Xist RNA Is a Potent Suppressor
of Hematologic Cancer in Mice
Eda Yildirim,1,2,6James E. Kirby,7Diane E. Brown,3,5Francois E. Mercier,4,8Ruslan I. Sadreyev,1,2,5,6David T. Scadden,4,8
and Jeannie T. Lee1,2,5,6,*
1Howard Hughes Medical Institute
2Department of Molecular Biology
3Center for Comparative Medicine
4Center for Regenerative Medicine
5Department of Pathology, Harvard Medical School
Massachusetts General Hospital, Boston, MA 02114, USA
6Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
7Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
8Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
X chromosome aneuploidies have long been associ-
ated with human cancers, but causality has not been
established. In mammals, X chromosome inactiva-
tion (XCI) is triggered by Xist RNA to equalize gene
expression between the sexes. Here we delete Xist
in the blood compartment of mice and demonstrate
that mutant females develop a highly aggressive
myeloproliferative neoplasm and myelodysplastic
syndrome (mixed MPN/MDS) with 100% penetrance.
Significant disease components include primary
myelofibrosis, leukemia, histiocytic sarcoma, and
vasculitis. Xist-deficient hematopoietic stem cells
(HSCs) show aberrant maturation and age-depen-
dent loss. Reconstitution experiments indicate that
MPN/MDS and myelofibrosis are of hematopoietic
rather than stromal origin. We propose that Xist
loss results in X reactivation and consequent
genome-wide changes that lead to cancer, thereby
causally linking the X chromosome to cancer in
mice. Thus, Xist RNA not only is required to maintain
XCI but also suppresses cancer in vivo.
X chromosome inactivation (XCI) transcriptionally silences one X
chromosome in the female mammal to balance gene expression
between the sexes (Lyon, 1961; Payer and Lee, 2008; Lee, 2011;
Wutz, 2011). The random form of XCI occurs only once during
development, on embryonic days 4.5–5.5 (E4.5–5.5), when the
epiblast consists of 10–20 cells. Beyond E5.5, the inactive X
(Xi)enters into a ‘‘maintenance phase’’ in whichthe sameX chro-
mosome is propagated as Xi in subsequent cell divisions for the
remainder of female life. Initiation of XCI depends on Xist, the 17
kb ‘‘X-inactive specific transcript’’ that targets and tethers
Polycomb-repressive complexes to the X chromosome in cis
(Brown et al., 1992; Clemson et al., 1996; Zhao et al., 2008;
Jeon and Lee, 2011). Current viewshold that, while Xist isessen-
tial for initiation of XCI both in an embryonic stem (ES) model
(Penny et al., 1996) and in mice (Marahrens et al., 1997), Xist is
dispensable once the Xi is established. Indeed, deleting Xist
in vitro in post-XCI fibroblasts and somatic cell hybrids does
not cause immediate X reactivation (Brown and Willard, 1994;
Rack et al., 1994; Csankovszki et al., 1999). Furthermore, in ES
models carrying autosomal Xist transgenes, switching off Xist
after autosomal silencing does not lead to reactivation (Wutz
and Jaenisch, 2000).
Nonetheless, Xist is continuously expressed throughout
female life. Notably, recent studies have uncovered stochastic
single-gene reactivation and a loss of Polycomb repression
when Xist is conditionally deleted in mouse fibroblasts (Zhang
et al., 2007). Moreover, inappropriate silencing of human XIST
et al., 2008; Anguera et al., 2012; Mekhoubad et al., 2012).
Whereas Xist has been investigated extensively in cell culture,
in vivo studies have been limited (Marahrens et al., 1997; Savar-
ese et al., 2006; Kalantry et al., 2009; Namekawa et al., 2010).
These findings and the limited exploration of in vivo models led
us to suspect that Xist may have as yet undiscovered functions
after XCI is established in the early embryo. Does X reactivation
occur, and would potential X-overdosage have undesirable
Intriguingly, supernumerary X chromosomes have long been
associated with human cancers (Moore and Barr, 1955; Liao
et al., 2003; Pageau et al., 2007). For example, breast and
ovarian cancer cells frequently lose the Barr body (the Xi)
and duplicate the active X (Xa). The association between X and
cancer also holds in men. For instance, XXY men have a 20- to
50-fold increased risk of breast cancer in a BRCA1 background
(Fentiman et al., 2006), and testicular germ cell tumors often
acquire supernumerary Xs (Kawakami et al., 2003). Neverthe-
less, an association between X and cancer has remained strictly
correlative. Here, we explore the long-standing association in
Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc. 727
Sx7E9 Xist RNA
Xist∆/∆ 8 1-1
Xist∆/+ 27 1-1
Stage 1Stage 2
Spleen:Body weight ratio (x10-2)
2 4 5+
Figure 1. Deleting Xist in the Blood Compartment Results in Female Specific Lethality
(A) Map of the Xist2loxand XistDalleles and FISH probes used to distinguish the two alleles. Xist RNA is detected by using (Cy5-Sx9 probe, cyan). Representative
RNA/DNA FISH are shown (n R 50 each). D, deletion. X, XbaI site.
(B) Xist RNA FISH of splenocytes (n R 100). Xist RNA: green, FITC-Sx9 probe.
(C) Female specific lethality: Kaplan-Meier kill curves plotted over 750 days were generated using Prism (GraphPad software). There were no differences
between any control group: Vav-Cre, Xist2lox/+, or Xist2lox/Xist2loxfemales or corresponding male controls. These control genotypes were combined: females,
‘‘Xist2lox/+’’; males, ‘‘Xist2lox/Y’’.
