c-Myc controls the balance between
hematopoietic stem cell self-renewal
Anne Wilson,2,4Mark J. Murphy,1,4Thordur Oskarsson,1Konstantinos Kaloulis,1
Michael D. Bettess,1Gabriela M. Oser,1Anne-Catherine Pasche,1Christian Knabenhans,3
H. Robson MacDonald,2and Andreas Trumpp1,5
1Genetics and Stem Cell Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges,
Switzerland;2Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland;
3School of Life Sciences, Swiss Federal Institute of Technology (EPFL), 1015 Lausanne, Switzerland
The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue
turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while
concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function
of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc
activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs
self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired
differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM
niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors,
suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc
expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of
differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term
HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and
differentiation, presumably by regulating the interaction between HSCs and their niche.
[Keywords: Hematopoietic stem cell (HSC); stem cell niche; self-renewal; c-Myc; N-cadherin]
Received June 14, 2004; revised version accepted September 13, 2004.
Many adult tissues, including skin epidermis, gastroin-
testinal epithelia, or the hematopoietic system, are re-
generative and self-renewing. In such tissues, mature
cells in the stratum corneum of the skin, differentiated
enterocytes at the tip of the intestinal villi, or blood
erythrocytes have limited lifespans, and must be con-
tinuously replaced to replenish those steadily lost by
shedding or apoptosis (Watt and Hogan 2000; Fuchs et al.
2004). Life-long production of differentiated progeny re-
lies on rare long-lived adult tissue stem cells (TSC) that
have the ability to both perpetuate themselves through
self-renewal and to generate all mature cell types of a
particular tissue through differentiation (Weissman
2000). Probably the most well-characterized adult stem
cell is the hematopoietic stem cell (HSC), which has the
clonal capacity to provide life-long reconstitution of all
hematopoietic lineages after transplantation into le-
thally irradiated mice (Till and McCulloch 1961; Cantor
and Orkin 2001; Kondo et al. 2003). Using various pro-
tocols, murine HSCs can be purified close to homogene-
ity by fluorescent-activated cell sorting (FACS) (Smith et
al. 1991; Goodell et al. 1996). All functional repopulating
HSCs are contained within the c-Kit+lin−Sca-1+(KLS-
HSC) bone marrow (BM) population (Uchida and Weiss-
man 1992; Weissman et al. 2001). Under steady-state
conditions, the total number of HSCs is kept constant,
whereas differentiated progeny are continuously gener-
ated to replace mature cells. However, in response to
injury (e.g., blood loss), disease (e.g., anemia, cancer), or
BM stress (e.g., myelotoxic chemotherapy), total stem
cell number can vary significantly, indicating that the
balance between stem cell self-renewal and differentia-
tion can adapt to physiological needs (Morrison et al.
1997; Uchida et al. 1997; Watt and Hogan 2000; Weiss-
man 2000). This flexibility is most likely achieved by
reciprocal intercellular interactions between stem cells
and their specific microenvironment called the niche.
The stem cell niche is defined as a subset of tissue cells
and extracellular substrates that can harbor one or more
stem cells controlling their self-renewal and progeny
production in vivo (Trentin 1970; Schofield 1978; Watt
and Hogan 2000; Spradling et al. 2001; Lin 2002; Fuchs et
4These authors contributed equally to this work.
E-MAIL email@example.com; FAX 41-21-6526933.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
GENES & DEVELOPMENT 18:2747–2763 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org2747
al. 2004). Retention of stem cells in the niche is thought
to be accomplished by stem cell niche and stem-cell ex-
tracellular matrix (ECM)-ligand interactions. It has been
shown in the Drosophila ovary that DE-cadherin-medi-
ated anchoring of germ line and somatic stem cells to the
niche is essential for their maintenance (Song and Xie
2002; Song et al. 2002). Putative niches have also been
identified in vertebrates, including the bulge region in
the skin epidermis and the stem cell-bearing base of in-
testinal crypts (Cotsarelis et al. 1990; Potten and Loeffler
1990; Rochat et al. 1994). In the BM, HSCs are located at
the endosteal lining of the BM cavities, and recent stud-
ies show that specialized spindle-shaped N-cadherin+os-
teoblasts (SNO) are a key component of the BM stem cell
niche. HSCs are thought to be anchored to SNO cells via
a homotypic N-cadherin interaction (Whetton and Gra-
ham 1999; Nilsson et al. 2001; Visnjic et al. 2001; Calvi
et al. 2003; Zhang et al. 2003; Zhu and Emerson 2004).
However, the molecules that regulate stem cell niche
interactions and how these may influence the balance
between self-renewal and differentiation are currently
The proto-oncogene c-myc encodes an unstable nu-
clear factor c-Myc, which has been implicated in regula-
tion of a wide variety of biological processes, including
control of division, apoptosis, cellular growth, angiogen-
esis, and differentiation (Amati et al. 1998; Schuhmacher
et al. 1999; Grandori et al. 2000; Jain et al. 2002; Pelen-
garis et al. 2002b; Bellmeyer et al. 2003; Watnick et al.
2003). c-Myc dimerizes in the cell with the ubiquitous
protein Max, and Myc/Max heterodimers control two
large independent sets of target genes. One set of target
genes is activated by binding of Myc/Max to E-boxes in
target gene promoters, inducing recruitment of com-
plexes containing histone acetyl-transferase activity (Ei-
senman 2001; Frank et al. 2001). The second set of target
genes is repressed by binding of Myc/Max to the tran-
scriptional activator Miz-1 that binds to INR elements at
the transcriptional start site of target genes (Staller et al.
2001; Wanzel et al. 2003). Various approaches have led to
the identification of a large number of putative Myc tar-
get genes (http://www.myc-cancer-gene.org/index.asp).
We and others have previously shown that deletion of
c-myc by gene targeting in mice causes midgestation le-
thality, and mutant embryos fail to develop a primitive
hematopoietic system (Trumpp et al. 2001; Baudino et
al. 2002). Here, we have utilized conditional gene target-
ing, as well as a gain-of-function approach, to investigate
the role of c-Myc during adult hematopoiesis. Our data
indicate that c-Myc, in addition to being essential for
proliferation of lineage-committed hematopoietic cells
in vivo, plays an unexpected role in controlling the bal-
ance between HSC self-renewal and differentiation.
Loss of c-Myc during adult hematopoiesis
causes severe cytopenia
Previous studies have shown that c-Myc is required for
primitive hematopoiesis (Trumpp et al. 2001). To study
the function of c-Myc during maintenance of adult hema-
topoiesis, mice homozygous for the conditional c-mycflox
allele were crossed with mice harboring the IFN?-in-
ducible Mx–Cre transgene (Fig. 1A; Kuhn et al. 1995;
Trumpp et al. 2001) to produce MxCre;c-mycflox/floxmice
(mutant) and their MxCre-negative littermate controls
(control). Postnatal induction of Mx–Cre after poly-I–
poly-C (pI–pC) treatment results in close to 100% deletion
of alleles flanked by loxP sites in liver, spleen, and BM
(Kuhn et al. 1995; Radtke et al. 1999). Conversion of the
c-mycfloxinto the c-myc?ORFrecallele, and therefore, dele-
tion of the coding region, was monitored by real-time
Taqman PCR and found to occur in 93% ± 6% of purified
c-Kit+, linneg, Sca-1+cells (KLS-HSCs). In parallel, c-myc
transcription was decreased in mutant KLS-HSCs and to-
tal BM 13- and 22-fold, respectively. The Myc family mem-
ber L-myc was not expressed in purified KLS-HSCs from
normal or mutant mice (data not shown). N-myc expres-
sion has been reported to be expressed in embryos, embry-
onic stem cells, as well as in fetal and adult BM HSCs
(Ivanova et al. 2002; Ramalho-Santos et al. 2002; Sperger et
al. 2003). In agreement with these reports, N-myc tran-
scripts were detectable in control and mutant-purified
KLS-HSCs. The N-myc expression level was more than
100-fold below that detected in E10.5 embryos used as
positive controls (data not shown). Importantly, no signifi-
cant compensatory up-regulation of N-myc transcripts
was detected in c-Myc-deficient KLS-HSCs by real-time
PCR (data not shown) or in KLSF-HSCs by Affymetrix mi-
croarray analysis (W. Blanco, M.J. Murphy, and A. Trumpp,
By 4 wk post-deletion, MxCre;c-mycflox/floxmice de-
velop acute anemia as apparent by their white feet and
ears and a hemoglobin concentration in peripheral blood
(PBL) as low as 1.6 g/dL compared with 14 g/dL in con-
trols (Fig. 1B; data not shown). Analysis of PBL subsets
shows that hematopoietic cell lineages are decreased
four- to sevenfold (data not shown). All hematopoietic
cell types normally present in the blood are generated
from precursors located principally in the BM, spleen,
and thymus. Analysis 8 wk post-c-myc-deletion shows
that all three organs are greatly reduced in absolute cell
number, particularly the thymus and BM, where the re-
duction is 50- and 20-fold, respectively (Fig. 1C). Because
the BM is the primary site of adult hematopoiesis, the
cellular composition of this organ was analyzed further.
The long bones of mutants appear white (Fig. 1B), and
kinetic analysis of BM cellularity demonstrates a con-
tinuous loss down to ?5% of control BM numbers by
8–9 wk post-deletion of c-myc (Fig. 1D). To determine
whether all BM cell lineages are affected, four-color
FACS analysis was performed. Eight weeks post-dele-
tion, differentiating and mature lineage-positive (linpos)
BM cells are decreased >30-fold (Fig. 1E). This decrease is
observed in all mature subsets tested (including B lym-
phocytes (B220), granulocytes (Gr-1), macrophages (Mac-
1), and erythroblasts (TER-119) (Fig. 1E).
