing
HSCs with intact p53 were able to sustain HSC self-renewal,
establishing that the negative impact of disabled p53 on HSC
self-renewal must be due to impairment of apoptosis-independent
p53 function. Taken together, these results indicate that p53 has
discrete functions in human HSCs that are balanced to facilitate
genome stability and optimal self-renewal: one is apoptosis
dependent and serves to negatively regulate HSCs, especially
after IR, whereas the other is apoptosis independent, positively
regulating HSCs especially during serial transplantation.
The major pathway for DSB repair in quiescent human cells is
nonhomologous end joining (NHEJ), a rapid but error-prone
method of DSB repair responsible for the majority of genomic
structural variants and chromosome translocations in human
cancers (Raphael et al., 2008). In mammalian cells, the protein
components of NHEJ begin to assemble at DSB sites seconds
after IR (Mari et al., 2006), and NHEJ-mediated repair and rejoin-
ing of DSBs is detected within 10 min of DSB induction (Gulston
et al., 2004). For quiescent human fibroblasts, "50% of DSBs
are rejoined within 30 min post-IR (Stenerlow et al., 2003).
However, no rejoining of DSBs occurred in the first 30 min

Cell Stem Cell 7, 186–197, August 6, 2010 ª2010 Elsevier Inc. 193
 Cell Stem Cell
DNA Damage Response in Hematopoietic Stem Cells

Figure 6. Effect of p53 Inactivation and Bcl-2 Overexpression on DNA Damage Accumulation and HSC Self-Renewal
(A) gH2AX foci distribution in Lin! human hematopoietic cells isolated from primary and secondary recipients of cells transduced with MA, GSE56, and Bcl-2
lentiviruses. Percent of cells with <1, 1–5, and >5 foci per nucleus is depicted.
(B and C) Comparison of human engraftment in the injected femurs between primary and secondary recipients. Nonirradiated (B) and irradiated (C) cells were
retransplanted to assay HSC self-renewal ability. Horizontal lines represent median for each group, *p < 0.05 in comparison with relevant experimental group.

post-IR in quiescent HSC/MPP populations, indicating that the
distinct DDR of HSCs/MPPs begins immediately post-IR with
the initial ‘‘decision’’ to delay NHEJ repair of DSBs. The resolu-
tion of gH2AX foci was also delayed in the HSC/MPP fraction.
The persistence of these DDR protein foci could be the result
of increased numbers of residual DSBs in the HSC/MPP fraction
compared to Progenitor cells. However, at 6 hr, the differences in
DSB rejoining between the two populations are minor, suggest-
ing instead that the persistence of gH2AX foci after rejoining of
their associated DSBs represent another aspect of the altered
DDR of HSCs. These data also provide a note of caution regard-
ing extrapolating DDR results from one cell type to another (e.g.,
fibroblasts and HSCs) and point to the importance of investi-
gating the DDR in cell types that make up hierarchically orga-
nized tissues.
Although the detailed mechanisms that account for the
cell type-specific activation of DDR are still elusive, our results
identify ASPP1 as one endogenous factor that accounts for the
high susceptibility of HSCs/MPPs to p53-dependent apoptosis.
ASPP1, a p53-interacting protein stimulating p53 apoptotic
potential (Samuels-Lev et al., 2001), was expressed at higher
levels in HSC/MPP fraction, and upon its silencing, only HSC/
MPP, but not Progenitor cells, were rescued from IR-induced
apoptosis. Although the BM microenvironment may impact
HSC function after DNA damage (Wouters et al., 2007),
the HSC/MPP subset still showed increased susceptibility to
IR-induced apoptosis after in vivo IR. Thus, our studies offer
a first step in the exploration of extrinsic and intrinsic factors
that might dictate the relative apoptotic sensitivity of different
cells in a tissue.
