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Stem Cell Reports
Ar ticle
Identification of Multipotent Progenitors that Emerge Prior to Hematopoietic
Stem Cells in Embryonic Development
Matthew A. Inlay,
1,5,
*
Thomas Serwold,
2
Adriane Mosley,
1
John W. Fathman,
1,3
Ivan K. Dimov,
1
Jun Seita,
1
and Irving L. Weissman
1,4
1
Institute for Stem Cell Biology and Regenerative Medicine (ISCBRM), Stanford University, Stanford, CA 94305, USA
2
Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA
3
Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA
4
Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
5
Present address : Sue and Bill Gross Stem Cell Research Center, Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine,
CA 92697, USA
*Correspondence: minlay@uci.edu
http://dx.doi.org/10.1016/j.stemcr.2014.02.001
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
SUMMARY
Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within
which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-
renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at
the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryo-
genesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a
defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-
gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and
AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.
INTRODUCTION
In the mammalian blood system, all mature blood lineages,
including erythrocytes, platelets, and all innate and adap-
tive immune cells, are generated from hematopoietic
stem cells (HSCs). In adults, HSCs reside almost exclusively
in the bone marrow. In the embryo, however, hematopoi-
esis is characterized by distinct yet overlapping waves of
blood development, appearing in multiple sites, with prim-
itive erythroid-biased waves succeeded by definitive waves
with increasing lineage potential and functionality. The
functional properties that define adult HSCs do not appear
at once during development but emerge gradually over the
course of several days.
In the mouse embryo, the first blood-forming cells
appear approximately 7.5 days into gestation (embryonic
day [E] 7.5) within the blood islands that line the extraem-
bryonic yolk sac (YS) (Moore and Metcalf, 1970). These
‘‘primitive’’ blood-forming cells appear to be lineage-
restricted, form primarily large nucleated erythrocytes,
and express embryonic globins (Palis et al., 1999). They
also lack the ability to engraft when transplanted intrave-
nously into lethally irradiated adult mice, a hallmark prop-
erty of fully functional adult bone marrow HSCs (Mu
¨
ller
et al., 1994). After the establishment of a circulatory system
at e8.5, ‘‘definitive’’ erythromyeloid progenitors appear
within the YS (Palis et al., 1999), the placenta (PL)
(Alvarez-Silva et al., 2003), and the embryo proper (EP).
The earliest intraembryonic hematopoietic progenitors
are found within the para-aortic splanchnopleura (p-Sp),
which develops into the aorta-gonad-mesonephros
(AGM) that contains the dorsal aorta (Cumano et al.,
1996; Godin et al., 1993, 1995; Medvinsky et al., 1993).
Hematopoietic progenitors with the ability to self-renew
appear within the YS and AGM at e9.0 and appear within
the fetal liver (FL) a day or two later (Yoder and Hiatt,
1997). e9.5 YS cells lack the ability to home to the bone
marrow when transplanted into adult mice, but their
long-term self-renewal activity can be revealed in vivo by
transplantation into the liver or facial vein of sublethally
irradiated newborn mice (Yoder and Hiatt, 1997; Yoder
et al., 1997a, 1997b) or alternatively by first coculturing
with reaggregated AGM tissue (Taoudi et al., 2008)oron
the OP9 bone marrow stromal line (Rybtsov et al., 2011),
indicating that progenitors residing within the YS can
mature into functional HSC. These embryonic progenitors
were thought to be precursors to HSCs, or ‘‘pre-HSCs,’’ and
whereas not precisely defined, pre-HSCs expressed markers
associated with endothelial (VE-cadherin) and hematopoi-
etic (CD41 then CD45) cells (Rybtsov et al., 2011). At e10.5,
fully functional HSCs have been isolated from the dorsal
aorta of the AGM region (Mu
¨
ller et al., 1994), the extraem-
bryonic YS, PL (Gekas et al., 2005), and from the vitelline
and umbilical vessels (de Bruijn et al., 2000). At e11.5,
HSCs are also found within the FL, which then becomes
the predominant site of hematopoiesis until the formation
Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors 457
A
B
C
D
EF
Figure 1. Development of an In Vitro Clonal Multipotency Assay
(A) Lentiviral constructs used to generate a tetracycline-inducible Dll1 OP9 stromal line (ODT). Construct C329 (top) drives constitutive
expression of the tetracycline-inducible transactivator rtTA3. Construct C388 allows induction of Dll1 expression when the transactivator
is activated in the presence of doxycycline (DOX).
(B) Strategy for clonal assay. ODT stroma is plated the day prior to cell sorting (day 1). At day 0, cells are clone sorted directly onto ODT
stroma and cytokines are added (SCF, TPO, EPO, Flt3L, IL-7, and IL-15). At day 3, hematopoietic colonies are counted. At day 5, cells are fed
and Flt3L, IL-7, IL-15, and DOX (1 mg/ml) are added. At day 9 or 10, colonies are harvested and analyzed by FACS.