As c-Myc has been shown to be rate limiting for pro-
liferation of fibroblasts and peripheral T cells (Trumpp et
al. 2001), the cell cycle status of committed cell types in
Wilson et al.
2748GENES & DEVELOPMENT
the BM (linposcells) was quantitated. Surface staining to
define subsets was combined with intracellular staining
for DNA content (Hoechst 33342) and the proliferation
marker Ki67, which is expressed in all actively dividing
cells, but not in resting (G0) cells (Brown and Gatter
2002). Whereas 95% of control linposcells are found in
the active phases of the cell cycle (G1+ S + G2/M), most
of the few remaining c-Myc-deficient linposcells accu-
mulate in G0(Fig. 1F). These results suggest that c-Myc
activity is required to maintain proliferation of lineage-
committed cell types present in the BM, and provide an
initial explanation for the severe cytopenia observed in
response to loss of c-Myc.
Phenotypic LT-HSCs accumulate
in c-Myc-deficient bone marrow
In contrast to the decrease in linposcells, lineage-nega-
tive (linneg) precursors defined by their lack of surface
expression of lineage markers (CD3, CD4, CD8, B220,
Mac-1, Gr-1, Ter119, and NK1.1) are increased around
threefold in the mutants 3 wk after c-myc deletion (Fig.
2A). In addition to early committed precursors of all he-
matopoietic lineages, the linnegpopulation also contains
the c-Kit+(CD117) Sca-1+subpopulation highly enriched
for hematopoietic stem cells (KLS-HSC) (Uchida and
Weissman 1992; Weissman et al. 2001). Unexpectedly,
c-Myc-deficient BM is enriched >20-fold in KLS-HSCs
compared with control BM (Fig. 2B). Due to the net de-
crease in BM cells, this enrichment corresponds to a two-
to threefold increase in the absolute number of KLS-
HSCs (Fig. 2E). As it has recently been shown that func-
tional LT-HSC activity is exclusively contained within
the Flk2/Flt3Rneg(CD135neg) subset of the KLS popula-
tion (c-Kit+linnegSca1+Flk2neg(KLSF) (Adolfsson et al.
2001; Christensen and Weissman 2001), the proportion
of KLS cells expressing this receptor was determined. As
shown in Figure 2C, the relative ratio of Flk2neg/Flk2+
KLS cells increases from 1:1 (50%/50%) to >7:1 (88%/
12%) in the c-Myc-deficient KLS population compared
with controls. In addition, a larger percentage of c-Myc-
deficient KLS-HSCs express low levels of CD90 (Thy1),
further confirming their primitive undifferentiated char-
acter (Fig. 2D; Christensen and Weissman 2001). Quan-
titation of KLSF cells 3 wk post-deletion reveals a four-
fold net increase compared with controls (Fig. 2F). These
results show that cells with a LT-HSC phenotype accu-
mulate, whereas committed progenitors and differenti-
ated cell types are lost after elimination of c-Myc activ-
ity in the BM.
Proliferation and survival of HSCs
in vivo is c-Myc independent
To determine the mechanism by which c-Myc-deficient
KLSF-HSCs accumulate in the BM, the effect of c-Myc
loss on proliferation and survival of KLS-HSCs was de-
termined. c-Myc is known to play an important role in
the control of proliferation and survival of various cell
entiated progenitors. (A) MxCre-mediated conversion of the c-mycfloxallele into the c-myc?ORFrecallele by deletion of DNA between
the two loxP sites in the c-mycfloxlocus. This includes the entire c-myc ORF (yellow). Exons 1–3 are indicated. (Red triangles) loxP
sites. Cre expression is induced by INF? or pI–pC. (B) Hind-paws (top) and femurs (bottom) of control (left) and MxCre;c-mycflox/flox
(right) mice 8 wk after pI–pC injection. (C) Cellularity of control (blue) and mutant (red) lymphoid organs 8 wk after deletion. (BM) Bone
marrow; (Thy) thymus; (Spl) spleen. Results are mean ± SD from 12 (control) and 15 (mutant) mice. (D) Kinetics of BM cellularity from
3 to 9 wk after c-myc deletion. (E) Quantitation of linpos(left) BM cells and BM subsets (right) in controls (blue) and mutants at 3 wk
(red) or 8 wk (orange) post-deletion. Between three and 12 mice were analyzed at each time point. (F) Cell cycle analysis of linposBM
cells. Total BM was surface stained to define the linpossubset, then fixed, permeabilized, and stained with Hoechst 33342 and Ki-67
(icKi67) for FACS analysis. Different cell cycle phases are indicated on the left.
Induced deletion of c-myc in the adult bone marrow results in severe cytopenia and a decrease in proliferation of differ-
c-Myc function in hematopoietic stem cells
GENES & DEVELOPMENT2749
types (Grandori et al. 2000; Trumpp et al. 2001; Pelen-
garis et al. 2002a). Either process, if altered, could pro-
vide a possible explanation for the accumulation of
HSCs in c-Myc-deficient BM. However, the apoptotic
rate of c-Myc-deficient KLS-HSCs (as determined by An-
nexin V/7AAD staining) is unchanged in mutant com-
pared with control cells, further supporting the sugges-
tion that the observed accumulation is not due to in-
creased survival of c-Myc-deficient KLS-HSCs (data not
shown). In contrast to linposBM cells that require c-Myc
for proliferation (Fig. 1F), no change in the cell cycle
profile as analyzed by DNA content is observed in mu-
tant compared with control KLS-HSCs. In addition, the
size of the quiescent (G0) pool in the KLS-HSC popula-
tion as determined by the absence of expression of the
proliferation marker Ki67 was unchanged (Ki67neg2n
DNA) (Fig. 2G). To determine the turnover rate of KLSF-
HSCs in vivo, the kinetics (2 h, 15 h, 3 d, 5 d) of BrdU
uptake was determined by five-color flow cytometry. No
significant difference could be detected at any time
point, suggesting that normal and mutant KLSF-HSCs
have a very similar turnover rate in vivo (Fig. 2H). Taken
together, these data show that the accumulation of c-
Myc-deficient HSCs cannot be explained by increased
proliferation or survival. It is interesting to note that
HSCs proliferate in a c-Myc-independent manner in
vivo. It is possible that HSCs, like ES-cells, may lack a
functional G1–S checkpoint, and thus, would not require
Myc activity (Burdon et al. 2002). Alternatively, this
could simply be due to the fact that N-Myc is still ex-
pressed in mutant HSCs. This issue needs to be ad-
dressed by a conditional c-Myc/N-Myc double knockout.
(blue) and mutant BM either 3 wk (red) or 8 wk (orange) post-deletion. Results are mean ± SD of 12 (control) or six (mutant) mice. (B)
BM was stained for lineage markers, gated on the linnegsubset, and further stained for c-Kit (CD117) and Sca-1. Numbers on the plot
are the frequency of cells in the indicated regions. Hematopoietic stem cells are contained within the c-Kit+linnegSca-1+(KLS cells)
population. Control (left) and mutant (right). An unusual linneg, c-Kitlow, Sca-1lowpopulation (which is also CD45+, IL-7Rneg, CD135neg
and expressing high levels of the integrins ?2, ?4, ?1, and ?2) appears in the mutants (data not shown). Despite extensive analysis, this
population does not correspond to any characterized cell type. (C) c-Kit (CD117) vs. CD135 (Flk2/Flt3R) expression on linnegSca-1+BM.
Numbers on the plot are the frequency of cells in the indicated (c-Kit+linnegSca-1+) regions. Control (left) and mutant (right). (D)
CD117 vs. CD90 (Thy1) expression linnegSca-1+BM. (E) Total number of KLS cells per two femurs 3, 4, and 5 wk post-c-myc-deletion
in control (blue) and mutant (red) BM. Results are mean ± SD of five mice per time point. (F) Number of KLSF (KLS Flk2−cells) 3 wk
post-deletion. Results are mean ± SD of three mice each. (G) Cell cycle analysis of KLS cells. Cells were stained with Hoechst 33342
and Ki67 (icKi67) and analyzed by FACS. The different cell cycle phases are indicated in the scheme at the left. Numbers are the
proportion of cells in each phase. (H) In vivo BrdU-labeling kinetics of KLSF-HSCs isolated from control (blue) and mutant (red) mice
3–4 wk post-deletion of c-myc. Results are mean ± SD from three mice of each genotype per time point.
Accumulation of hematopoietic stem cells (HSC) in c-Myc-deficient BM. (A) Quantitation of the linnegpopulation in control
Wilson et al.