Our findings that p53 and Bcl-2 are important mediators of
IR-induced apoptosis in primitive human hematopoietic cells
extends prior observations made in mice (Domen et al., 1998;
Lotem and Sachs, 1993; Wu et al., 2005). Interestingly, despite
effective abrogation of apoptosis by p53KD and Bcl-2 overex-
pression measured at early time points, immediate clonogenic
survival still decreased by 80%, indicating that some CFCs either
died or became senescent at a later time through p53- and Bcl-
2-independent mechanisms. By contrast, in two independent
assays for primitive cells (long-term in vitro cultures and serial
repopulation of NOD/SCID mice), irradiated cells with inactive
p53 or overexpression of Bcl-2 regenerated myelopoiesis or
repopulated mice. These results demonstrate that not only do
HSCs and progenitors differ in apoptosis induction, they also
differ in the extent to which the p53/Bcl-2 pathway is involved.
After p53 inactivation and IR, the higher degree of relative HSC
protection compared to more mature CFC (5.7- and 1.7-fold,
respectively), combined with their ability to repopulate primary

194 Cell Stem Cell 7, 186–197, August 6, 2010 ª2010 Elsevier Inc.
Cell Stem Cell
DNA Damage Response in Hematopoietic Stem Cells
recipients, argues that HSCs utilize this pathway to a greater
extent than progenitors.
Importantly, neither p53 disruption nor Bcl-2 overexpression
rescued long-term HSC function to the nonirradiated level, sug-
gesting that additional DDR alterations may contribute to the
increased radiosensitivity of HSCs. Survival after IR exposure
is the integrated result of multiple p53/Bcl-2-dependent and
-independent factors, including the extent and quality of DNA
repair, the initiation and release of cell cycle checkpoints, and
the threshold for triggering apoptosis. The delays in DSB rejoin-
ing and persistent gH2AX foci we observe in human HSCs/
MMPs may be among those p53/Bcl-2-independent DDR alter-
ations that help determine HSC radiosensitivity. Such alterations
may be part of an overall HSC strategy that emphasizes accu-
racy of DNA repair over efficiency, with HSCs harboring unre-
paired damage eliminated by p53-mediated apoptosis.
One implication of the distinct effects that p53 and Bcl-2
exhibit in serially transplanted mice, despite providing similar
protection from apoptosis, is that p53 has both a positive and
negative roles in the regulation of HSC function under both
homeostatic and stress conditions. When p53 is disabled, the
balance between these roles collectively results in a net negative
effect on HSC self-renewal. As seen in the primary transplanted
mice, p53 inactivation decreases apoptosis sensitivity and
increases HSC frequency and migration—each is a positive
component to HSC function and self-renewal. However,
disabling p53 comes at a price for the HSC self-renewal. It is
possible that increased gH2AX foci numbers in secondary grafts
of p53KD cells reflect the engagement of the complex network of
DDR proteins that reduce HSC self-renewal. Therefore, the net
consequence of these numerous (and sometimes antagonistic)
functions of intact p53 is reflected in self-renewal capacity.
The genetic bypass of p53-dependent apoptosis by Bcl-2 over-
expression established that the effect of p53 on genome integ-
rity, and thus on HSC self-renewal, was apoptosis independent.
Our results may argue that p53 apoptosis-independent
mechanisms become activated to prevent damaged HSCs
from self-renewing.
Because transplantation itself is a nonphysiological situation
for HSCs, our findings point to a role for the p53/Bcl-2 pathway
in transplant-mediated cellular stress. Although the nature of
such stress is ill defined, the accumulation of spontaneous
gH2AX in hematopoietic progenitors provides an important
novel marker for the study of this type of stress. The opposing
roles of p53 inactivation and Bcl-2 overexpression on the rate
of gH2AX foci accumulation, which became evident only after
a second round of transplantation, provide for the first time
insight into dynamics, stability, and mechanisms that control
the human HSC genome upon BM transplantation, albeit in
a xenotransplant setting.