(C) Representative hematopoietic output at day 10 of culture of unsorted e12.5 FL. Representative examples of multipotent output from
single-cell cultures can be found in Figure S1.
(D–F) Testing multipotency assay on adult bone marrow (BM) stem/progenitor cells. (D) Sort gates for KIT
+
Lin
SCA-1
+
(KLS) cells and
subpopulations of KLS, including HSCs (CD34
, SLAMF1
+
) and three fractions of multipotent progenitors (MPP) are shown. (E) The
(legend continued on next page)
458 Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
of a bone-marrow cavity several days later ( Gekas et al.,
2005; Mu
¨
ller et al., 1994). Thus, the maturation of blood-
forming cells takes place in discrete steps and likely at
several different sites.
A fundamental unresolved question is whether definitive
hematopoietic cells derive directly from the primitive
precursors that first appear in the YS blood islands (Moore
and Metcalf, 1970) or instead emerge separately from a
hematoendothelial precursor in the dorsal aorta called
hemogenic endothelium (Dzierzak and Medvinsky, 1995;
Nishikawa et al., 1998). A large body of evidence supports
the de novo generation of HSCs within the dorsal aorta,
including ex vivo tissue explants of the dorsal aorta prior
to circulation (Cumano et al., 1996, 2001; Medvinsky and
Dzierzak, 1996). Also, time-lapse imaging of AGM sections
in culture reveals the emergence of hematopoietic clusters
from within the luminal wall of the dorsal aorta in mice,
which express several HSC markers, such as KIT, SCA-1,
and CD41 (Boisset et al., 2010). Definitive hematopoietic
progenitors also exist within the YS (Huang and Auerbach,
1993; Kumaravelu et al., 2002). However, early studies
could not exclude the possibility that such progenitors
originated elsewhere and then migrated to the YS. Evidence
supporting a distinct YS origin of definitive hematopoiesis
comes from lineage-tracing experiments that used a
Runx1 Cre-estrogen receptor (ER) reporter to exclusively
label YS-derived hematopoietic cells; subsequent anal-
ysis of these mice revealed labeling of adult HSCs
(Samokhvalov et al., 2007). Similarly, inducible rescue of
Runx1 expression in Runx1 knockout embryos demon-
strated that definitive hematopoiesis could only be rescued
at the developmental stages when Runx1 expression was
restricted to the YS (Tanaka et al., 2012). In Ncx1
/
embryos, which lack a heartbeat and thus circulation, all
hematopoietic cells are found within the YS and PL
prior to embryonic lethality at e10.5 (Lux et al., 2008;
Rhodes et al., 2008). Additionally, transplantation of YS
cells from e8 to e9 allogeneic donors into the YS cavities
of e8 to e9 hosts in utero led to YS blood-island engraft-
ment and, when analyzed several months after birth,
gave rise to donor-derived spleen colony-forming myeloer-
ythroid cells and thymic and peripheral T cells (Weissman
et al., 1977, 1978). Therefore, maturation of early YS stem/
progenitors to adult HSC was demonstrated, but the
cellular identity of HSC precursors, their sites of matura-
tion, and the molecular mechanisms involved remain a
mystery.
Identification of the key populations that give rise to
each wave of embryonic hematopoiesis may provide
critical insights into the relationship between primitive
and definitive hematopoiesis. However, the surface
markers used to isolate adult HSCs and downstream stages
have proven unreliable for identifying the equivalent
embryonic populations (Cumano and Godin, 2007). As a
result, the cells that initiate each hematopoietic wave
remain poorly defined. Multipotency in hematopoiesis
refers to the ability of a progenitor to give rise to all blood
lineages: myeloid cells including erythrocytes, platelets,
monocytes, tissue macrophages, and granulocytes, as well
as lymphocyte lineages including T, B, natural killer (NK),
and dendritic cells. Multipotency also distinguishes defini-
tive hematopoiesis from more primitive cells with limited
lineage potential. Conclusive evidence of multipotency
requires a single-cell assay to prevent the false positives
that may occur when mixtures of lineage-committed
progenitors collectively produce all lineages. In the em-
bryo, multipotency was first observed within the AA4.1
+
p-Sp population at e9.5 (Godin et al., 1995). YS AA4.1
+
WGA
hi
cells possess myeloid and lymphoid potential
in vitro by e10.5 and robustly by e11.5, though this was
not demonstrated clonally (Huang and Auerbach, 1993).