2750GENES & DEVELOPMENT
BM devoid of c-Myc activity reconstitutes HSCs,
but not more mature hematopoietic lineages
Mice lacking c-Myc become cytopenic and accumulate
cells with an HSC phenotype in the BM. Because prolif-
eration and survival are unchanged in mutant HSCs, an
alternative explanation for their observed accumulation
may be a defect in HSC differentiation. To directly test
this possibility, c-Myc-deficient HSCs were analyzed for
their reconstitution capacity in vivo. Groups of four le-
thally irradiated mice were injected with a range of doses
of total BM isolated from c-Myc-deficient or control ani-
mals. While as few as 0.63 × 106control BM cells were
able to fully reconstitute 100% of irradiated hosts, no
survivors were observed with doses of up to 2.5 × 106
c-Myc-deficient BM (data not shown). This inability to
reconstitute suggests that c-Myc is required for either
HSC function or progenitor expansion, or both. To dis-
tinguish between these possibilities and to eliminate any
contribution from a mutant BM microenvironment,
competitive mixed BM chimeras, in which the fate of
mutant HSCs could be followed in mice harboring a
functional hematopoietic system with a wild-type BM
microenvironment were prepared. Lethally irradiated
CD45.1+mice were transplanted with a mixture of
CD45.1+wild-type BM, together with either mutant or
control CD45.2+BM. Analysis of chimeric mice 1–4 mo
after transplantation revealed very poor contribution of
c-Myc-deficient donor cells in peripheral organs (Fig. 3A;
data not shown). However, while control donor HSCs
generated normal proportions of stem and progenitor cell
types, phenotypic LT-HSCs (KLS CD4negCD11bneg), ST-
HSCs (KLS CD4negCD11blow); and MPPs (KLS, CD4low
CD11blow) of c-Myc-deficient origin accumulate (Fig.
3B,C). Quantitation of these precursor populations in
mixed chimeras shows an ∼20-fold increase in LT-HSCs
in c-Myc-deficient BM compared with control. After nor-
malization of input KLS cells (twofold increase in mu-
tant compared with control at 3 wk post-deletion, Fig.
2E), the net increase in donor derived c-Myc-deficient
LT-HSCs after reconstitution remains elevated by a fac-
tor of 10 (Fig. 3C). Both ST-HSCs and MPPs are also
increased, but to a lesser extent (Fig. 3C). In contrast,
CMPs (c-KithiSca-1negCD34+and CD16/32+) and CLPs
(c-KitlowSca-1+CD127+), as well as differentiated cell
types such as granulocytes and B cells of mutant origin,
are virtually absent (Fig. 3A,C; Kondo et al. 1997; Akashi
et al. 2000). These results directly show that transferred
c-Myc-deficient BM cells are unable to reconstitute early
committed progenitors or mature hematopoietic lin-
eages, whereas at the same time, HSCs with a predomi-
nant LT-HSC phenotype accumulate in situ.
This failure to generate expanding progenitors is simi-
lar to what is observed in MxCre;c-mycflox/floxmutants;
however, the accumulation of HSCs is significantly
more pronounced in the mixed chimeras. Most impor-
tantly, in the latter situation, mutant HSCs specifically
accumulate in a wild-type mouse, which develops a nor-
mal hematopoietic system and carries a normal stromal
microenvironment. Together with the finding that KLS-
HSC proliferation and survival are c-Myc independent
(Fig. 2G,H), this provides compelling evidence that the
failure to generate early progenitors is due to the inabil-
ity of mutant HSCs to differentiate. Instead, they accu-
mulate, suggesting that the balance in these cells is
shifted toward self-renewal at the expense of differentia-
tion (Fig. 7A, left, see below). Thus, c-Myc activity is
essential to maintain functional HSCs in vivo.
c-Myc-deficient HSCs can differentiate ex vivo
The balance between self-renewal and differentiation of
stem cells is thought to be controlled by interaction of
stem cells with their niches (Watt and Hogan 2000; Sprad-
ling et al. 2001; Fuchs et al. 2004). To test whether the
effect of c-Myc on HSC differentiation in vivo is direct or
is dependent on their location in the BM microenviron-
ment/niche, KLS-HSCs were isolated and bulk cultured
in vitro in the presence of a differentiation permitting
cytokine cocktail containing IL-3, IL-11, SCF, TPO, Flt-
3L, GM-CSF, EPO (Akashi et al. 2000). After 1 wk, cells
were harvested and analyzed for the appearance of linpos
cells (Fig. 3D). In control cultures, differentiation into
several different lineages is accompanied by a >100-fold
increase in cell number (50,000 → 5,000,000). In con-
trast, only minimal or no expansion occurred in c-Myc-
deficient cultures, consistent with the fact that c-Myc is
required for proliferation of differentiating transient-am-
plifying (TA) cells. However, despite this observed lack
of expansion, morphologically differentiated cells ex-
pressing myeloid and lymphoid markers such as Gr-1
and CD11b or B220 and CD19, were present in c-Myc-
deficient cultures (as in control cultures), indicating that
c-Myc-deficient HSCs can differentiate in vitro in con-
trast to what is observed in vivo (Fig. 3D). To rule out
that c-Myc-deficient cells were overgrown with rare cells
that escaped Cre-mediated deletion, cultured cells were
genotyped by PCR and found to be c-myc?ORFrec/?ORFrec
(Fig. 3E). Interestingly, single-cell sorted c-Myc-deficient
KLS-HSCs fail to divide, suggesting that c-Myc is essen-
tial for division in vitro, whereas HSCs in the BM pro-
liferate in a c-Myc-independent manner (M.J. Murphy
and A. Trumpp, unpubl.).
To show that mutant KLS-HSCs can be rescued by
ectopic expression of c-MYC, mutant, and control, KLS-
HSCs were sorted and infected with a mouse stem cell
virus (MSCV) expressing human c-MYC in conjunction
with a huCD2 reporter gene (MYC–IRES–huCD2) or
with a control virus expressing only huCD2 (Deftos et al.
1998). After culturing the transduced KLS-cells for 9 d as
described above, cells were counted and differentiation
was determined by expression of the lineage markers
Ter119, Gr1/CD11b, and CD19. Whereas the differentia-
tion potential of c-Myc-deficient HSCs was similar in
the presence or absence of ectopic c-MYC (Fig. 3F), mu-
tant cultures expressing c-MYC expanded about 20
times compared with those infected with the control vi-
rus (Fig. 3F). This result shows that the failure of mutant
c-Myc function in hematopoietic stem cells
GENES & DEVELOPMENT 2751
KLS-cells to proliferate in vitro can be rescued by ectopic
c-MYC. In addition, these experiments show that mu-
tant KLS-HSCs are not an abnormal population of cells
expressing stem cell markers by chance. Most impor-
tantly, the ability of c-Myc-deficient HSCs to undergo
multilineage differentiation (but not expansion) in vitro
suggests that the defect in vivo (no normal differentia-
tion, but proliferation/self-renewal) is indirect and po-
tentially due to a misregulated interaction with the local
c-Myc-deficient HSCs home to the stem
cell niche at the BM endosteum
BM stem cell niches have recently been shown to be part
of the endosteal lining of the internal bone cavities (Nils-
son et al. 2001; Askenasy et al. 2002). In addition, it has
recently been demonstrated that osteoblastic cells are a
key component of the BM niche (Calvi et al. 2003; Zhang
et al. 2003). One explanation for the dichotomy in dif-
ferentiation capacity of c-Myc-deficient HSCs observed
Myc-deficient HSCs in vivo or in vitro. (A)
The development of HSCs in vivo was de-
termined by injecting control or mutant
CD45.2+BM cells into groups of lethally
irradiated wild-type CD45.1+host mice to-
gether with competing wild-type CD45.1+
BM cells. (Left) After 2 mo, splenocytes
were analyzed by flow cytometry for the
contribution of each donor population by
expression of CD45.1 and CD45.2. The
(granulocytes, Gr1+; and B lymphocytes,
B220+) derived from CD45.2+donor cells
(control, blue; and mutant, red) was quan-
titated. (Right) Results are mean ± SD
Sca-1/CD117 phenotype of LinnegCD45.2+
BM in control and mutant chimeras 2 mo
post-reconstitution. While the majority of
mutant cells show a KLS-HSC phenotype,
levels of c-Kit (see also Fig. 2B). (C)
Number of different HSC and progenitor
donor cells in the chimeras shown in A
and B, and defined as CD117+Sca1neg
man 2000). Mutant donor BM 3 wk post-
deletion contains twofold more KLS cells
compared with control BM (Fig. 2E); there-
fore, the net increase in HSCs is about half
that indicated. (D) Differentiation poten-
tial of KLS cells in vitro. FACS sorted KLS
cells from control (left) and mutant (right)
BM were isolated and grown in stem cell
medium containing a cytokine cocktail in-
cluding mSCF, mTPO, mFlt3L, IL-6, IL-7, Il-11, GM-CSF, and EPO. After 7 d, cultures were photographed using phase contrast. FACS
analysis of expression of lineage markers on bulk cultured KLS cells. (Top) Gr-1 (granulocytes) versus CD11b (macrophages). (Bottom)
B220 (B lymphocytes) (control, blue; mutant, red). Numbers on plots are the proportion of cells in indicated regions. (E) Genotyping
by PCR of cultured control (C) and mutant (M) cells from D after 7 d. Control cells were positive for the c-mycfloxallele (flox) and the
DNA control (18s), whereas the mutant (M) cells were negative for the c-mycfloxallele (flox), but positive for the c-myc?ORF/rec(?) allele
as expected. (F, left) In vitro differentiation of purified KLS cells. LinnegCD117+Sca1+BM cells were FACS sorted from control and
c-Myc-deficient mice and cultured for 9 d after transfection with either MYC–IRES–huCD2 or huCD2 control viruses. Expression of
mature hematopoietic cell markers (Gr1, CD11b, Ter119, and CD19) is shown on huCD2+cells in control (huCD2 alone virus in
control cells, dark-blue line), c-Myc-deficient cells transfected with the huCD2 control virus (solid red histogram), or c-Myc-deficient
cells transfected with the MYC–IRES–huCD2 virus (light-blue line). (Right) Expansion of c-Myc-deficient (KO) or control (C) KLS-HSC
cultures 9 d after infection with the huCD2 control virus or with the MYC–IRES–huCD2 virus (c-MYC). Data is expressed as fold
increase over input cell number. This is a representative example of one of two experiments giving similar results.