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728 Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc.
mice and demonstrate a direct causal link between X and
To address consequences of losing Xist in vivo, we used the
Vav-Cre transgene to excise Xist conditionally in murine hemato-
poietic stem cells (HSC) as they emerge at E10.5, which is after
the establishment of XCI (Mu ¨ller et al., 1994; Csankovszki et al.,
1999; de Boer et al., 2003). All XistD/+, XistD/XistD, and XistD/Y
pups were born at expected frequencies and without obvious
abnormalities. The Xist deletion was confirmed in splenocytes
by RNA-DNA fluorescence in situ hybridization (FISH), in which
the two alleles could be distinguished using probes Sx7 (present
on both alleles) and E9 (absent in deleted allele) (Figure 1A). In
XistD/XistDfemales, Xist was deleted from Xi in 100% of cells
(Figure 1B). In XistD/+ females, Xist was deleted from Xi in
?50% of cells and from Xa in ?50%, consistent with the random
nature of XCI. For two years, we monitored the mutant animals
alongside of control littermates and performed experiments in
accordance with the relevant regulatory standards of animal
policies of MHG Institutional Animal Care and Use Committee
and, at the 2 year mark, only 10% of XistD/+ and XistD/XistDre-
mained alive (Figure 1C). By contrast, XistD/Y males and control
littermates (Xist+/+; Xist2lox/+; Xist2lox/Y; Vav-Cre) remained
was female specific, with XistD/+ and XistD/XistDfemales
behaving indistinguishably. These data demonstrate a striking
in vivo effect of deleting Xist after XCI is established.
Splenomegaly and Multilineage Hyperproliferation
The female mice typically perished after a period of illness char-
acterized by wasting, shaking, rapid breathing, and lethargy.
Nearly all mice showed distended abdomens and/or necks in
the terminal stage (Figure 1D). Upon necropsy, XistD/+ and
(enlargement of spleen). Splenomegaly was progressive and
specific to female mutants (Figures 1D and 1E, p < 0.01).
Histologic analysis revealed that, prior to 2 months, the spleens
were relatively normal (Figure S1, available online). But in stage 1
of the disease (2–5 months), staining for the proliferation
marker, Ki67, showed general hyperplasia of all splenic com-
partments (Figures 1F and S2), including red pulp (where
erythroids [Ter119+], myeloids [MPO+], monocytes/macro-
phages [Mac2+], and megakaryocytes reside [Schmitt et al.,
2001]), and white pulp (where B cells [B220+] and T cells
[CD3+] reside). In later stage 1, the germinal centers (B220+,
a B cell marker) became enlarged (Figure S2). These changes
suggested marked extramedullary hematopoiesis (EMH), the
formation of new blood cells outside of the bone marrow. In
stage 2 (beyond 4–5 months), myeloid cells (MPO+) and mono-
cell types and eradicated the white pulp and germinal centers
(Figure 1F). XistD/+ and XistD/XistDfemales behaved similarly,
whereas male mutants and female controls exhibited no
pathology (Figure S2). Thus, deleting Xist resulted in hyperprolif-
eration of all hematopoietic lineages, but myeloid cells have
a competitive advantage.
Bone Marrow Dysfunction: Myelofibrosis,
Myeloproliferation, and Myelodysplasia
The adult mouse spleen may be an active site of hematopoiesis
and account for ?30% of total hematopoiesis (Suttie, 2006;
vastly exceeded physiological levels and was accompanied by
defects in central hematopoiesis. Reticulin staining of the bone
marrow revealed progressive myelofibrosis (Figure 2A), a patho-
logical condition of unknown etiology in which the marrow is
replaced by fibrotic tissue. In mutant females of % 2 months of
age, marrow cellularity and spleen size were normal. In stage 1,
myeloid hyperplasia became a prominent feature (Figures 2B
and 2C). In stage 2, marrow cellularity decreased dramatically
as myelofibrosis became exuberant (Figure 2A). These changes
paralleled development of splenomegaly (Figures 1E and 1F)
and EMH not only in spleen but also in organs not normally
associated with EMH, such as liver, kidney, lymph nodes, and
cardiac muscle (Figure 2D). With concurrent marrow pathology,
EMH might be partly compensatory.
Importantly, hematopoiesis at neither primary nor extramedul-
lary sites was normal. In XistD/+ and XistD/XistDfemales (but
not in any mutant males or control females), bone marrow aspi-
rates showed multilineage proliferative and dysplastic changes
(Figure 2B and data not shown). For example, findings included
binucleated erythroid precursors, siderocytes containing nonhe-
moglobin iron (revealed by Prussian blue staining), and
increased phagocytosis of red and white blood cells (RBC,
WBC) by macrophages, as well as increased numbers of fibro-
blasts consistent with myelofibrosis. Although hyperproliferative
in early stages (increased Ki67 staining), end-stage animals
demonstrated pancytopenia (loss of all lineages; Figure 2C)
concurrent with exuberant myelofibrosis.
Circulating cells also demonstrated dysplastic changes (Fig-
ure 3A). In the myeloid lineage, we observed hypogranularity of
neutrophils, atypical condensation of chromatin, and abnormal
lobation in neutrophils and myelocytes (e.g., pseudo-Pelger-
Huet anomaly). Marked leukocytosis or leukopenia, erythropha-
gocytosis, and increased numbers of circulating immature mye-
lomonocytic cells (e.g., bands, metamyelocytes) were seen. In
the platelet lineage, we noted megaplatelets with retained nuclei
(D) Female-specific splenomegaly. Top panels: representative mutant females and age-matched controls are shown. Note abdominal swelling in 27-1-1 (XistD/+,
6.3 months old at death) and cervical mass in 8-1-1 (XistD/XistD, 7 months old at death). Animal ID numbers are shown with genotype throughout the figures.
Bottom: representative spleens from each male and female genotype are shown.
(E) Age-dependent increase in the spleen-to-body weight ratio. Ratios taken at 2 (n = 2–6), 4 (n = 6), and 5 to 16 (n = 14–36) months of age. Means ± SEM are
shown. Significance of the differences between mutant and WT spleen were calculated using the Student’s t test. **p % 0.01; ***p % 0.001.
(F) Temporal progression in spleen pathology. Immunostains are as indicated. Scale bars, 100 mm. See also Figures S1 and S2.
Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc. 729
Figure 2. Bone Marrow Insufficiency
(A) Reticulin staining of bone sections reveals progressive hypocellularity and myelofibrosis (black stains representing reticulin fibers) in mutant females. XistD/+
cases are shown. Scale bars, 50 mm.
(B) Representative bone marrow cytology from WT and mutant females, with anomalies indicated. All were Wright-Giemsa stained except siderocytes stained by
Prussian blue to reveal pathological presence of nonhemoglobin iron.
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730 Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc.
and clustered, atypical granulation in the cytoplasm occurring
in the context of either thrombocytopenia or thrombocytosis
(decreased or increased platelet counts, respectively) (Figures
3A and 3B). In spite of thrombocytopenia in some animals,
megakaryocytic hyperplasia was evident in the marrow and at
sites of EMH, suggesting aberrant platelet maturation.