Thus, whereas much is known about the timing of appear-
ance of progenitor potentials within the early embryo, the
clonal identities of the cells with multipotent potentials are
less clear. Therefore, we sought to define the earliest clon-
ally multipotent cells and to determine their distribution
within the sites of early hematopoiesis.
RESULTS
Development of an In Vitro Clonal Multilineage Assay
In order to identify clonally multipotent populations
throughout embryonic development, we developed a
single-cell multipotency assay (Figure 1). The bone-
marrow-derived OP9 stromal line can be used to generate
most hematopoietic lineages in culture, with the notable
exception of T lymphocytes (Kodama et al., 1994). Alterna-
tively, a modified OP9 stromal line that expresses Dll1
(OP9-DL1) can promote T lineage development but
inhibits B cell development (Schmitt and Zu
´
n
˜
iga-Pflu
¨
cker,
2002). Because of the importance of detecting lymphocyte
potential in distinguishing definitive hematopoietic cells
from their primitive myeloid-restricted counterparts, we
distribution of lineage potential of colonies from adult BM KLS cells, showing cells that produced a single branch (MegE in red, GM in green,
and L in blue), two branches (MegE + GM in orange, GM + L in yellow, and MegE + L in purple), and all three branches (MegE + GM + L in
white). Cells that produced lineages from all three branches (white) were scored as multipotent. Cells that gave rise to colonies that did not
survive to day 10 are shown in black. The number of hematopoietic colonies scored (n) is indicated. (F) Distribution of lineage potential in
colonies derived from sorted KLS subpopulations. Note that only HSCs gave multipotent (white) readout.
Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors 459
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
sought a method to generate both B cells and T cells from a
single cell within a single well. We previously published an
assay whereby common lymphocyte progenitors could be
cultured and expanded on OP9 stroma without commit-
ting to either the B or T cell lineages (Karsunky et al.,
2008). We therefore generated an OP9 stromal line with
an inducible Dll1 expression cassette (Figure 1A). In this
line, which we call ODT, addition of doxycycline (DOX)
rapidly induces surface expression of Dll1 , driving uncom-
mitted lymphocyte progenitors to the T cell lineage,
whereas B-committed progenitors resist T cell induction
and continue to produce B cells (M.A.I. and I.L.W., unpub-
lished data). Hematopoietic progenitors were plated on
ODT stroma along with a combination of hematopoietic
cytokines (SCF, TPO, EPO, Flt3L, interleukin [IL]-7, and
IL-15); DOX was added 4 to 5 days into the culture (Fig-
ure 1B), leading to eight hematopoietic lineages (erythro-
cytes, platelets, macrophages, granulocytes, dendritic cells,
natural killer cells, B cells, and T cells), representing the
three major branches of hematopoiesis: megakaryocyte/
erythrocyte (MegE), granulocyte/monocyte (GM), and
lymphoid (L) (Figure 1C). Representative fluorescence-
activated cell sorting (FACS) plots of single-cell-derived
colonies scored as multipotent can be found in Figure S1
available online. To validate this assay, we sorted adult
bone marrow (BM) KIT
+
Lin
SCA-1
+
(KLS) cells, a popula-
tion that contains HSCs and multipotent progenitors, and
analyzed their clonal lineage potential (Figures 1D and 1E).
Individual KLS cells produced a variety of different lineage
outcomes: some cells yielded only a single lineage, others
generated multiple lineages, and about 15% of colonies
produced lineages representing each of the three major
hematopoietic branches. These colonies we scored as mul-
tipotent. We next separated the KLS population into four
subsets based on expression of CD34, SLAMF1, and FLK2,
markers that identify HSCs and other multipotent progen-
itor populations, and repeated the assay (Figures 1D and
1F). Whereas each population could collectively produce
all lineages, we found that only the HSC population
(CD34
FLK2
SLAMF1
+
KLS) contained multipotent cells,
despite the fact that all KLS subpopulations are known to
be multipotent in vivo. Thus, this assay can reveal multipo-
tency of individual cells, although not all multipotent cells
are revealed.
Nearly All Hematopoietic Colony-Forming Cells
Reside in the KIT
+
CD43
+
Fraction
In our assay conditions, hematopoietic stem and progeni-
tor cells give rise to distinct colonies by day 3, and thus
we could also use this assay to identify markers for these
cells. We initially screened eight markers (KIT, CD43,
CD34, CD41, SCA-1, CD45, AA4.1, and SLAMF1) from
YS, AGM, and FL tissues from e9.5 to e12.5 to identify
which markers could consistently enrich or deplete for
colony-forming activity (Figure S2). For most markers,
colony-forming activity was found in both the positive
and negative fractions. For example, whereas the hemato-
poietic marker CD41 could enrich hematopoietic colony-
forming ability at e9.5, it became downregulated at later
time points, resulting in colony-forming activity within
the CD41
fraction (Figure S2A). However, two markers,
KIT and CD43, nearly uniformly marked all colony-form-
ing cells in all tissues from e9.5 to e12.5.