Differentiation potential of c-
Wilson et al.
2752 GENES & DEVELOPMENT
in vitro and in vivo, and for their increased self-renewal
and consequent accumulation in vivo, could be their re-
tention in the differentiation preventive microenviron-
ment of the niche. This hypothesis would require that
c-Myc-deficient stem cells be localized in the stem cell
niche in vivo. Thus, immature purified progenitors
(linnegcells) were labeled with the fluorescent dye CFSE
and injected i.v. into sublethally irradiated wild-type re-
cipients. HSCs present in this precursor population are
predicted to home to stem cell niches located at the BM
endosteum (Nilsson et al. 2001). Therefore, long bones
were isolated 15 h later, and CFSE+cells were localized
on decalcified trabecular bone sections using fluores-
cence microscopy. Bone sections were stained for osteo-
pontin, an extracellular matrix phosphoglycoprotein se-
creted by osteoblasts (Fig. 4A, panels 1,2) and BMPR1?
(Fig. 4A, panels 3,4), which is expressed on the surface of
osteoblasts (Denhardt and Guo 1993; ten Dijke et al.
1994; Zhang et al. 2003). As shown in Figure 4A, control
and mutant progenitor/stem cells can home to the end-
osteum of the trabecular bone and are in close proximity
or in direct contact with osteoblasts. Zhang et al. (2003)
has shown that a subset of spindle-shaped N-cadherin-
expressing osteoblasts (SNO) are an essential component
of the stem cell niche, and has further suggested that
N-cadherin-expressing HSCs are anchored to SNO via
N-cadherin-mediated homotypic interactions (Zhang et
al. 2003). To visualize SNO-cells and N-cadherin-ex-
pressing early progenitors present at the endosteum (pre-
sumably HSCs), trabecular bone sections were stained
for N-cadherin expression. As shown in Figure 4B, panels
7–15, c-Myc-deficient N-cadherin-expressing early pro-
genitors can home to the endosteum similar to control
progenitors (Fig. 4B, panels 1–6). Most mutant progeni-
tors at the endosteum are strongly stained for N-cad-
herin, while control cells are only weakly stained or not
at all, suggesting that N-cadherin expression is up-regu-
lated on stem cells in the absence of c-Myc. Importantly,
the majority of mutant N-cad+cells are in direct contact
with SNO cells (Fig. 4B, panels 9,12,15). While not quan-
titative, these data show that c-Myc-deficient early pro-
genitors are able to home to the BM endosteal stem cell
niche and are in direct contact with SNO niche cells
putatively via increased levels of N-cadherin.
Up-regulation of adhesion molecules on
Since our data indicate that c-Myc-deficient HSCs may
have a deregulated interaction with their BM stem cell
niche, surface expression of 31 adhesion molecules was
compared on control and mutant KLS-HSCs by four-
color FACS analysis (Table 1). Interestingly, both the
percentage of N-cad+cells, as well as the expression level
of N-cadherin increased significantly in mutant KLS-
HSCs compared with controls (Fig. 5A), supporting the
bone immunohistochemistry shown in Figure 4. In con-
trast, E-cadherin is not expressed on KLS cells, and only
a minor population (5%) express VE-cadherin at similar
levels in normal and mutant cells (Table 1). In addition
to N-cadherin, members of the integrin family of adhe-
sion receptors have also been implicated in homing and
mobilization of HSCs into and out of the BM stem cell
expressing bone marrow stem cell niche. Linnegprecur-
sors from control (Co) and c-Myc-deficient (Mu) BM
were stained with CFSE (green, *) and i.v. injected into
sub-lethally irradiated wild-type mice. (A) Trabecular
bone sections are stained with DAPI (blue) and anti-os-
teopontin (panels 1,2, red) or anti-BMPRI? (panels 3,4,
red) to visualize osteoblasts. White lines trace the edge
of the endosteum. Yellow arrowheads denote osteopon-
tin deposits. (BM) bone marrow (B, middle row) Trabecu-
lar bone sections are stained with anti-N-cadherin (red)
to visualize SNO-cells (spindle-shaped N-cadherin+os-
teoblasts) in the niche. (Bo) Bone; (En) endosteum. White
lines trace the edge of the endosteum. Arrowheads de-
note CFSE+cells (green). (Bottom row) Images were
merged using Adobe Photoshop software. (Panels 1–3)
Control precursors localized at the N-cadherin+endos-
teum. (Panels 4–6) Control cell with low expression of
N-cadherin localized in the center of the bone marrow.
(Panels 7–12) Two frames showing c-Myc-deficient pre-
cursor cells expressing high levels of N-cadherin local-
ized to the N-cadherin+endosteum. (Panels 12–15) Two
CFSE+mutant precursor cells attached to the N-cad-
herin+endosteum. Whereas the cell at the top expresses
N-cadherin only at low levels, the cell at the bottom
(panels 14,15, and inset in panel 15) expresses high levels
of N-cadherin and is in direct contact with a SNO-cell.
Localization of precursors in the N-cadherin-
c-Myc function in hematopoietic stem cells
GENES & DEVELOPMENT2753
niche (Whetton and Graham 1999). Leukocyte function
antigen 1 (LFA-1), a heterodimer consisting of the ?L-
integrin light chain (CD11a) and ?2-integrin heavy chain
(CD18) (Hogg et al. 2002) is consistently up-regulated
around threefold at both mRNA (data not shown) and
surface protein levels in the absence of c-Myc (Fig. 5B;
Table 1). In addition, c-Myc-deficient HSCs express
slightly increased levels of ?2-integrin (CD49b), ?5-inte-
grin (CD49e), and ?1-integrin (CD29) (Fig. 5B). As listed
in Table 1, many other adhesion molecules tested were
either not detected on the surface of HSCs or were un-
changed in the mutants. The greatly increased levels of
some adhesion molecules on HSCs lacking c-Myc activ-
ity is consistent with the hypothesis of deregulated in-
teraction of mutant HSCs with SNO niche cells, and
suggests that the mutants fail to differentiate due to
their inability to detach from the differentiation preven-
Deregulated overexpression of c-Myc in HSCs
causes repression of cell-adhesion molecules
and loss of self-renewal activity
Loss of c-Myc function results in failure of differentia-
tion, presumably by retention of HSCs in the BM niche
due to up-regulated expression of adhesion molecules.
Therefore, HSCs unable to down-regulate c-Myc should
increase differentiation at the expense of self-renewal ac-
companied by down-regulation of adhesion molecules.
To test this hypothesis, wild-type linnegprecursors were
infected with MYC–IRES–huCD2 or with a control virus
expressing only huCD2. After 4 d, cells were harvested
and surface expression of N-cadherin in the linnegSca-
1+huCD2+population was assessed. As predicted, N-cad-
herin levels were reduced almost to background levels in
c-MYC-transduced cells (Fig. 6A), showing that c-MYC
alone is sufficient to repress N-cadherin expression on
HSCs. Similarly, c-MYC overexpression also caused
down-regulation of both chains of the LFA-1 receptor,
?L- and ?2-integrins, whereas ?1- and ?4-integrin ex-
pression were essentially unchanged (Fig. 6B). These data
show that c-Myc represses cell-adhesion molecules in-
volved in the interactions between HSCs and SNO niche
cells, and furthermore, suggest that c-Myc expression
levels might be crucial for the control of these interac-
To address whether c-Myc overexpression results in
defects in stem cell function, wild-type CD45.2+linneg
BM was infected with the MYC–IRES–huCD2 or huCD2
control viruses and used to generate mixed BM chimeras
together with wild-type CD45.1+BM cells. Initially, the
contribution of c-MYC-expressing cells was similar to
that of control grafts, as determined by the percentage of
huCD2+cells in peripheral blood cells (PBLs) 2 wk after
transplantation (Fig. 6C). By 4 wk post-transplantation,
donor-derived c-MYC overexpressing PBLs
comprised 45.8% ± 10.2% Ter119+(erythroid lineage),
22.8% ± 11.4% Gr1+(granulocytes), and 38.2% ± 14.9%
B220+(B cells), similar to huCD2+control donor-derived
BM (data not shown). However, compared with 2 wk post-
transfer, the proportion of c-MYC-expressing huCD2+
cells was significantly decreased (Fig. 6C), and became
virtually undetectable (<1%) in BM, spleen, and PBLs of
the CD45.1+recipients after 12 wk (Fig. 6C; data not
shown). Moreover, huCD2+linnegSca-1+donor cells
overexpressing c-MYC were absent in BM after 12 wk
(data not shown). As expected, engraftment of the cells
infected with the huCD2 control virus remained stable
even after 12 wk post-transplantation (Fig. 6C). These
results directly demonstrate that although enforced ex-
pression of c-MYC allows homing and multilineage dif-
ferentiation, long-term self-renewal activity of HSCs is
Whereas the kinetics of HSC loss supports a premature
differentiation mechanism, it is still formally possible
that c-Myc-overexpressing HSCs are lost due to apopto-
sis. To address the effects of c-Myc overexpression on
HSC survival in vitro, wild-type linnegBM cells were
infected with the MYC–IRES–huCD2 or huCD2 control
Expression of cell-adhesion molecules on KLS-HSCs
Four-color FACS analysis of control and c-Myc deficient KLS-
cell (gated as linnegcKit+Scal+). (NE) not expressed; (U) un-
changed; (+/−) low expression on a % of cells; (+) intermediate to
high expression on most cells; (↑%) change in the proportion of
positive cells; (↑*%) expected increase due to enrichment of
LT-HSC in mutants; (↑ or ↑↑) up-regulated in both % and mean
fluorescence intensity (MFI).