The findings presented here establish the importance of
experimentation with primary human cells to extend studies on
p53-null mice. Although resistant to IR-induced apoptosis,
mouse p53-deficient hematopoietic cells become immortalized
in vitro (Palacios et al., 1996) and undergo leukemic transforma-
tion beginning at 10 weeks posttransplantation (Marusyk et al.,
2010; Yoshida et al., 2002). By contrast, we saw no evidence
of immortalization in long-term cultures and none of the 46 sec-
ondary recipients transplanted with p53-inactivated cells devel-
oped hematological malignancies (irrespective of IR exposure)
during 14–18 weeks of observation. Collectively, these results
underscore the differences in tumor suppressor mechanisms
between species. Moreover, studies that focus on modulation
of p53 and Bcl-2 in primary human cells may be more directly
relevant for potential therapeutic manipulation to enhance HSC
expansion and transplantation potential without promoting
transformation.
EXPERIMENTAL PROCEDURES
Sample Collection and Purification
Lineage depletion and CD34+ enrichment of cord blood (CB) samples were
achieved by negative selection with the StemSep system according to the
manufacturer’s protocol (Stem Cell Technologies, Canada). Sorting experi-
ments and antibodies are described in detail in Supplemental Experimental
Procedures.
Immunofluorescence and Neutral Comet Assay
Cells were labeled with anti-gH2AX (1:3000, Upstate, MA) and secondary
antibodies.
The number of gH2AX foci as well as ‘‘Olive Tail Moments’’ were estimated
as detailed in Supplemental Experimental Procedures.
Viral Constructs and Transduction
Full-length human Bcl-2 cDNA (kindly provided by Dr. L. Penn, Ontario Cancer
Institute, Toronto, Canada) and dominant-negative p53 peptide GSE56 cDNA
(Ossovskaya et al., 1996) were subcloned into bidirectional lentiviral construct
MA1.
p53 and ASPP1 shRNAs were designed with the Dharmacon algorithm
(http://www.dharmacon.com). 19 nucleotide sense siRNA sequences are
detailed in Supplemental Experimental Procedures.
Viral particles pseudotyped with VSV-G were generated by transient trans-
fection and titrated on HeLa cells as described (Mazurier et al., 2004).
Thawed Lin! CB cells were incubated with lentivirus, at multiplicity of infec-
tion of 50–100, in X-VIVO 10 (BioWhittaker, Waldersville, MD) medium supple-
mented with 1% BSA and SCF (100 ng/ml), FLT3L (100 ng/ml), TPO (15 ng/ml),
G-CSF (10 ng/ml), and IL-6 (10 ng/ml) for 16 hr. The cells were washed and
resuspended in the new transduction medium for additional 60–72 hr. At the
end of gene transduction procedure, cells were sorted for EGFP-expressing
cells, irradiated (if required) on ice with the indicated dose, and used for
experiments.
Ex Vivo Experiments
Annexin V Apoptosis Detection Kit (BD PharMingen) and SYTOX Blue Dead
Cell Stain (Invitrogen) were used according to manufacturer’s protocols.
For long-term cultures, Lin-CB cells sorted after transduction were seeded
in IMDM, 10% FCS (Sigma) supplemented with FLT3L (50 ng/ml), TPO (20 ng/
ml), SCF (50 ng/ml), and IL-6 (10 ng/ml) at the density of 1 3 105 cells/ml as
described in Piacibello et al. (2002). Every week cells were counted, washed,
and resuspended at the density of 1 3 105 cells/ml in fresh medium and growth
factors. Harvested cells were also used to assay clonogenic cell content in
methylcellulose (Guenechea et al., 2001) and for immunophenotype by flow
cytometry.
NOD/SCID Mouse Repopulation
All animal experimental protocols were approved by the Animal Care
Committee of the University Health Network (Toronto, Canada). Cells were
transplanted intrafemorally (IF) as described previously (McKenzie et al.,
2007) into 8- to 10-week-old NOD/LtSz-Prkdcscid (NOD-SCID) mice which
were irradiated (2.5 Gy) and injected intraperitoneally with 200 mg of anti-
CD122 antibodies 24 hr before transplantation.