Emergence of Multipotent KLS Cells during
Embryogenesis
Based on our initial screen, we restricted the search for
multipotent cells to subpopulations within the KIT
+
CD43
+
fraction. We stained tissues with a variety of anti-
bodies to identify candidate populations within the KIT
+
CD43
+
fraction that we could test for clonal multilineage
potential (Figures 2 and S3). In the adult, all multipotent
stem and progenitors are found in the KLS fraction. We
identified a similar population that is KIT
+
CD43
+
and
SCA-1
+
, which appears in e9.5 YS and AGM and e11.5 FL
(Figure 2). This population is highly enriched for colony-
forming activity (between 35% and 50% of plated cells
gave rise to colonies) and, as a population, gives rise to all
lineages in vitro (data not shown). We refer to this embry-
onic population as KLS (KIT
+
Lin
SCA-1
+
) to reflect its
similarity to the analogous KLS population in adult BM,
although embryonic KLS cells also are defined by expres-
sion of CD43, and the only required lineage marker
for negative gating is the red-blood-cell marker TER119
(Figure S3A).
In adult BM, myeloid progenitors are found within the
KIT
+
SCA-1
(MYP) fraction, which contains common
myeloid progenitors (CMP), granulocyte-monocyte pro-
genitors (GMP), and megakaryocyte-erythrocyte pro-
genitors (MEP) ( Akashi et al., 2000). In the embryo, we
identified a similar MYP population, from which emerged
CMP and then MEP and GMP, both by surface phenotype
and in vitro lineage potential (Figure S3B; data not shown).
CD11A Expression Divides Embryonic KLS into Two
Distinct Fractions
The surface markers SLAMF1, FLK2, and CD34 are known
to subdivide adult KLS into HSCs and downstream multi-
potent progenitors (Figure 1D). We examined these
markers in embryonic KLS and found that their expression
levels varied from time point to time point and from tissue
to tissue, making the use of these markers unreliable to sub-
divide embryonic KLS prior to e11.5 (Figure S4). However,
we did find that the KLS population could be consistently
subdivided based on expression of CD11A (Figure 3A).
CD11A (Itgal) is a component of the leukocyte adhesion
460 Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
complex lymphocyte function associated 1 (LFA-1), an
integrin involved in several immune system functions
including leukocyte trafficking and lymphocyte activation
(Hibbs et al., 1991; Hogg et al., 2011). In a related
manuscript, we show that adult BM HSCs can also be
subdivided based on CD11A expression and only the
CD11A
fraction contains functional HSCs (J.W.F., M.A.I.,
Nathaniel B. Fernhoff, J.S., and I.L.W., unpublished data).
CD11A
KLS cells uniformly expressed high levels of the
endothelial adhesion molecule VE-cadherin (VE-CAD),
whereas CD11A
+
KLS cells expressed variable levels of VE-
cadherin (Figure 3A). We also examined the expression of
several other markers including the endothelial-associated
markers TIE2 and endoglin and the hematopoietic markers
CD45 and MAC-1 (Figure S5). Whereas the expression
patterns of these markers were less consistent than
CD11A and VE-cadherin across all tissues and time points,
in general we found that endothelial markers were
more highly expressed at earlier time points and on
CD11A
KLS cells, whereas hematopoietic markers were
more highly expressed at later time points and on
CD11A
+
KLS cells.
We next tested CD11A
and CD11A
+
KLS cells for clonal
multilineage potential (Figures 3B and 3C). In adult BM,
the KLS population contains all multipotent cells (Figures
1D–1F). At the population level, both CD11A
and
CD11A
+
KLS cells were able to give rise to all lineages,
including lymphoid B, T, and NK cells (data not shown).