Wilson et al.
2754GENES & DEVELOPMENT
viruses. After 72 h in culture, the percentage of apoptotic
cells within the huCD2+KLS-HSC populations was de-
termined by 7-AAD/Annexin V staining. No increase in
apoptotic (Annexin V+) cells was observed in c-MYC-
overexpressing KLS-HSCs in comparison to controls. In
fact, these cells showed slightly better survival, suggest-
ing that c-MYC overexpression does not induce apopto-
sis in cultured KLS-HSC cells (data not shown).
c-Myc-induced apoptosis has been previously shown
to be blocked by expression of BCL-2 (Pelengaris et al.
2002a; Nilsson and Cleveland 2003). To further exclude
the possibility that c-MYC-overexpressing HSCs are lost
in vivo due to apoptosis, we generated mixed BM chime-
ras in which H2K–BCL-2 transgenic linnegBM cells in-
fected with the MYC–IRES–huCD2 or huCD2 control
viruses were transferred together with wild-type total
BM. In these chimeras, repopulating virus-transduced
(but not wild-type) HSCs express a BCL-2 transgene
(Domen et al. 2000). Two weeks post-transfer, huCD2+
cells were detected in PBLs of both experimental and
control mice (data not shown). At 6 wk post-transfer, the
BM was analyzed for the proportion of huCD2+KLSF-
HSCs. As shown in Figure 6D, 14.6% ± 2% of control
KLSF cells were huCD2+. In contrast, only around
1.5% ± 0.5% of c-MYC/BCL-2-expressing KLSF cells
were huCD2+, showing that even in the presence of BCL-
2, c-MYC-overexpressing HSCs fail to long-term self-re-
new and are eventually lost (Fig. 6D). In addition, 5/8
experimental mice developed pre-B-cell lymphomas
originating from cells coexpressing c-MYC and BCL-2,
similar to what has been previously reported (Strasser et
al. 1990; Cory et al. 1999). However, it is important to
note that c-Myc-expressing KLSF cells are virtually ab-
sent whether tumors develop or not. Thus, despite initial
engraftment (differentiation) and inhibition of apoptosis,
c-Myc overexpressing HSCs apparently fail to self-re-
new, strongly suggesting that this is due to premature
Expression of endogenous c-myc is induced
upon onset of LT-HSC differentiation
Our genetic studies show that total loss of c-Myc or de-
regulated gain of c-MYC activity shift the balance toward
HSC self-renewal or differentiation, respectively (Fig.
7A). In a homeostatic situation, division of a wild-type
HSC generates two daughter cells, with one remaining
an HSC, whereas the other initiates differentiation. If
endogenous c-Myc is involved in this decision process in
normal HSCs, its expression should be negative/low in
the HSC remaining in the niche, but would be higher in
the daughter cell that starts to differentiate. To test this
hypothesis, c-myc transcripts were quantified in FACS-
sorted HSC subsets by real-time RT–PCR. The results
show that although c-myc is expressed at low levels in
KLSF-HSCs (LT-HSCs), it is expressed in FLK2+KLS-
HSCs (ST-HSCs/MPPs) at
higher levels. Collectively, our results indicate that the
balance between HSC self-renewal and differentiation is
controlled by c-Myc expression levels (Fig. 7A). The ef-
fects of c-Myc throughout hematopoiesis are summa-
rized in Figure 7B.
2.3-fold ± 1.3(P = 0.008)
In this study, we provide genetic evidence confirming
the expected involvement of c-Myc during the expansion
of committed progenitors in the adult hematopoietic
system. In addition, and in contrast to progenitors, we
KLS-HSCs as determined by FACS. (A, filled histogram) c-Myc-deficient KLS-HSCs; (solid line overlay) control KLS-HSCs; (dotted line
overlay) negative control (omission of N-cadherin antibody). Horizontal arrow indicates positive N-cadherin staining. (B) Up-regula-
tion of LFA-1 (?L?2 integrin), ?5 and ?1 integrins on c-Myc-deficient KLS-HSCs. Histogram analysis of CD11a (?L integrin), CD18 (?2
integrin), CD29 (?1 integrin), CD49d (?4 integrin), CD49b (?2 integrin), CD49e (?5 integrin), CD62L (L-selectin), and CXCR4. (Filled
histogram) Mutants; (overlaid line) control.
Up-regulation of N-cadherin and several integrins on c-Myc-deficient HSCs. Expression of N-cadherin on c-Myc-deficient
c-Myc function in hematopoietic stem cells
GENES & DEVELOPMENT2755
have also uncovered a novel role for c-Myc during the
first steps of HSC differentiation. This function of c-Myc
is only evident in HSCs located within BM niches, but
not if grown in vitro, where stem cell niches are absent.
c-Myc controls (represses) the expression of specific in-
tegrins and the HSC anchor N-cadherin, suggesting a
model in which c-Myc controls the balance between self-
renewal and differentiation by modulating migration
and/or adhesion of HSCs to the niche (Fig. 7A).
c-Myc function in stem-cell differentiation
Although the model shown here proposes a function for
c-Myc in the first differentiation steps of stem cells, the
data may also be explained (at least at first glance) by a
role of c-Myc in proliferation alone. In this study, we
demonstrate that, whereas HSCs proliferate in a c-Myc-
independent manner, late and potentially also early pro-
genitors require c-Myc activity for cell cycle progression.
Thus, early progenitors would not expand in the absence
of c-Myc, making them virtually invisible, and generat-
ing the false impression of HSCs not undergoing differ-
entiation. In such a situation, there is no a priori reason
for HSC numbers to increase. However, deletion of c-
Myc leads to a specific increase of LT-HSCs. Because the
mutants develop severe anemia, one could predict feed-
back mechanisms that may lead to HSC accumulation.
However, in mixed BM chimeras, where c-Myc-deficient
HSCs develop in the context of a normal hematopoietic
system that includes wild-type stem cell niches, an in-
crease of mutant HSCs is still observed. In fact, the in-
crease in HSCs is even more pronounced in BM chimeras
compared with mutant mice. Thus, we strongly favor a
model in which c-Myc has a dual role. Initially, it is
essential to induce the first differentiation steps in
HSCs, whereas in committed progenitors, c-Myc func-
tion is required for cell cycle progression and expansion
Recently, data obtained in other systems also provided
evidence of c-Myc influencing progenitor differentiation
independent of its function in division and survival. For
example, during Xenopus, neural crest induction knock
down of c-Myc results in the failure to induce expression
of early neural crest markers, causing a subsequent block
in the formation of neural crest-derived structures
(Bellmeyer et al. 2003). Development and maintenance
of intestinal and colonic mucosa are controlled by the
canonical Wnt/?-catenin/TCF4 pathway. Recent studies
in colorectal carcinoma cell lines suggest that c-Myc and
its downstream target gene p21CIP1are key effectors of
this pathway, consequently controlling self-renewal, ex-
pansion, and differentiation of mucosal progenitors (He
pression in response to ectopic expression of c-
MYC. (A) Wild-type linnegBM infected with
MYC–IRES–huCD2 virus (c-MYC overexpres-
sion, filled histogram) or huCD2 control virus
(wild-type, solid line). Dotted overlay shows a
negative control-omitting N-cadherin antibody.
Histograms are gated on huCD2+linnegSca-1+
cells after 4 d in culture. (B) Integrin expression
in response to ectopic expression of c-MYC.
Wild-type linnegcells were isolated by flow cy-
tometry and either infected with MYC–IRES–
huCD2 (filled histogram), or huCD2 control vi-
rus (overlaid line). After 7 d, cells were harvested
and the huCD2+linnegsubset analyzed for ex-
pression of indicated integrins by FACS. (C) Ec-
topic expression of c-MYC in vivo. Wild-type
LinnegBM, highly enriched for KLS-HSCs, was
isolated and infected as in A and B. c-MYC over-
expression (MYC–IRES–huCD2) (dotted lines), or
control expressing huCD2 alone (solid lines). In-
fected BM was transferred together with wild-
type BM into lethally irradiated recipients to gen-
erate mixed BM chimeras. At times indicated,
the percent huCD2+cells in donor phenotype
PBLs or BM was assessed by FACS analysis. Each
line represents data from an individual mouse.
(D) Percent huCD2+cells in KLSF cells after BM
reconstitution in vivo as in C, except that start-
ing linnegBM cells were obtained from H2K–
BCL-2 transgenic mice in which HSCs express
BCL-2. Control chimeras (BCL-2) and c-MYC
shown are from three (BCL-2) and four (c-Myc/
Down-regulation of N-cadherin ex-
Wilson et al.