Inthe erythroid lineage, weobserved poikilocytosis and aniso-
cytosis (RBC of abnormal shape and variable size), circulating
(C)Quantitation ofbone marrow cells inseven mutant(Mut1–Mut7) and fourWT (WT1–3and 7)females intabular (top)and histogram (bottom)form. M:E,myeloid
to erythroid ratio. Mac, macrophages. Means ± SEM are shown. **p % 0.01; *p % 0.05.
(D) Nonphysiological EMH in multiple organs (17-1-4, XistD/XistD, 6 months old at death; 8-1-1, XistD/XistD, 7 months old at death). All sections were H&E stained.
Scale bars, 50 mm.
Figure 3. Multilineage Dysplasia and Myeloproliferative Neoplasia
(A) Representative peripheral blood smears from WT and mutant females, Wright-Giemsa stained unless noted.
(B) Hematologic analysis of end-stage mutants and age-matched control.
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immature erythroids (binucleated rubricytes and metarubricytes;
nucleated RBC, up to 40% circulating in stage 2) (Figure 3B),
nuclear-cytoplasmic dysynchrony, dacrocytes (oddly shaped
RBC characteristic of myelofibrosis), and increased numbers
of Howell-Jolly bodies (condensed DNA remnants in mature
RBC) (Figure 3A). Circulating erythroid precursors also had cyto-
plasmic vacuolation and nuclear irregularity. Nonhemoglobin
iron revealed by Prussian blue staining (abnormal ring sidero-
blasts, siderocytes) and positive PAS staining (periodic acid-
Schiff) suggested features of erythroleukemia. Anemia was
characteristic, with the shift toward immature forms signaling
an ineffectual regenerative response consistent with lethargy
and rapid breathing in end-stage animals. We conclude that
deleting Xist in the blood compartment results in multilineage
defects, with features characteristic of myelofibrosis, myelopro-
liferation, and myelodysplasia.
indicates a hematological cancer termed myeloproliferative
neoplasm. Although the animal presentations varied, general
trends were evident and were indistinguishable between XistD/+
and XistD/XistDmice. The possibility of cancer is supported by
several coexisting conditions. Primary manifestations related
to two types of leukemia, chronic myelomonocytic leukemia
persistent monocytosis, neutrophilia, pseudo-Pelger-Huet cells
(0.5%–15.0% of circulating WBC), leukocytosis of up to 70,000
WBC/ml (Figures 3A and 3B), and increased numbers of circu-
lating promonocytes (<20%) and immature cells with myelomo-
nocytic features. In leukemic animals (e.g., 3-3-3 [XistD/D,
13.7 months], 27-1-1 [XistD/+, 6.3 months], and 23-2-1 [XistD/+,
9.6 months]), leukocytosis occurred together with splenomegaly
and general lymphadenopathy (Figure 4A). Large germinal
centers showed increased numbers of B cells, disorganized
T cells, histiocytes, and mitotic rates (Figure 4B). A coexisting
erythroleukemia-like syndrome was supported by erythrodys-
plasia, anemia, and expanded circulating erythroid precursors
with positive cytoplasmic PAS and Prussian-Blue staining (e.g.,
24-1-4, XistD/+, 11.9 months; 4-2-3, XistD/XistD, 12 months)
(Figures 3A and 3B).
Mutant animals also demonstrated a histiocytic sarcoma (HS,
also called malignant histiocytosis), a rapidly fatal cancer of
unknown but presumed hematologic origin. The HS was female
multinucleated giant cells, marked hemophagocytosis, and a
high mitotic rate (Figure 4D; see inset). Approximately 20% of
withmyelodysplasia and myeloproliferation
animals showed widespread HS in bone marrow, pancreas,
lung, liver, kidney, and/or lymph nodes (Figure 4D). Local
invasion led to spinal compression and limb paralysis (animal
4-2-1, XistD/XistD, 9.4 months); renal failure resulted from kidney
infiltration. Immunohistochemistry demonstrated a histiocytic
but nonLangerhans origin, confirming the diagnosis of meta-
static HS (Figures 4E and 4F: Mac2+, F4/80+, S100?, MPO?,
B220?, and CD3?).
A third manifestation was an unusual vasculitis, comprised not
of neutrophils but of lymphocytes and plasma cells (Figures 4B
and S2O). For example, in animal 8-1-1 (XistD/XistD, 7 months),
a massive cervical lymph node (>1 cm diameter, Figure 1D)
exhibited necrosis, dystrophic calcification, a disorganized
capsule, and a striking lymphoplasmacytic infiltrate around
blood vessels (Figure 4B, bottom panel). Multiple organs were
usually affected (Figures 4D and S2O), with accompanying
vessel destruction and organ damage (e.g., glomerulonephritis).
This rare vasculitis can be associated with lymphoma, though no
lymphoid malignancy was obvious.
Leukemia, HS, and vasculitis coexisted to varying degrees
and accounted fordifferent clinical presentations andoutcomes.
static masses consistent with HS in ?22%, and vasculitis in
?40%. These features suggested a diagnosis of ‘myeloprolifer-
ative neoplasm’ (MPN) with features of ‘myelodysplastic
syndrome’ (MDS), classified by the World Health Organization
(WHO) as ‘‘mixed MPN/MDS’’ (Tefferi, 2011; Vainchenker
et al., 2011). Indeed, primary myelofibrosis, CMML, and erythro-
leukemia are significant components of human MPN/MDS. We
conclude that deleting Xist results in a fulminant, highly lethal
Origin in Blood Cells Rather Than Stroma
topoietic cells, we performed three types of transplantation (Tp)
experiments: (1) mutant into wild-type (mutant-to-WT) Tp, where
lethally irradiated WT recipients were reconstituted with bone
marrow (n = 12 mice) or splenic cells (n = 6 mice) from mutant
females with overt disease, to determine whether the disease
could be conferred by hematopoietic cells alone (hematopoietic
3 mice), to determine whether the mutant host could induce
disease in genetically WT hematopoietic cells (hematopoietic
cell nonautonomous); and (3) WT-to-WT Tp to control for effects
of transplantation (n = 5 mice). At 1 month post-Tp with either
mutant bone marrow or splenic cells, recipients showed full
bone marrow reconstitution by donor cells, as determined by
Figure 4. Histiocytic Sarcoma and Lymphoplasmacytic Vasculitis
(A) Enlarged lymph nodes from end-stage mutants shown with a WT control. Cervical lymph nodes of 27-1-1 (XistD/+, 6.3 months old) and brachial lymph node of
31-2-1 (XistD/+, 5.8 months old) are shown.