However, only the CD11A
fraction was able to simulta-
neously give rise to MegE, GM, and lymphoid cells at the
single-cell level, indicating the presence of multipotent
cells within this population (Figures 3B and 3C). We found
multipotent CD11A
KLS cells in all tissues examined,
from e9.5 to e11.5. Conversely, in the CD11A
+
KLS
010
3
10
4
10
5
0
10
3
10
4
10
5
1.99
25
010
3
10
4
10
5
0
10
3
10
4
10
5
12.4
47.6
010
3
10
4
10
5
0
10
3
10
4
10
5
4.9319.7
010
3
10
4
10
5
0
10
3
10
4
10
5
1.43
55.6
010
3
10
4
10
5
0
10
3
10
4
10
5
15.546.5
010
3
10
4
10
5
0
10
3
10
4
10
5
7.38
10.5
010
3
10
4
10
5
0
10
3
10
4
10
5
3.53
39.5
010
3
10
4
10
5
0
10
3
10
4
10
5
24.1
43.5
010
3
10
4
10
5
0
10
3
10
4
10
5
11.5
10.6
010
3
10
4
10
5
0
10
3
10
4
10
5
14.6
5.21
Adult BM
e9.5 e10.5 e11.5
AGM
FL
YS
SCA-1
KIT
KLS MYP
Gate: CD43
+
TER119
-
Figure 2. KIT versus SCA-1 Expression of CD43
+
Cells in YS, AGM, and FL from e9.5 to e11.5
KIT (y axis) and SCA-1 (x axis) expression of CD43
+
adult BM is shown in the upper left corner. Gates of KIT
+
SCA-1
+
‘‘KLS’’ and KIT
+
SCA-1
myeloid progenitors ‘‘MYP’’ are shown, with the percent of KLS and MYP populations among CD43
+
TER119
cells indicated. See Figure S3A
for CD43/TER119 plots and gates.
Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors 461
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
A
B
C
(legend on next page)
462 Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
population, we found bipotent MegE/GM and GM/L col-
onies but never all three branches at once, regardless of
time point or tissue (Figure 3C). Thus, the CD11A
KLS
cells population contains all the multipotent cells from
e9.5 to e11.5 and appears in both extra- and intraem-
bryonic regions around e9.5.
YS Contains the Most Multipotent Cells from E9.5 to
E11.5
Our assay also allowed us to estimate the numbers of multi-
potent progenitors in each tissue from e9.5 to e11.5, based
on colony-forming activity (Figure 4A). We found that,
from e8.5 to e10.5, the YS contained the majority of
Figure 3. Clonal Analysis of CD11A
and CD11A
+
KLS
(A) VE-cadherin (VE-CAD; y axis) versus CD11A (x axis) expression of embryonic KLS from e9.5 to e11.5.
(B) Percent of multipotent colonies from clone-sorted CD11A
and CD11A
+
KLS subsets. The percent of multipotent colonies arising from
adult BM KLS is indicated in black and was duplicated from Figure 1E. For e9.5 and e10.5, CD11A
and CD11A
+
KLS cells derived from the
entire embryo proper (EP) were analyzed. The number of multipotent colonies observed out of total colonies scored is listed in parentheses.
(C) Lineage distribution of CD11A
and CD11A
+
KLS colonies. The distribution of lineages that resulted from clone-sorted CD11A
and
CD11A
+
KLS cells onto ODT stroma from e9.5 to e11.5 is shown. A description of the scoring system can be found in the legend to Figure 1E.
The percentage of colonies scored as multipotent is shown as white bars and is identical to the data shown in (B).
AB
CD
E
Figure 4. Numeric Analysis of Hemato-
poietic Progenitor Cells in Embryonic
Development
(A) Absolute number of colony-forming
cells per embryo from YS (blue squares),
AGM (red downward triangles), and FL
(green diamonds) from e8.5 to e11.5. At
e8.5 and e9.5, the whole embryo proper (EP;
orange upright triangles) was cultured
instead of AGM and FL. Unsorted tissues
were plated onto ODT stroma at serial
dilutions and colonies counted at day 3. The
number of litters analyzed (n) for each time
point is indicated.
(B–D) The absolute numbers of CD11A
KLS
(B), CD11A
+
KLS (C), and MYP (D) cells were
calculated for YS (blue), AGM (red), and FL
(green) from e9.5, e10.5, and e11.5. The
numbers shown are per tissue, per embryo.
Error bars are SD. The asterisk (*) in (B)
indicates a statistically significant differ-
ence (unpaired t test) between the absolute
number of CD11A
KLS cells in the YS and
AGM at e10.5 (p < 0.002). The number of
litters analyzed (n) for each tissue (YS in
blue, AGM in red, FL in green) is indicated.
(E) Estimation of the absolute number of
clonally multipotent cells per tissue per
embryo. These numbers were generated by
multiplying the absolute number of CD11A
KLS (from B) times the percent of multi-
potent cells contained within (from Fig-
ure 3B).
Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors 463
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Mouse Embryonic Multipotent Hematopoietic Cells
A
BC
(legend on next page)
464 Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors
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Mouse Embryonic Multipotent Hematopoietic Cells
progenitors, peaking around 1,000 colony-forming cells at
e10.5 and e11.5. By e11.5, the FL had surpassed the YS
and became the predominant source of hematopoiesis
from then on. At all time points examined, the AGM con-
tained the fewest colony-forming cells.