2756 GENES & DEVELOPMENT
et al. 1998; van de Wetering et al. 2002). A role for c-Myc
in intestinal stem cell driven maintenance of mucosa is
further suggested by expression profiling of microdis-
sected early progenitor populations from intestinal
crypts in which a number of genes either controlling or
controlled by c-Myc have been identified (Stappenbeck
et al. 2003). Complementary to the BM results presented
here, where forced expression of c-MYC in HSCs leads to
their differentiation and subsequent loss, over-expres-
sion of c-MYC in the stem cell-containing basal layer of
the murine epidermis results in severe epidermal defects
(including epidermal loss) thought to be caused by the
loss of epidermal stem cells (Arnold and Watt 2001;
Waikel et al. 2001). The latter result is in direct contrast
to a different c-Myc-overexpressing transgenic mouse
line, in which c-Myc was targeted to a more differenti-
ated progenitor population. In this second model, precan-
cerous epidermal lesions develop (Pelengaris et al. 1999).
These contrasting results in the skin are consistent with
our model, suggesting that c-Myc has distinct roles in
stem and progenitor cell types (Fig. 7B). Collectively,
these data suggest that c-Myc is not only involved in the
first differentiation steps of HSCs, but may have similar
roles in other self-renewing tissues, including the intes-
tinal mucosa and the skin epidermis.
Role of c-Myc in HSC–niche interactions
The differentiation defect of c-Myc-deficient HSCs in
the BM does not appear to be intrinsic, as in vitro-cul-
c-Myc-controlled adhesion to the stem cell niche. (Top)
A quiescent HSC expressing low c-Myc levels is re-
tained in the stem cell niche consisting of spindle-
shaped N-cadherin+osteoblasts (SNO) (Zhang et al.
2003) embedded in stromal fibroblasts. HSCs are an-
chored to SNO cells via homotypic N-cadherin and
LFA-1/ICAM interaction. In addition, expression of
?2?1-integrin and ?5?1-integrin connects HSCs to the
specialized extracellular matrix (ECM) in the niche. In
response to mitogenic signals, HSCs enter the cell cycle
and generate two daughter cells. (a) In the absence of
c-Myc-induction integrins and other putative cell-adhe-
sion molecules remain highly expressed in both daugh-
ter cells retaining them in the niche, thereby promoting
expansion of HSCs at the expense of differentiation. (b)
Induction of c-Myc in only one of the daughter cells
causes down-regulation of cell-adhesion molecules and
generates asymmetry with one HSC retained in the
niche and one leaving the niche, promoting differentia-
tion into committed progenitors (CP). In this homeo-
static situation, the stem cell pool is maintained while
differentiated progeny are produced. (c) High c-Myc ex-
pression in both daughter cells (e.g., ectopic c-MYC ex-
pression in HSCs) results in repression of cell-adhesion
molecules and departure of both cells from the niche.
This leads to the production of two CPs and hence pro-
gressive exhaustion of the stem cell pool due to differ-
entiation. (B) Model for the regulation of hematopoiesis
by c-Myc. (a) The wild-type (WT) hematopoietic system.
Under homeostatic conditions, long-term hematopoi-
etic stem cells (LT-HSCs) express low levels of c-Myc,
ensuring self-renewal. In a subset of LT-HSCs, c-Myc
expression increases, inducing differentiation toward a
short-term hematopoietic stem cell (ST-HSC) fate. Con-
tinued differentiation leads to the loss of self-renewal
activity, and early progenitor cells of different hemato-
poietic cell types become transient-amplifying cells
(TA-cells), which rapidly expand through proliferation
while continuing to differentiate into more and more
(A) Model for the regulation of HSC fate by
lineage-restricted progenitors. High c-Myc levels are maintained in TA-cells, ensuring continued cell cycle progression. At the onset
of terminal differentiation, c-Myc is down-regulated to allow permanent cell cycle exit and progression toward terminal differentia-
tion. (b) Induced elimination of c-Myc in the fully developed adult hematopoietic system results in two distinct effects. First, TA-cells
(progenitors) stop expanding, as c-Myc is required to maintain cells in an active cell cycle. Second, c-Myc-deficient LT-HCSs self-
renew, but fail to initiate normal differentiation, leading to their accumulation as long as niche space is available. Together, this leads
to loss of all hematopoietic cell types with the exception of HSCs. (c) Enforced expression of c-Myc in LT-HSCs promotes differen-
tiation at the expense of self-renewal, resulting in stem cell exhaustion. In the absence of stem cell activity, all hematopoietic cell
types are lost over time due to normal cellular turnover.
c-Myc function in hematopoietic stem cells
GENES & DEVELOPMENT2757
tured FACS-purified HSCs were able to differentiate
along myeloid and lymphoid lineages. Interestingly, this
occurs in the absence of significant proliferation, a phe-
nomenon previously reported by Fairbairn et al. (1993),
who showed that multipotent progenitors can differ-
entiate without proliferation if cultured in the absence
of cytokines, but in the presence of BCL-2. Re-expres-
sion of constitutive MYC activity in mutant HSCs
rescued the in vitro proliferation defect without affect-
ing multilineage differentiation. The contrasting behav-
ior of HSCs under- or overexpressing c-Myc in vitro
and in vivo reported in this study, together with the
identification of c-Myc-regulated specific cell adhesion
molecules strongly suggests that c-Myc controls interac-
tion between HSCs and their stem cell niche environ-
While the concept of the stem cell niche was first sug-
gested several decades ago (Trentin 1970; Schofield
1978), experimental evidence for the location and cellu-
lar composition of stem cell niches in higher organisms
is still in its infancy. Genetic studies in the Drosophila
ovary have demonstrated that germ-line stem cells are
required to remain attached via an adhesion anchor (DE-
cadherin) to the niche cells (Cap cells). Detachment from
the niche induces stem cell differentiation and loss of
self-renewal (Spradling et al. 2001; Song and Xie 2002;
Song et al. 2002). BM stem cell niches are thought to be
located in the endosteal lining of trabecular BM cavities,
with ostoblastic cells being a crucial component of the
stem cell-maintaining niche (Nilsson et al. 2001; Calvi
et al. 2003; Visnjic et al. 2004). Zhang et al. (2003) have
further demonstrated that spindle-shaped N-cadherin+
osteoblasts (SNO) fulfill the function of BM niche cells.
Because a fraction of HSCs also express N-cadherin, it is
likely (in analogy to the situation in the Drosophila ovar-
ian niche) that HSCs are anchored to SNO cells via ho-
motypic N-cadherin junctions (Song and Xie 2002; Song
et al. 2002; Zhang et al. 2003). Our findings that c-Myc-
deficient HSCs are in direct contact with SNO cells, and
furthermore express increased levels of surface N-cad-
herin, strongly support the notion that mutant stem
cells are retained in the niche, and c-Myc-mediated
down-regulation of adhesion molecules is necessary for
HSCs to exit the stem cell niche. Our model suggests
that upon division of a c-Myc-deficient HSC, both
daughter cells maintain an HSC fate, thereby contribut-
ing to expansion of the stem cell pool at the expense of
differentiated cell types (Fig. 7A). This model is further
supported by the fact that HSCs that cannot down-
regulate c-MYC display no surface N-cadherin and
c-MYC+N-cadherinnegHSCs progressively lose their self-
renewal capacity, presumably due to failure of retention
in the BM niche. It is interesting in this respect that the
Angiopoietin-1 (Ang-1)/Tie2-signaling pathway main-
tains repopulating quiescent HSCs in the niche, presum-
ably by activating cell adhesion molecules such as N-
cadherin (Arai et al. 2004). This raises the possibility that
c-Myc (and N-Myc) may be negative mediators down-
stream of the Tie2 pathway repressing N-cadherin and
activating the cell cycle. In ES-cells, it has been shown
that lack of c-Myc causes a marked up-regulation of
Ang-1 (Baudino et al. 2002). If this were also the case in
HSCs, one could hypothesize that the Myc and the Ang-
1/Tie2-signaling pathways are intricately linked in the
control of HSC–niche interactions, ultimately regulating
In addition to the more recent implication of cadherins
in stem-cell niche interactions, there is ample evidence
to support an important role for integrins in adult stem
cell function (Jones and Watt 1993; Pruijt et al. 1998;
Whetton and Graham 1999; Hynes 2002; Lapidot and
Petit 2002; Velders et al. 2002; Watt 2002). In the hema-
topoietic system, integrins have been particularly impli-
cated in controlling HSC migration from niches into the
circulation (mobilization) and from the circulation into
niches (homing). For example, HSCs deficient in ?1-in-
tegrin fail to engraft lethally irradiated mice due to im-
paired homing and retention in the BM (Potocnik et al.
2000). Experiments using blocking antibodies have also
implicated ?L, ?4, ?5, and ?2 integrins in the complex
mobilization process (Pruijt et al. 1998; Asaumi et al.
2001; Velders et al. 2002). It is intriguing, in this respect,
that in addition to controlling N-cadherin expression on
HSCs, c-Myc also represses expression of several mem-
bers of the integrin family of adhesion receptors, namely
VLA-5 (?5?1 integrin, fibronectin receptor), ?2?1 inte-
grin (collagen receptor), and LFA-1 (?L?2 integrin). Inte-
grins and cadherins have been previously shown to syn-
ergistically regulate migration and mobility of cells
(Huttenlocher et al. 1998). Whether these adhesion mol-
ecules also cooperate in controlling the function of
HSCs, and whether they only function as adhesion re-
ceptors or also as signaling molecules, is still an open
question. In addition, the exact function of N-cadherin
in HSC–niche interactions remains to be elucidated by
tissue-specific knockouts, as embryos lacking N-cad-
herin fail to develop past midgestation (Radice et al.