(B) Top: sections of enlarged lymph node from 21-3-1 (XistD/+, 8.8 months old at death) with follicular B and T cell and intra- and interfollicular histiocytic
expansion are shown. Bottom: sections of enlarged lymph node from 8-1-1 (XistD/XistD, 7 months old at death) with a plasmacytoma-like infiltrate are shown.
Lymphoplasmacytic vasculitis is also shown. See also Figure S2O.
(C) Masses containing metastatic histiocytic sarcoma (arrows) in end-stage liver and kidney.
(D) H&E stains of metastatic histiocytic sarcoma in multiple organs. Scale bars represent 100 mm.
(E) Immunohistochemistry confirms histiocytic sarcoma. Scale bars represent 100 mm.
(F) Histiocytic sarcoma in bone marrow. Scale bars represent 100 mm. Note positive F4/80 staining for intralesional giant cells (inset).
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Ly-5.2+state of donor cells (FACS analysis, not shown). Signifi-
cantly, between 1 and 4 months post-Tp, multiple mice in the
mutant-to-WT Tp group became ill and recapitulated mixed
MPN/MDS (n = 11 mice) (Figures 5D, 5E, S3A, and S4).
First, monthly monitoring of peripheral blood demonstrated
progressive anemia and thrombocytopenia (Figures 5A and
S3A). Second, leukemia (17,000–64,000 WBC/ml) with high
monocyte and granulocyte counts was observed in multiple
donor-recipient combinations in the mutant-to-WT group (n = 7
mice), matching the CMML-like disease of Xist mutants (Fig-
ure S3A). Third, dysplastic changes identical to those in mutant
donors occurred in recipient blood and bone marrow, including
hypogranular neutrophils, binucleated RBC, pseudo-Pelger-
Huet cells, erythroid and megakaryocytic dysplasia, histiocyto-
sis, and multinucleated giant cells (n = 10 mice) (Figure 5B).
Fourth, necropsies of all dead and/or moribund animals revealed
splenomegaly in the mutant-to-WT Tp group (n = 8 mice) (Fig-
ure 5C). Fifth, metastatic HS was recapitulated in multiple recip-
ients (n = 3 mice). For example, bone marrow and splenic cells
from XistD/XistDdonor 12-1-2 separately gave rise to HS in three
different recipients, all reaching terminal stage within 40 days of
Tp from widespread metastases (1433, 1434, and 3944; Figures
5C, 5E, S4B, and S4C). Marrow effacement by histiocytes and
multinucleated giant cells and immunohistochemistry of infiltra-
tivemasseswere consistent with HS(Figures 5Eand S4).Finally,
exuberant myelofibrosis in the recipient occurred in multiple
donor-recipient combinations (n = 8 mice) (Figures 5E and
S4A), indicating that myelofibrosis could be induced solely and
rapidly (<2 months) by hematopoietic cells. None of the WT-to-
WT Tp controls showed these abnormalities (n = 5 mice) (Figures
5F and S3A).
Intriguingly, in the reverse Tp experiments (WT-to-mutant),
mutant phenotypes were rescued by WT donor hematopoietic
cells (n = 2). There was a normalization of spleen-to-body
mass ratios (Figure 5F) and peripheral blood profiles (Fig-
ure S3C). Moreover, myelofibrosis, tumor infiltration, and other
findings indicate that myelofibrosis and MPN/MDS could be
corrected by transplanting WT blood cells after radiation condi-
tioning of recipients. Thus, the Xist-deficient host environment is
not sufficient to confer disease. We conclude that blood cells—
rather than stroma—play the primary role in the pathogenesis of
A Primary HSC Defect
The multilineage hematopoietic phenotypes pointed to an HSC
‘‘LSK+’’ cells) and committed progenitor HSCs (Lin?SCA-1?
c-KIT+; ‘‘LSK?’’ cells) (Okada et al., 1992) revealed both qualita-
tive and quantitative defects (Figures 6A–6C). In mutant bone
marrow cells, an increased LSK+/LSK?ratio (Figures 6A and
lymphoid, and erythroid lineages were present (Figures S5C
and S5D), dysplastic changes (Figures 2 and 3) indicated that
maturation was incomplete. To test the differentiation capacity
of Xist-deficient HSCs, we performed competitive repopulation
assays (Harrison, 1980), in which we mixed mutant (n = 5) or
WT bone marrow cells (n = 4) with WT congenic bone marrow
cells (Ly-5.1+) at 1:1 ratio and transplanted them into lethally
irradiated recipients to test relative reconstitution potential.
Intriguingly, in all experiments, WT cells showed a statistically
significant competitive advantage over mutant cells in peripheral
blood (Figure 6D) and bone marrow (Figure 6E). Thus, mutant
cells were compromised in their ability to repopulate irradiated
hosts. Consistent with this, the numbers of each cell type
were generally decreased in mutant marrows (Figure 6F).
These results demonstrated a defect of mutant HSC to engraft
or mature. Interestingly, whereas none in the control group
died, three of five animals receiving mutant competitors
perished within 4 months. Thus, the maturation defect paralleled
the lethal tumorigenic potential of mutant cells, even in the pres-
ence of WT cells.
On further evaluation, time-course analysis showed an
age-dependent loss of SLAM-enriched long-term HSCs, as
evidenced by a decrease in the LSK+CD48?CD150+(SLAMs
[Kiel et al., 2005]) and LSK+CD34?Flt3?(Adolfsson et al.,
2005) populations (Figures 6A, 6B, and S5B), while the popula-
tion remained stable or even increased 2.5-fold over time in
WT mice (Figure 6C). This loss was consistent with progressive
myelofibrosis and pancytopenia in late-stage disease (Figures
2 and 3). Although the deficiencies were most apparent in late
stages, the increased LSK+/LSK?ratio and maturation defects
were already evident in predisease and early-disease mice (Fig-
ure S5). Furthermore, the defects were recapitulated in HSCs
derived from the spleen, a site of EMH (Figures 6G–6I, S6A,
Finally, reciprocal transplantation experiments showed that
WT recipients of mutant bone marrow cells recapitulated
the maturation defects (elevated LSK+/LSK?ratios) and loss
of long-term HSCs at 2 months post-Tp (Figures 6J and S3D).