We also estimated the absolute number of CD11A
KLS,
CD11A
+
KLS, and MYP (Lin
CD43
+
KIT
+
SCA-1
) popula-
tions in each tissue (Figures 4B–D). Collectively, these three
populations contain all KIT
+
CD43
+
cells and, thus, likely
all hematopoietic stem/progenitors. We found that the
YS contained approximately 300 CD11A
KLS cells per
embryo at e10.5, roughly six times as many as in the
AGM at this time point (Figure 4B). Furthermore,
CD11A
KLS numbers appeared to decrease in the YS at
e11.5 and increase in the AGM and FL. At e11.5, the FL
contained the most CD11A
+
KLS and MYP (Figures 4C
and 4D). Because CD11A
KLS cells contain all multipotent
activity at e11.5, this suggests that the bulk of the cells that
initially seed the FL are CD11A
+
KLS and MYP, which we
hypothesize are downstream of CD11A
KLS.
With our estimates of the absolute number of CD11A
KLS cells (Figure 4B) and the frequency of multipotent cells
within each tissue (Figure 3B), we could estimate the
absolute number of multipotent cells (Figure 4E). We
found that e10.5 YS contains the most multipotent cells
of all tissues and time points examined, and at e11.5, the
YS still contained more multipotent cells than AGM or
FL. Taken together, our examination of the absolute num-
ber of CD11A
KLS and multipotent cells suggests that
the YS is a major source of multipotent cells at the stages
when fully functional HSCs first appear in embryonic
development.
EPCR Marks a Subset of CD11A
KLS and Can Partially
Enrich for Multipotency
The protein C receptor EPCR (CD201; Procr) has previously
been reported to be expressed in adult HSCs, as well as
embryonic FL e12.5 HSCs (Balazs et al., 2006; Iwasaki
et al., 2010). We found that EPCR is expressed on a subset
of KLS cells as early as e9.5 and remains expressed
throughout gestation (Figure 5A). EPCR expression corre-
lates with high VE-CAD expression and lower CD11A
expression. Using the clonal multipotency assay, we
observed multipotent colony formation from both EPCR
+
and EPCR
subsets of CD11A
KLS cells, though multipo-
tency appeared greater in the EPCR
+
subfraction overall
(Figures 5B, 5C, and S1). We also identified CD11A
and
CD11A
+
KLS cells in the placenta (Figure S6), a region
known to be a niche for hematopoietic stem cells (Gekas
et al., 2005; Ottersbach and Dzierzak, 2005), and included
placental populations in the analysis. Although we did
not observe multipotency in e10.5 PL CD11A
KLS, we
did find it within the EPCR
+
CD11A
KLS at e11.5 in the
PL. Taken together, our data suggest that EPCR can partially
enrich for multipotent cells, though not all multipotent
cells are EPCR
+
.
Lineage-Tracing KLS Cells In Vivo
To better understand the lineage relationship between
CD11A
and CD11A
+
KLS cells, we examined two line-
age-tracing reporter mouse strains (Figure S7). CD11A
KLS cells generally express higher levels of TIE2 than
CD11a
+
KLS cells (Figure S5A); thus, we crossed Tie2
Cre
mice (Kisanuki et al., 2001) to the mT/mG reporter strain
(Muzumdar et al., 2007), which permanently switches
from Tomato to GFP fluorescence in any cell that expresses
Cre (Figure S7A). In the Tie2
cre
3 mT/mG embryos, we
found that all KLS cells were GFP
+
and thus derived from
TIE2-expressing precursors (Figure S7B). However, other
cell types express TIE2, including hemogenic endothelium,
so we next crossed VE-cadherin
CreER
mice (Monvoisin et al.,
2006; Zovein et al., 2008) to mT/mG reporters (Figures
S7C–S7E). In this cross, when tamoxifen is administered,
all cells that express VE-cadherin during the 24 hr window
when tamoxifen is active will permanently express GFP in
themselves and in their progeny. Because VE-cadherin
expression is associated with earlier time points and more
multipotent cells, we hypothesized that VE-CAD
+
cells
would precede VE-CAD
cells. We injected tamoxifen
into pregnant females and examined embr yos at 24, 48,
and 72 hr after injection to monitor the distribution of
GFP-expressing KLS cells. We found that, at 24 hr, the
majority of labeled KLS cells were VE-CAD
+
(both
CD11A
and CD11A
+
), but at 48 and 72 hr, the fraction
of labeled VE-CAD
KLS cells increased and the frequency
of labeled CD11A
KLS cells decreased, consistent with
the notion that VE-CAD
+
KLS cells are giving rise to
VE-CAD
KLS cells. Whereas neither reporter conclusively
demonstrates that CD11A
KLS cells give rise to CD11A
+
KLS cells, our data support our contention that early
Figure 5. Clonal Multipotent Analysis of EPCR
+
and EPCR
KLS Subsets
(A) VE-CAD (y axis) versus EPCR (x axis) expression of KLS cells in YS, AGM, FL, and PL from e9.5 to e11.5. Gates and percentages of EPCR
+
and EPCR
KLS cells are indicated. Nearly all EPCR
+
cells were CD11A
(data not shown).