1997). However, the connections between c-Myc, Ang-
1/Tie2, and N-cadherin, as well as that of c-Myc and
integrin signaling, raises the intriguing possibility of a
complex network linking adhesion to specific signaling
cascades in stem cells attached to their niche (Jamora
and Fuchs 2002; Schwartz and Ginsberg 2002; Perez et al.
2003; Arai et al. 2004).
The total number of existing stem cell niches in the
BM, as well as their individual size, is currently un-
known. In this context, it is interesting to note that KLS-
HSC numbers in mutants increase only two- to three-
fold, whereas an accumulation of up to 10-fold is ob-
served in mixed BM chimeras. The difference may be
explained by the fact that whereas stem-cell niches are
wild type in chimeras, they are partially c-Myc-deficient
in mutants, due to MxCre activity in BM stromal cells
(M.J. Murphy and A. Trumpp, unpubl.). The concept that
c-Myc may also be required for (long-term) BM niche
maintenance is currently under investigation. It is also
possible that c-Myc-deficient HSCs positively impact on
(wild-type) niche size. In any event, our data suggest that
stem cell niches in the BM are limiting and crucial for
maintaining HSCs in the undifferentiated state, a con-
Wilson et al.
2758 GENES & DEVELOPMENT
clusion also recently reached by others (Akashi et al.
1999; Calvi et al. 2003; Zhang et al. 2003).
Although c-myc is the first proto-oncogene described to
control stem cell homeostasis, some of its target genes
and proteins that collaborate with Myc during tumori-
genesis have recently been implicated in stem cell func-
tion. For example, the polycomb protein Bmi-1 collabo-
rates with c-Myc during lymphomagenesis, and has been
shown to be essential for maintenance of adult HSCs
(Jacobs et al. 1999; Park et al. 2003). The CDK inhibitor
p21CIP, which is repressed by c-Myc, controls HSC pro-
liferation, and is furthermore required for maintenance
of long-term self-renewal (Cheng et al. 2000; van de We-
tering et al. 2002). This suggests that part of c-Myc‘s
effects in HSCs could be mediated by p21 repression.
Because c-Myc has been postulated to be an effector of
canonical Wnt signaling and also appears to be con-
nected to the Ang-1/Tie2-signaling pathway, this protein
is now evolving as a ringmaster in regulating adult stem
cell function in vivo (Baudino et al. 2002; van de Weter-
ing 2002; Arai et al. 2004). It is thus crucial to elucidate
which signaling pathways are responsible for the tight
control of c-Myc expression in stem/progenitor cells. Ir-
respective of what niche signals fine tune c-Myc expres-
sion during the constantly changing conditions of BM
homeostasis in vivo, it appears that this oncoprotein is a
key element that fulfils the function of a homeostat,
determining the balance between stem cell self-renewal
Materials and methods
Generation of c-Myc-deficient mice
c-mycflox/flox(control) mice as previously described (Trumpp et
al. 2001) were crossed with the Mx–cre transgenic mice (Kuhn
et al. 1995) to obtain MxCre;c-mycflox/flox(mutant) mice. IFN?-
induced deletion was effected by five i.p. injections of polyI–
polyC (pI–pC), each 2 d apart as previously described (Radtke et
al. 1999). Unless otherwise stated, all control mice were litter-
mates of mutant mice and were all treated with pI–pC. Deletion
was assessed by Southern Blot analysis and Taqman PCR on
total BM and FACS-sorted KLS BM as previously described
(Trumpp et al. 2001). Taqman RT–PCR was also performed to
analyze c-myc, L-myc, and N-myc expression in the BM and
KLS subsets. Primers and conditions were as described (Trumpp
et al. 2001), except for L-myc, where the following primers were
used: LmycF (ACGGCACTCGTCTGGAA) and LmycR (GT
GACTGGCTTTCGGATGTC). LFA-1 was detected using the
following primers: LFA-1F (ATTTTCCTGGCGCTCTACAA)
and LFA-1R (TCCATTTGGAACACCTCCAT). All cDNAs
were normalized using ?2microglobulin (?2mF [GTGTATGC
TATCCAGAAAACCC] and ?2mR [TCACATGTCTCGATC
CCAGTAG]) expression. All DNA samples were normalized
using the 18s rRNA gene (probe: 5?6-FAM/3?TAMRA, GTG
TATGCTATCCAGAAAACC; m18sF, ATTAAGTCCCTGCC
CTTTGTACAC; m18sR, CCGAGGGCCTCACTAAACC).
Analysis of endogenous c-myc expression
in HSCs by real-time RT–PCR
RNA from wild-type LT-HSCs and ST-HSCs (sorted as de-
scribed below from pools of six mice) was extracted using the
RNeasy mini kit (Qiagen). cDNA was generated using Stratas-
cript Reverse Transcriptase (Stratagene). Subsequently, samples
were normalized using ?-2 microglobulin, and c-myc levels
were determined using the primers/probes described above on
both a Light Cycler (Roche) and a Taqman GeneAmp5700 Se-
quence Decector (Applied Biosystems). This experiment was
performed five times on different days and the mean ± SD de-
termined (both Light Cycler and the GeneAmp5700).
For FACS analysis of PBLs, five drops of blood were collected
into a tube containing Heparin, diluted in PBS, and centrifuged
over a Lympholyte M gradient (Cedarlane Laboratories). PBLs
were harvested from the interface, washed, and FACS stained as
described below. Hemaglobin concentration was determined on
PBLs by the Veterinaermedizinisches Labor of the University of
Zurich, using a CELL-DYN Haematology Instrument.
BM preparation, analysis, and culture
BM was taken from the long bones of hind- and forelegs and
prepared by standard procedures. All cell suspensions were fil-
tered through a nylon mesh filter (70 µm) prior to FACS analysis
to prevent clumps. LinnegBM was prepared by first staining
with a cocktail of FITC or biotin-conjugated mAbs against lin-
eage markers (CD3, CD4, CD8, CD11b, CD161, B220, Gr1, and
Ter119). After washing, labeled cells were removed by incuba-
tion with Sheep anti-Rat IgG-coated M450 Dynabeads (Dynal
Biotech) at a bead-to-target cell ratio of either 10 or 20:1. Linneg
cells were harvested and purity checked by FACS. A second
round of bead depletion was performed when required, other-
wise remaining linposcells were gated out on the FACS during
analysis or sorting. To isolate KLS cells (linneg, cKit+Sca1+), lin-
depleted BM was subsequently stained with mAbs to Sca1 and
cKit (see below), and FACS sorted using a FACS Star+Flow
Cytometer (Becton Dickinson), at 1000 per well directly into
96-well flat-bottomed culture plates containing Stem Pro-
34SFM medium (GIBCO Invitrogen Corp.) supplemented with
L-glutamine (GIBCO), mSCF (50 ng/mlL, mTPO (25 ng/mL),
mFlt3L (30 ng/mL), and IL-6 (105µ/mL) all purchased from Re-
search Diagnostics Inc. Individual wells were observed micro-
scopically daily for 14 d, and cell number and morphology was
scored. Medium was partially exchanged every 3 d. For in vitro
bulk cultures, KLS-HSC cells were sorted (as above) and seeded
at between 10 and 40 K per well in 96 flat-well plates in stem-
cell medium (as above) supplemented with additional cytokines
as follows: IL-11 (20 ng/mL), GM-CSF (50 ng/mL), and EPO (3
ng/mL) (Research Diagnostics). Cells were cultured for 7–9 d
and subsequently harvested, surface stained, and analyzed by
four- or five-color FACS. For infection, KLS-HSCs or LinnegBM
were prepared and infected as described below. Medium was
changed 12 h after infection, and cells were cultured for 5–9 d
prior to FACS analysis. To isolate LT-HSCs and ST-HSCs, wild-
type LinnegBM was prepared from 7-wk-old C57BL/6 mice (Har-
lan Olac), and stained as described above with mAbs to CD117,
Sca-1, and CD135. cKit+Sca1+CD135+(ST-HSC) and cKit+Sca1+
CD135−(LT-HSC) were sorted on a five-color FACS DIVA Flow
Cytometer (Becton Dickinson). For each individual experiment,
the BM of six mice was pooled.