By contrast, mutant recipients of WT bone marrow cells
showed a correction of the maturation defect and number of
long-term HSCs at 2 months post-Tp, in comparison to their
pre-Tp profiles (Figures 6K and S3E). We conclude that there
are intrinsic quantitative and qualitative defects in the mutant
Figure 5. Transplantability of MPN/MDS Suggests a Hematopoietic Rather Than Stromal Origin
(A) Schematic of transplantation experiments.
(B) Dysplasia in peripheral blood and bone marrow of mutant-to-WT transplants. See also Figures S3A–S3C.
(C) Histiocytic sarcoma recapitulated in WT recipients of mutant bone marrow or splenocytes. Recipient livers and spleen (1434) were pale from anemia. White
masses in recipient spleen contain histiocytic sarcoma (arrow). Mice succumbed within 40 days after transplantation.
(D) H&E stain of EMH in matched donor and recipient livers and spleens, as indicated.
(E) Immunohistochemistry confirms histiocytic sarcoma in donor and recipient (middle panels). Reticulin stain of bone marrow shows exuberant myelofibrosis in
donor (at necropsy) and recipient (40 days after transplantation).
(F) WT-to-mutant transplantations reversed disease. WT-to-WT transplantation controls were normal. Scale bars represent 100 mm. See also Figure S4.
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Genetic Pathway Reconstruction
To reconstruct a genetic pathway toward cancer, we carried out
expression profiling before disease (2 months old) and during
overt MPN/MDS and CMML-like disease (27-1-1, XistD/+,
6 months; and 4-2-3, XistD/XistD, 12 months) and compared
profiles of purified bone marrow HSC (LSK+CD34?), splenic B
cells (B220+), myeloid cells (CD11b+), and erythroid cells
(TER119+) against those of corresponding cell types from age-
matched WT females. Significant changes were observed in all
cell types before and during overt disease (Tables S1A–S1D).
Compared to the autosomal average, the average X-linked
gene expression was significantly higher (Figure 7A and Table
S1A; enrichment of upregulated genes on X compared to
autosomes; hypergeometric p z 0.010). In the predisease
state, upregulated X-linked genes included Tlr8, Gria8, Gyk,
and Gm14636. During disease, upregulated X-linked genes in
all cell types included Gata1, a crucial hematopoiesis factor
frequently mutated in leukemia (Zheng and Blobel, 2010), and
Kif4, a mitotic positioning motor protein aberrantly expressed
in erythroleukemia (Mazumdar et al., 2011). Thus, deletion of
Xist results in significant X reactivation, arguing that Xist is
required not only for initiation but also for maintenance of XCI
Significant genome-wide changes occurred before (Fig-
ure S1B) and during (Figures S1C and S1D) disease. Commonal-
ities and differences were also apparent between cell types
(Figure S7 and Tables S1E–S1G). Comparing predisease and
disease states, we observed a general increase in expression
changes across and within cell types, probably reflecting
disease progression. Notable autosomal deviations included
Cenpl (centromere protein L); Erc6l (kinetochore assembly
gene); Bmx (hematopoietic differentiation); Cxcr3 (leukocyte
trafficking cytokine); Med14, Hdac8, and Hmgn5 (transcription
and/or chromatin regulation); Pola1 (DNA replication); and
Lamp2 (tumor metastasis). Using the DAVID tool (Huang et al.,
2009), analysis of functional gene annotations among all differ-
entially expressed genes revealed significant enrichment of
genetic pathways involved in DNA replication, cell-cycle regula-
tion, hematopoiesis, and primary immunodeficiency (Benjamini-
Hochberg FDR % 6.67 3 10?5).
To investigate further, we performed correlation analysis and
hierarchical clustering of differentially expressed genesin Tables
S1B–S1D and uncovered additional functional categories (Fig-
ure 7B). In addition to pathways involved in DNA replication,
cell-cycle regulation, and hematopoiesis, we also identified
those in Ca+2-dependent leukocyte signaling, Toll-like receptor
signaling, MAPK kinase signaling, p53 regulation, and acute
myeloid leukemia (AML) and/or chronic myeloid leukemia
(CML) progression. When cross-referenced to the list of known
oncogenes and tumor suppressors (Higgins et al., 2007),
multiple upregulated oncogenes were identified (Table S1H),
such as Clspn (cell-cycle arrest), Espl1 (chromosome segrega-
tion), Pparg (nuclear receptor signaling), Myb (megakaryocyte
proliferation and maturation), and Csf1 (macrophage differentia-
tion factor), and several tumor suppressor genes and hemato-
poiesis regulators were downregulated (Table S1I), such as
Flt3 and c-Kit(Huang etal.,1998;Li etal.,2011).Several of these
genes were previously implicated as single-gene mutations in
MDS, MPN, and myeloid leukemias (e.g., Runx3, Kit, Foxo1,
Flt3, Rap2a, p53, Ezh2, and Rras [Pellagatti et al., 2010; Bejar
et al., 2011]; Aurka and Fos [Pang et al., 2011]; and Idh1, Tet2,
and CebpA [Brecqueville et al., 2012; Muramatsu et al., 2012]).
Taken together, our data indicate that MPN/MDS is initiated by
an Xist deletion that leads to X reactivation, which in turn results
in a series of autosomal changes causing DNA replication and
mitotic anomalies, genome instability, and dysregulation of the
hematopoiesis pathway (Figure 7C). Our data causally link the
X chromosome to cancer.
Here we have demonstrated that Xist suppresses hematologic
cancer. Deleting Xist, once thought to be essential only for
dosage compensation, is sufficient to induce an aggressive,
lethal blood cancer. The cancerisfemale specific and fully pene-
trant. Not a single mutant female—either heterozygous or
Figure 6. A Primary Defect in the HSC
(A) Representative FACS analysis of bone marrow cells from a 5-month-old female mutant and a WT littermate mouse showing increased LSK+/LSK?ratio and
decreased number of SLAM-enriched long-term HSCs (LSK+CD48?CD150+) in mutant mice.