(B and C) Percent of multipotent colonies in EPCR
+
and EPCR
KLS subsets in e10.5 (B) and e11.5 (C) CD11A
and CD11A
+
KLS cells. For
CD11A
+
KLS, only VE-CAD
+
cells were analyzed. ND, not detected; NA, not analyzed. The number of multipotent colonies observed out of
total colonies scored is indicated in parentheses. FACS analyses of representative multipotent colonies from e11.5 EPCR
+
CD11A
KLS cells
can be found in Figure S1.
Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors 465
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
hematopoietic progenitors that express endothelial-
associated markers generally precede those that express
hematopoietic markers.
Multipotency Moves into the CD11A
+
KLS Subfraction
at E12.5
By e12.5, the FL is by far the dominant site of hemato-
poiesis and is known to produce fully functional HSCs by
transplantation (Morrison et al., 1995). When we exam-
ined e12.5 FL KLS, nearly all (95%) of KLS cells were
CD11A
+
(Figure 6A). When we examined both CD11A
and CD11A
+
KLS fractions for clonal multilineage poten-
tial, we now found multipotency in the CD11A
+
KLS
fraction (Figures 6B–6D). Interestingly, only the VE-CAD
lo
subset of CD11A
+
KLS cells showed multipotency, whereas
the VE-CAD
CD11A
+
KLS subset, which represents over
A
C
B
D
Figure 6. Clonal Analysis of E12.5 FL KLS Subsets
(A) Gating of CD11A
and CD11A
+
KLS cells in e12.5 FL. Only CD43
+
CD34
+
TER119
cellsare shown. Percentof cells within each gate are shown.
(B) Distribution of clonal lineage potential in e12.5 FL EPCR
+
and EPCR
CD11A
and CD11A
+
KLS cells. The legend is described in Figure 1E.
Only CD11A
KLS and VE-CAD
+
CD11A
+
KLS cells were analyzed.
(C) Percent of multipotent colonies in e12.5 FL CD11A
and CD11A
+
KLS subsets. The number of multipotent colonies observed out of total
colonies scored is indicated in parentheses.
(D) Estimation of the absolute number of multipotent colonies per embryo in e12.5 FL CD11A
and CD11A
+
KLS cells, calculated as in
Figure 4E.
466 Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
3
/
4
of CD11A
+
KLS cells, lacked multipotency. By e12.5, the
multipotent CD11A
KLS population was exceedingly rare
compared to the robustly expanding CD11A
+
KLS
population (Figures 6A and 6D). Thus, it appears that the
primary source of multipotent cells shifts from CD11A
to CD11A
+
KLS cells by e12.5.
Only CD11A
KLS Cells Produce All Hematopoietic
Lineages In Vivo
The in vitro multipotency assay demonstrates that, from
e9.5 to e11.5, only the CD11A
KLS population contains
clonal multilineage potential. We next compared the in vivo
lineage potential of CD11A
and CD11A
+
KLS cells by
competitive transplantation (Figure 7). We sorted CD11A
and CD11A
+
KLS cells from cyan fluorescent protein
(CFP)
+
and CFP
embryos and mixed CFP
+
CD11A
KLS
cells with CFP
CD11A
+
KLS cells, and vice versa, to directly
compare engraftment and lineage potential between these
two populations in the same recipient animals. We also co-
transplanted 100 adult BM KLS cells as an internal control.
Cells were sorted from e11.5 YS, AGM, FL, and the vitelline/
umbilical (VU) region, which is also known to contain pre-
HSCs and HSCs at this time point (de Bruijn et al., 2000;
Gordon-Keylock et al., 2013). Because e11.5 hematopoietic
progenitors have poor engraftment potential when trans-
planted into adult animals, we instead transplanted intrave-
nously into irradiated newborn nonobese diabetic-severe
combined immunodeficiency-gc
/
(NSG) recipients.
When we examined donor chimerism in the blood of recip-
ient mice, we found substantially more donor cells derived
from CD11A
KLS cells than from CD11A
+
KLS cells in all
recipient mice (Figures 7A and 7B), despite transplanting
nearly a 5-fold excess of CD11A
+
KLS cells (Figure 7A).
Whereas the level of donor chimerism varied considerably
from recipient to recipient, in every case, the contribution
of CD11A
+
KLS cells was either minor or absent, indicating
these cells are much less effective at engraftment than
CD11A
KLS cells. We also examined the lineage distribu-
tion of donor-derived cells (Figures 7C and 7D). Whereas
all recipients contained donor-derived lymphocytes, only
two had significant donor myeloid cells (FL #1 and
VU #1). We focused on the recipient ‘‘FL #1’’ and found
that CD11A
KLS cells produced robust B cells, T cells,
NK cells, granulocytes, and macrophages (Figure 7C).