c-Myc function in hematopoietic stem cells
GENES & DEVELOPMENT 2759
Monoclonal antibodies and FACS analysis
The following monoclonal antibody conjugates were purchased
from eBiosciences: CD117 (c-kit R, ACK2)-PE, -PE-Cy5, -PE-
Cy7, and -APC-Cy7; CD127 (IL7R? chain, A7R34)-biotin, -PE
and -PE-Cy5; CD135 (Flt3R/Flk2, A2F10)-PE; CD11b (M1/70)-
PE-Cy5; Sca-1 (Ly-6A/E, D7)-FITC, -PE, and -APC; Ter 119-PE;
B220 (RA3-6B2)-PE-Cy5; CD49b (?2 integrin, HMa2)-biotin;
CD49d (?4 integrin, R1-2)-biotin; CD51 (?V integrin, RMV-7)
protein; CD34 (RAM34)-biotin; CD43 (?IIb integrin, S7)-biotin;
CD54 (ICAM-1, YN1/1.7.4)-biotin; and CD106 (VCAM-1, 429)-
biotin. Anti-E-cadherin-FITC (36), CD9 (KMC8)-biotin; CD29
(Ha2/5)-biotin and purified; CD49a (?1 integrin, HA3/18)-bio-
tin; CD49e (?5 integrin, 5H10-27)-biotin; CD61 (?3 integrin,
2C9.G2)-biotin; CD104 (?4 integrin, 346.11A)-biotin; Sca-1
(2B8)-biotin; ?7 integrin (M293)-biotin; CD49f (?6 integrin,
GoH3)-PE; and CD184 (CXCR4, 2B11)-PE were purchased from
BD Biosciences. B220-PE was purchased from Caltag. Gr-1 (Ly-
6G, RB6-8C5)-FITC, biotin and -Alexa 647, Ter 119-FITC, B220
(RA3-6B2)-FITC and biotin, CD11b-FITC and biotin, CD4
(clone GK1.5)-FITC and biotin, CD8? (53.6.7)-FITC and biotin,
CD161 (NK1.1, PK136)-FITC and biotin, CD3? (17A2)-FITC and
biotin, CD11a (?L integrin, FD44.8)-biotin, CD18 (?2-integrin,
FD18.5)-biotin, CD45.1 (A20.1)-FITC, -biotin, -PE or -Alexa 647,
CD45.2 (ALI-4A2)-FITC, -biotin, -PE or -Alexa 647, CD24 (HSA,
M1/69)-biotin, and CD44 (Ly24/PgP1, IM.781)-biotin were pu-
rified and conjugated in this laboratory following standard
protocols. Alexa 647 conjugates were prepared using the appro-
priate Alexa protein labeling kits (Molecular Probes). CD144-
purified protein (Rat anti-mouse VE-cadherin, 11D4.1) was pur-
chased from BD Biosciences and revealed with Goat anti-Rat
Ig-PE (Caltag), followed by blocking with Rat Ig (Jackson Immu-
noResearch) prior to staining with lineage cocktail-FITC,
CD117-PECy5, and Sca 1-APC. Affinity-purified rabbit anti-hu-
man N-cadherin (YS) antibody was purchased from Immuno-
Biological Laboratories and revealed with Goat anti-Rabbit Ig-
biotin (purified and conjugated in this laboratory). Blocking and
further staining was performed as described for VE-Cadherin. In
the experiments in which c-MYC was overexpressed using ret-
roviral constructs, N-cadherin was detected using the Rabbit
Zenon-biotin labeling kit (Molecular Probes). Streptavidin-APC
(Molecular Probes), Streptavidin PE-Cy5 and PE-Cy5.5 (eBiosci-
ences), or Streptavidin PE-Cy7 and Streptavidin-PE (Caltag)
were used to reveal biotin conjugates. Four- and five-color FACS
analysis (FITC, PE, PE-Cy5, or PE-Cy5.5, PE-Cy7, and APC or
Alexa 647) were performed using a FACSCalibur Flow Cytom-
eter (Becton Dickinson) or a FACS Canto Flow Cytometer (Bec-
ton Dickinison) respectively, and data was analyzed using Cel-
lQuest software. Five-color FACS sorting (FITC, PE, PE-Cy5,
APC, and APC-Cy7) was performed on a FACS DIVA Flow Cy-
tometer (Becton Dickinson).
Cell cycle analysis and BrdU uptake
Simultaneous cell cycle analysis and four-color surface staining
was performed. Briefly, BM cells were labeled with mAb conju-
gates (FITC, PE, PE-Cy5, and PE-Cy7) to surface markers, fixed
in 2% paraformaldehyde in PBS, washed, permeabilized with
0.5% Saponin in PBS/3% FCS, and stained with Hoechst 33342
(Molecular Probes) at 20 µg/mL for 5 min. After washing, cells
were analyzed on a LSR Flow Cytometer (Becton Dickinson).
Doublets were eliminated using the DDM unit. To distinguish
between G1and G0phases of the cell cycle, surface labeling
using either anti-Ki67-FITC or an isotype control FITC conju-
gate was added with the Hoechst, and the incubation prolonged
to 30 min. BrdU uptake studies were performed as previously
described (Wilson et al. 2001) using the BrdU staining kit (BD
Bone marrow chimeras
CD45.2+C57BL/6 female mice (Harlan Olac) and CD45.1+
C57BL/6 female mice (The Jackson Laboratory) were purchased
and maintained in the ISREC animal facility. H2K–BCL-2 trans-
genic mice were obtained from Dr. J. Domen (Duke University
Medical Center, Durham, NC). Competitive BM reconstitution
was performed as previously described (Radtke et al. 1999).
Briefly, a mixture of 10 × 106MxCre;c-mycflox/floxBM (CD45.2+)
and 1 × 106wild-type BM (CD45.1+) was i.v. transferred into
CD45.1+lethally irradiated (1000 rads) recipient mice that had
been treated with anti-NK1.1 mAb 48 h previously. Mice were
maintained on antibiotic (Bactrim, Roche, Basel) containing wa-
ter and long-term reconstitution of peripheral blood, BM, and
lymphoid organs analyzed 2–3 mo later. For retroviral infec-
tions, CD45.2+C57BL/6 or H2K–BCL-2 transgenic mice re-
ceived 0.15 mg/g body weight 5-fluorouracil (Sigma-Aldrich) i.p.
3 d prior to sacrificing. LinnegBM highly enriched for KLS-HSCs
was prepared as described above, and cells were infected with
either pMI–IRES–huCD2 or pMI–MYC–IRES–huCD2 viruses.
Around 500 × 103cells were infected with virus using polybrene
(4 µg/mL, Sigma) in Stem Pro-34SFM medium (described above).
Cells were spun for 15 min at 1800rpm and incubated at 37°C
for a further 2 h. Cells were subsequently i.v. transferred into
lethally irradiated (1000 rads) CD45.1+recipients. A total of 15
h later, a second dose of 500 × 103-infected BM cells, which had
undergone a second round of viral infection with the same virus
and been maintained in stem-cell medium was injected i.v. A
further 24 h later, 0.5 × 106wild-type CD45.1+BM was trans-
ferred. PBLs from these chimeric mice were analyzed 2 and 4 wk
post-injection, and BM and lymphoid organs analyzed 12 wk
later for the presence of donor (CD45.2+) cells successfully in-
fected with virus (huCD2+).
Localization of progenitor/stem cells in situ
The localization assay was performed as described (Nilsson et
al. 2001). LinnegBM was prepared and resuspended to 106cells/
mL in DMEM/0.5% FCS containing 10 µM 5-(and-6)-carboxy-
fluorescein diacetate succinimidyl ester (CFSE) (Molecular
probes) and incubated at 37°C for 10 min (Trumpp et al. 2001).
Ice-cold PBS with 20% FCS was added to stop staining. After
washing in PBS, cells from one donor mouse were resuspended
in PBS/0.5% FCS and injected i.v. into one recipient that had
received 300 rads 24 h prior. Fifteen hours later, the mice were
sacrificed and muscle and tissue cleaned from the fore- and
hindlimbs before the bones were placed in PBS/4% PFA for
16–18 h at 4°C. After decalcification (Al.Cl2, Acetic acid, HCl
7%:5%.8.5% w/v) for 24–48 h at 4°C, the bones were embedded
in paraffin according to standard protocols. A total of 0–20 µm
longitudinal (for observation) or 5 µm cross-sections (for immu-
nostaining) were cut and mounted using Vectashield (Vector
Laboratories). Immunohistochemistry was performed as de-
scribed (Trumpp et al. 1992). Briefly, sections were deparaf-
finized and antigen retrieved at 70°C in 10 mM citrate buffer
(pH 6.0) for 3–4 h. Slides were cooled for 20 min, blocked in 2%
mouse serum, and 10% normal goat serum in PBS (blocking
buffer) for 30 min, incubated in polyclonal Rabbit anti-human
N-cadherin (as above) or monoclonal mouse anti-osteopontin
(clone Akm2A1) (Santa Cruz Biotechnology) or BMPRIa (ALK3)
(gift from Dr. C.H. Heldin, Ludwig Institute for Cancer Re-
search, Uppsala, Sweden) in a 1:100 dilution of blocking buffer
at 4°C overnight in a humidified chamber. Slides were rinsed
Wilson et al.
2760 GENES & DEVELOPMENT
three times in PBS/0.3% Triton X-100, incubated in goat anti-
rabbit Ig-Alexa-546 (Molecular Probes) for N-cadherin and
ALK3 staining, or with Goat anti-mouse Ig-Cy3 (Jackson Labo-
ratories) for Osteopontin for 1 h at room temperature in 1:100
blocking buffer. Slides were rinsed three to five times in water,
mounted in fluorescent mounting medium. Analysis was per-
formed under a DC200 Leitz LEICA DMIRB fluorescent micro-
scope equipped with filters for FITC (578 nm), Texas Red (610
nm). Cell nuclei were localized by staining with DAPI (Molecu-
We thank Céline Agosti for excellent technical assistance;
Christelle Dubey for animal husbandry and genetic screening;
Drs. Bettina Ernst and Anita Wolfer for initial help with setting
up bone marrow chimeras; Pierre Zaech and Frédéric Grosjean
for FACS sorting; Dr. Jos Domen for the H2K–BCL2 transgenic
mice; Dr. Michael Bevan for the huCD2 retroviral construct; Dr.
Carl Henrik Heldin for the ALK3 antibody; and Catherine
Pythoud, Estelle Sauberli, and the ISREC MIM core facility for
histology. We thank Drs. Michel Aguet and Freddy Radtke for
critical reading of the manuscript. This work was supported in
part by grants to A.T. from the Swiss National Science Foun-
dation, the Swiss Cancer League, and the Leenards Foundation.
A.T. is member of the EMBO Young Investigator Programme.
M.B. was supported by a fellowship from Roche and K.K. from
the UBS Optimus Foundation.
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