(B) Histograms of bone marrow LSK+/LSK?ratios and SLAM-enriched long-term HSCs suggest, respectively, a failure of maturation and loss of progenitors in
mutant (n = 12) female mice in comparison to WT female mice (n = 13). Means ± SEM shown; Student’s t test, ***p % 0.001.
(C) Progressive loss of SLAM-enriched long-term HSCs in bone marrow of mutant mice (n = 12) over time as compared to WT mice (n = 13). R2= 0.5317.
(D and E) Competitive repopulation assays reveal maturation defects in mutant cells. WT (Xist2lox/+) or mutant (XistD/+) cells were mixed at 1:1 ratio with cells
isolated from congenic WT Ly-5.1+mice and transplanted into WT hybrid Ly-5.1+/Ly-5.2+recipients (n = 4–5 per group). Peripheral blood was sampled for FACS
analysis repeatedly for 2.5 months (D), and bone marrow was sampled at 2.5 months (E). Means ± are SEM shown; *p < 0.05, **p % 0.01.
(F) FACS analysis of bone marrow cells in 4-month-old mutant and WT mice (n = 4 mice per group). Means ± SEM shown; *p < 0.05, **p % 0.01.
(G) Representative FACS analysis of splenocytes from a 5-month-old female mutant and a WT littermate mouse showing an increased LSK+/LSK?ratio and
a decreased number of SLAM-enriched long-term HSCs in mutant mice.
(H)HistogramsofsplenicLSK+/LSK?ratiosandSLAM-enriched long-termHSCssuggest,respectively,afailure ofmaturationandlossofprogenitorsovertimein
mutant females (n = 4) as compared to WT females (n = 6). Means ± SEM shown. Student’s t tests show *p % 0.05 and **p % 0.01.
(I) Progressive loss of SLAM-enriched long-term HSCs in spleen of mutant mice (n = 4) over time as compared to WT mice (n = 6). R2= 0.846.
(J) FACS analyses show that HSC maturation defects (elevated LSK+/LSK?ratios) and loss of SLAM-enriched long-term HSCs are recapitulated in the WT
recipients of mutant bone marrow cells at 2 months post-Tp.
(K) FACS analyses of reverse transplantation (WT-to-mutant), with WT-to-WT control. At 2 months post-Tp with WT bone marrow cells, mutant 39-1-2 (XistD/+,
8.2 months) shows a correction of LSK+/LSK?ratio and number of SLAM-enriched long-term HSCs (right panels), in comparison to pre-Tp profiles (left panels).
See also Figures S3, S5, and S6.
Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc. 737
(legend on next page)
738 Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc.
homozygous—has escaped to date (n > 87). Our analysis
suggests that upregulation of X-linked genes after deleting Xist
leads to genome-wide changes in key developmental and
homeostatic pathways, which in turn drive progression toward
cancer (Figure 7C). A role for Xist RNA in suppressing cancer
adds to the growing list of functions associated with long non-
coding RNA (Huarte and Rinn, 2010; Lee, 2012).
The clinical presentation, histopathology, and cellular defects
are most consistent with mixed MPN/MDS (Tefferi, 2011; Vain-
chenker et al., 2011). MPN and MDS have been proposed to
be overlapping diseases along a spectrum; when there is signif-
icant overlap, they are categorized as mixed MPN/MDS. Signif-
icant components of the Xist mutant disease include primary
myelofibrosis, HS, leukemia (CMML and an erythroleukemia-
like disease), and lymphoplasmacytic vasculitis. Although the
WHO does not currently recognize HS as a component of
MPN/MDS, HS often occurs in the context of CMML and other
monocytic, myeloid, and lymphocytic leukemias (e.g., [Doll
et al., 1987; Laurencet et al., 1994; Feldman et al., 2004]).
Thus, human HS may be linked to MPN/MDS. Furthermore,
although a lymphoid leukemia is not a manifestation of the
mouse disease, lymphoplasmacytic vasculitis is suggestive of
an inflammatory or autoimmune disease, if not outright malig-
nancy. The lymphoid lineage is therefore also clearly affected
by the Xist deletion.
These multilineage defects or cancers coexist to varying
degrees in each female mutant. Their relative dominance prob-
ably results from stochastic developmental differences that
influence disease course. Like human MPN/MDS, the Xist-defi-
cient disease has a variable course. In humans, death eventually
results from leukemic progression and comorbid conditions
(e.g., infection, bleeding [Tefferi, 2011; Vainchenker et al.,
2011]). The mouse disease is also characterized by leukemic
progression, widespread infiltration and/or invasion, end-organ
failure, and a resulting constellation of comorbid conditions
Our study argues that MPN/MDS arises from a primary, cell-
autonomous defect in hematopoietic cells rather than bone
marrow stroma. Which hematopoietic cell is the source of the
disease phenotype cannot be definitively stated, but several
lines of evidence point to HSC. First, Xist is conditionally deleted
when HSCs first appear at E10.5 (Figure 1) and expression
profiling reveals aberrant changes in this population even in pre-
disease animals (Figures 7 and S7 and Table S1). Second, prolif-
erative and dysplastic changes are present in all hematopoietic
cell types (Figures 3 and 4). Third, malignancies occur in myelo-
monocytic, erythroid, and histiocytic lineages (Figures 3 and 4).
Fourth, our analyses point to maturation defects of the HSC
and age-dependent loss of long-term HSCs (Figure 6). Finally,
reciprocal transplantation experiments between WT and mutant
mice reveal recapitulation of MPN/MDS after transplantation of
hematopoietic cells alone (Figures 5, 6, S3, and S4). These
experiments also demonstrate that primary myelofibrosis and
HS arise from hematopoietic cells.
The dramatic phenotypes probably reflect the extensive
network of X-autosome interactions (Figures 7 and S7 and Table
S1). Although the Xi is initially unaffected (Brown and Willard,
1994; Csankovszki et al., 1999), we have now shown that
deleting Xist leads to failure of long-term Xi maintenance in vivo.