Supplemental Experimental Procedures contains cell doses injected,
human engraftment flow cytometry analysis on FACScalibur or LSR II cytom-
eters (Becton Dickinson) and antibodies used, lineage depletion of human cells
from engrafted mice, and secondary transplantation procedure.
Cell Stem Cell 7, 186–197, August 6, 2010 ª2010 Elsevier Inc. 195

For in vivo IR experiments, preconditioned NOD/SCID mice were trans-
planted with 50,000 Lin! CB cells. 5–10 weeks after transplantation, the cohort
of the recipients was irradiated with 3 Gy and sacrificed in 1.5 hr. Lineage-posi-
tive cell depletion from human hematopoietic engraftment was performed and
cells incubated in IMDM, 10% FCS (Sigma) supplemented with FLT3L (50 ng/
ml), TPO (20 ng/ml), SCF (50 ng/ml), and IL-6 (10 ng/ml) for additional 18 hr
followed by staining of CD34, CD38, Annexin, and CYTOX Blue for flow cytom-
etry analysis.
Quantitative RT-PCR and Microarrays
RNA was extracted with the Trizol reagent (Invitrogen) and reverse transcribed
with SuperScript III (Invitrogen). Real-time PCR reactions were prepared with
SYBR Green PCR Master Mix (Applied Biosystems) in triplicates and analyzed
on Applied Biosystems 7900HT instruments. Absolute gene expression was
quantified with SDS software (Applied Biosystems) based on the standard
curve method and presented after normalization for GAPDH. Supplemental
Experimental Procedures contain list of primers. RNA from 10,000 or more
sorted cells was used for microarray analysis by Human HT-12 Expression
Beadchip (Illumina). Data were preprocessed and normalized with the Biocon-
ductor package lumi, which included log2 transformation.
Statistical Analysis
The significance of differences among groups was determined by Student’s
t test and Mann-Whitney test (SigmaStat software; Jandel).
ACCESSION NUMBERS
The microarray data are available in the Gene Expression Omnibus (GEO)
database (http://www.ncbi.nlm.nih.gov/gds) under the accession number
GSE21830.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and three tables and can be found with this article online at
doi:10.1016/j.stem.2010.05.016.
ACKNOWLEDGMENTS
We thank S. Harding and Dr. R.G. Bristow (Princess Margaret Hospital,
Toronto, Ontario) for helping with immunofluorescent imaging; P. Scheufler,
P. Savage, and the obstetrics unit of Trillium Hospital (Mississauga, Ontario)
for providing cord blood samples and co-op students for processing cord
blood samples; S. Zhao, L. Jamieson, and P.A. Penttilä (Hospital for Sick Chil-
dren, Toronto, Ontario) for sorting; and the J.E.D. lab members for critical
review of the manuscript. This work was supported by postdoctoral fellow-
ships from the European Molecular Biology Organization (EMBO) and Euro-
pean Hematology Association and Canadian Institutes for Health Research
(CIHR) (M.M.) and The Netherland Society for Scientific Research (NOW)
(K.G.H.) and grants from The Stem Cell Network of National Centers of Excel-
lence, Canadian Cancer Society Research Institute, Terry Fox Foundation,
Genome Canada through the Ontario Genomics Institute, Ontario Institute
for Cancer Research, Premier’s Summit Award funded from the province of
Ontario, Leukemia and Lymphoma Society, and Canadian Institutes for Health
Research, and a Canada Research Chair. This research was funded in part by
the Ontario Ministry of Health and Long Term Care (OMOHLTC). The views ex-
pressed do not necessarily reflect those of the OMOHLTC.
Received: November 18, 2009
Revised: March 24, 2010
Accepted: May 14, 2010
Published online: July 8, 2010
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