We propose that carcinogenesis is driven by a series of changes
occurring in the HSC and further accumulated in mature hema-
topoietic cells (Figure 7C). These changes are initiated by loss
of Xist, which leads to progressive X reactivation, which in turn
induces a cascade of unfavorable genome-wide changes that
includedysregulation ofgenesinvolved in DNAreplication, chro-
mosome segregation, cell-cycle checkpoints, and hematopoi-
esis. A failure of HSC maturation and loss of long-term HSC in
the marrow progressively shift hematopoiesis to extramedullary
sites resulting in EMH. The defects are recapitulated and ampli-
fied at sites of EMH. Together, aberrant hematopoiesis at central
and extramedullary sites result in MPN/MDS, with eventual
progression to myelofibrosis, leukemia, HS, and death from
Although loss of the Barr body has been correlated with
cancer for 60 years, here we have demonstrated direct causality
in mice. Our study implies that human hematologic cancers
may result from overdosage of X, either from XIST loss on Xi or
from duplication of Xa. Interestingly, MDS is more common in
women (Bennett and Orazi, 2009), with noted XIST deletions
and X chromosome duplications occurring in MPN, MDS, and
myeloid cancers (Dewald et al., 1989; Rack et al., 1994; Dier-
lamm et al., 1995; Paulsson et al., 2010). The association is not
restricted to women, however, as extra X chromosomes are
seen in ALL, AML, acute nonlymphoblastic leukemia (ANLL),
adult T cell leukemia, CML, erythroleukemia, and non-Hodgkin
lymphoma of both sexes (Sandberg, 1983); some 60% of child-
hood acute lymphoblastic leukemias (ALL) display extra X chro-
mosomes and an extra X may be the only aneuploidy in some
chronic myeloid leukemias (Heinonen et al., 1999; Yamamoto
et al., 2002). The Xist-deficient mouse provides a unique oppor-
tunity to study mixed MPN/MDS, HS, and primary myelofi-
brosis—one that can be approached from the perspective of
long noncoding RNAs and the sex chromosome so frequently
associated with human cancers.
Xist2lox/Xist2loxmice (129Sv/Jae strain) were a gift of R. Jaenisch (Csankovszki
et al., 1999). To generate XistD/+ and XistD/Y mice, we crossed Xist2lox/Xist2lox
females to Vav.Cre males [B6.Cg-Tg (Vav1-cre) A2Kio/J; Jackson Laboratory].
To generate homozygous mutants, we crossed Xist2lox/+;Vav-Cre females to
Xist2lox/Y males or Xist2lox/+ females to Xist2lox/Y;Vav-Cre males. Mice were
screened by PCR for Vav.Cre, XistWT, and Xist2loxalleles using the follow-
ing primer sets: Vav.Cre (522-1: 50-CTT CTC CAC ACC AGC TGT GGA-30,
Figure 7. Gene Expression Profiling by Microarray Analysis Reveals X Reactivation and Genome-wide Changes
(A) List of upregulated X-linked transcripts in blood cells of Xist mutants. See Figure S1A for details.
(B) Hierarchical clustering of transcripts that are differentially expressed between WT and mutant blood cells, as listed in Tables S1B,S1C, and S1D. Enriched
functional categories are indicated on left. See also Figure S7 and Tables S1A–S1I.
(C) A model for the pathogenesis and progression of cancer resulting from Xist loss.
Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc. 739
522-2: 50-GAC AGG CAG GGC CTT CTC TGAA-30; amplicon 580 bp) and Xist
(Xint3F: 50-GGC CAG TTT CTG ACA CCC TA-30, Xint3R: 50-CAC TGG CAA
GGT GAA TAG CA-30; XistWT200 bp, Xist2lox300 bp). Animal experiments
were approved by the MGH Institutional Animal Care and Use Committee
(IACUC). Dead and moribund animals (sacrificed per IACUC) were included
in the kill curve.
DNA and RNA FISH
RNA and DNA FISH were performed using established protocols (Takizawa
et al., 2008; Namekawa and Lee, 2011). Briefly, splenocytes were plated
bated at 37?C for 2 hr, and fixed with 4% PFA in 1xPBS containing 10% acetic
acid for 15 min at room temperature (RT). Cells were washed three times with
1xPBS at RT and kept in 70% EtOH at ?20?C until use. To detect Xist RNA, we
performed FISH using Alexa-fluor 488-dUTP-labeled Sx9 probe. We next
performed sequential RNA and DNA FISH, whereby RNA FISH was performed
first using Alexa-647-dUTP-labeled Sx9 probe, photographed, and then fol-
lowed by DNA FISH using Alexa-fluor-488-dUTP-labeled Sx7 and Cy3-
dUTP (Enzo Life Sciences)-labeled E9 probes. Fluorophore-labeled dUTPs
were from Molecular Probes unless noted. Images were obtained with a Nikon
Eclipse 90i microscope and a Hamamatsu CCD camera and analyzed using
Volocity Software (Improvision, Perkin-Elmer).
Tissues were fixed in 10% buffered Formalin (Fisher Scientific) and bones
were fixed and decalcified using Cal-Ex II (Fisher Scientific). Sections were
stained with hematoxylin and eosin (H&E) stain, Prussian blue, or reticulin
stains, as noted. For immunohistochemistry, B220 (550286; BD PharMingen),
Ki67 (VP-RM04; Vector), CD3 (ab16669; Abcam), Ter119 (550565; BD
PharMingen), MPO (sections from all organs, A0398; Dako; bone sections,
RB-373; Neomarkers), Mac2 (CL8942AP; Cedarlane), S100 (Z0311; Dako),
using a Nikon Eclipse90i microscope and a Q-imaging MicroPublisher RTV
color camera and analyzed using Volocity (Improvision, Perkin-Elmer).
Bone Marrow and Peripheral Blood Cytology
For live bone marrow aspiration, mice were anesthetized using isoflurane and
bone marrow cells aspirated using a 27 gauge needle from the femur through
the patellar tendon. The cells were then prepared for FACS analysis (below).
For bone marrow analysis at necropsy, cells were collected either by crushing
or flushing tibias and femurs with 3 ml of 1xPBS/5% FBS solution into a 50 ml
tube (Falcon) using a 22.5 gauge needle or by bone marrow brush prepara-
tions. Peripheral blood was collected by terminal cardiocentesis after CO2
administration or as survival bleeds using facial venipuncture into EDTA-
treated tubes (Becton, Dickinson and Company). Fresh blood and bone
marrow smears were stained with Wright-Giemsa (Fisher Scientific), Prussian
blue, and PAS (Sigma-Aldrich). Automated complete blood counts were
performed using the HemaTrue Veterinary Hematology Analyzer (Heska
Corporation, Loveland, CO).
Splenocytes or bone marrow cells were used for transplantation. Spleen
and bone (tibiae, femurs, iliac bones, humeri, and spine) were dissected,
crushed in cold 1xPBS with 2% FBS, and passed through a 40 mm filter.
Live mononuclear cells were isolated by gradient centrifugation using Ficoll-
Paque Plus (GE Healthcare). Blood cells were collected by retro-orbital
bleeds under anesthesia. The Ly-5.1 marker was used to distinguish donor
versus recipient. For mutant-to-WT or WT-to-WT transplantation,53 106cells
from mutant or WT littermate (Ly-5.2+) were transplanted into 6- to 12-week-
old lethally irradiated (9.5 Gy) WT Ly-5.1+/Ly-5.2+F1 hybrid recipients
(129/SvJ x B6.SJL-PtprcaPepcb/BoyJ; Jackson Laboratories). For reverse
transplantation (WT-to-mutant), 5 3 106cells from Ly-5.1+hybrids were
transplanted into lethally irradiated mutant recipients. For competitive trans-
plantation assays, 2 3 106cells from mutant or WT littermates were mixed
with 2 3 106cells from B6.SJL-PtprcaPepcb/BoyJ (Ly-5.1+) WT competitors
and transplanted into WT Ly-5.1+/Ly-5.2+hybrid recipients. Irradiated recipi-
ents were maintained on sterile water containing 0.5 g/l of enrofloxacin (Bayer,
Shawnee Mission, KS) for 2 weeks after irradiation.
All antibodies were from BD PharMingen unless noted. The following mono-
clonal antibodies were used for HSC analysis: SCA-1 (D7; Biolegend), CD34
(RAM34; Ebioscience), CD135 (A2F10.1), CD150 (TC15-12F12.2; Biolegend),
c-KIT (2B8), and CD48 (HM48-1). Biotinylated antibodies against CD11b
(M1/70), Gr-1 (RB6-8C5), CD3ε (145-2C11), B220 (RA3-6B2), CD8a (53-6.7),
CD4 (GK1.5), and TER-119 (TER-119) were used as lineage markers in combi-
nation with a Pacific Orange-Streptavidin conjugate (Invitrogen). Ly5.1 (A20;
Biolegend) was used for donor cell tracing. For lineage-specific markers, the
following were used: Erythroids (anti-Ter-119, TER-119 and anti-CD71, C2),
T cells (anti-CD3, 500A2), B cells (anti-B220, RA3-6B2), and myeloids (anti-
CD11b, M1/70, and anti-Gr-1, RB6-8C5). Biotin-labeled cocktails containing
CD3, B220, Gr-1, Ter119, and CD11b antibodies (BD-Biotin mouse lineage
panel, 559971) were used to differentiate LSK+and LSK?cells in combination
with SCA-1 (D7; Biolegend) and c-KIT (2B8) antibodies. SYTOX AADvanced
Dead Cell Stain Kit was used to exclude dead cells. FACS data were acquired
using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA) and was
analyzed with FlowJo version 8.8.6 for Mac.
B-lymphoid,myeloid, erythroid cells, and HSCs were isolated from predisease
XistD/+) and age-matched 37-1-1 (Xist2lox/+) were used. For leukemic cases,
27-1-1 (XistD/+, 6 months old, WBC 64 3 103cells/ml) and 4-2-3 (XistD/XistD,
12 months old, 35.7 3 103cells/ml) and age-matched control 27-1-3 were
used. To isolate LSK+HSCs, we depleted bone marrow cells of lineage-
specific cells using Mouse Lineage mixture Biotin conjugate, MLM15 (Invitro-
gen), and MyOne Streptavidin T1 Dynabeads (Invitrogen). LSK+CD34?HSCs
were then sorted by using SCA-1 (D7)-PE-Cy7, c-KIT (2B8)-APC, and CD34-
FITC (RAM34, eBioscience) antibodies. Between 3 3 103and 6 3 103HSCs
were acquired. Splenocytes from the same animals were sorted for B cells
(B220-APC and RA3-6B2), myeloids (CD11b-PE and M1/70), and erythroids
(Ter119-APC and Ter-119). For each, 105–106cells were obtained. Cells
were sorted using BD Sorp Vantage SE DiVa cell sorter (BD Biosciences). All
antibodies were from BD Biosciences unless noted. Total RNA was isolated
using TRIzol Reagent (Invitrogen). RNA was processed using NuGEN Ovation
Pico WTA System V2 paired with the Encore Biotin Module (NuGEN), and
labeled cDNA probes were hybridized onto Affymetrix mouse Gene 1.0ST
arrays (Affymetrix). Expression data were normalized by RMA (Irizarry et al.,
2003). Data from diseased 27-1-1 and 4-2-3 were normalized to control 27-
1-3; data from predisease 38-1-2 and 38-1-1 were normalized to control 37-
1-1. Differentially expressed genes in Table S1 differed by two-fold or more
between controls and mutants.
The GEO accession number for our microarray data set is GSE43961.
Supplemental Information includes seven figures and one table and can be
found with this article online at http://dx.doi.org/10.1016/j.cell.2013.01.034.
We are grateful to members of the Lee lab for stimulating discussions. We
thank R. Jaenisch for Xist2lox/Xist2loxmice, G. Pihan and A. Sohani for hemato-
pathology advice, H. Hock for advice on Vav-Cre mice, W. Press for mainte-
nance of Xist2lox/Xist2loxmice colony, the MGH CCM-Clinical Pathology labo-
ratory for assistance on hematological analysis, D. Dombkowski for FACS
assistance, and A.J. Zall for survival bleed collections. This work was sup-
ported by the MGH ECOR Medical Discovery Fund (E.Y.), Clinician-Scientist
Training Award from the Canadian Institutes of Health Research (F.E.M.),
740 Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc.
a National Institutes of Health grant (HL44851, D.T.S.), and the Howard
Hughes Medical Institute, where J.T.L. is an Investigator.
Received: February 28, 2012
Revised: December 4, 2012
Accepted: January 23, 2013
Published: February 14, 2013
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