Conversely, CD11A
+
KLS cells produced only a modest
number of B cells and Tcells, which faded over time (Figures
7C and 7D). Because erythrocytes and platelets do not
express CD45, a marker we used to distinguish donor and
recipient cells, we were unable to determine whether
CD11A
KLS could produce these lineages, but all other
major hematopoietic lineages were detectable and robust
at 15 weeks posttransplantation, suggesting that CD11A
KLS cells are multipotent both in vivo and in vitro.
DISCUSSION
The essential properties of adult HSCs include the capacity
to maintain themselves indefinitely (self-renewal), the
ability to differentiate into all hematopoietic lineages
(multipotency), and the ability to circulate from the blood
to niches in the bone marrow (engraftability). It remains
unclear whether HSCs develop from hematopoietic-
committed precursor cells that contain some, but not all,
three HSC properties or instead whether HSCs arise de
novo from a hematoendothelial precursor. Earlier studies
had suggested that hematopoietic precursors to HSCs
(i.e., pre-HSCs) exist within the fetus and that these pro-
genitors lacked the ability to home to the bone marrow
but could be matured in vitro into bona fide, bone-
marrow-homing HSCs (Gordon-Keylock et al., 2013; Rybt-
sov et al., 2011). We have adopted an alternative strategy to
identify such precursors to HSCs. We hypothesize that HSC
precursors have the property of multipotency, and we
developed a single-cell assay to reductively identify, quan-
tify, and characterize multipotent cells in the early embryo.
Our data indicate that multipotent cells are predominantly
localized to the YS during the stages when definitive, self-
renewing HSC precursors are thought to arise (e9.5–
e10.5). As the surface marker phenotype of multipotent
CD11A
KLS (VE-CAD
+
CD43
+
KIT
+
CD11A
) cells over-
laps with that of pre-HSCs (VE-CAD
+
CD41
+
CD45
or +
),
CD11A
KLS and pre-HSC populations likely contain the
same precursor cells. The ability of CD11A
KLS cells to
engraft in newborn mice and give rise to multiple hemato-
poietic lineages in vivo confirms the multilineage potential
of this population and strongly implicates CD11A
KLS
cells as a critical intermediate population between the
earliest primitive YS progenitors and fully functional HSCs.
It remains unclear whether HSCs arise from both intra-
embryonic (AGM and FL) and extraembryonic (YS and
PL) tissues or if they emerge from a single site and migrate
elsewhere. Our absolute cell number data indicate that the
YS contains by far the most multipotent cells during the
critical stages during which HSCs arise. Pre-HSCs or HSCs
may also emerge de novo from hemogenic endothelium
in the AGM, but our data suggest that the AGM region by
itself does not produce enough multipotent cells to ac-
count for all the multipotent cells in the embryo and that
likely the vast majority come from the extraembryonic
YS. This is consistent with previous work estimating the
absolute number of HSCs during gestation, suggesting
that both the YS and AGM may contribute to the FL HSC
pool (Kumaravelu et al., 2002). Additionally, our previous
work showing direct orthotopic synchronic transplanta-
tion of e8 to e9 YS cells showed that YS-derived cells can
indeed contribute to complete and lifelong adult hemato-
poiesis (Weissman et al., 1978).
Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors 467
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
A
B
C
D
(legend on next page)
468 Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
CD11A
KLS cells express multiple markers of endothe-
lial cells (EPCR, VE-cadherin, and CD34), suggesting that
they are recently derived from the endothelial lineage
and raising the question of whether they retain endothelial
cell potential and contain hemogenic endothelium.
Whereas we did not observe any endothelial cell differenti-
ation from CD11A
KLS cells in our in vitro cultures, we
were also unable to drive robust hematopoietic colony for-
mation from populations that should contain hemogenic
endothelium (VE-CAD
+
CD34
+
SCA-1
+
CD43
CD41
).
Thus, it is possible that the in vitro assay described here is
incapable of revealing hemogenic endothelium. Whereas
the CD11A
KLS population retains surface markers of
hemogenic endothelium, the defining indicator that the
CD11A
KLS population is hematopoietic is that these
cells can engraft upon intravenous transplantation into
neonatal mice and give rise to multiple hematopoietic
lineages.
Many of the surface markers we used to define embryonic
hematopoietic populations have functional roles in cell
adhesion and trafficking. VE-cadherin is an endothelial
adhesion molecule involved in facilitating the adherens
junctions between endothelial cells (Harris and Nelson,
2010). CD11A is part of a leukocyte adhesion complex
(LFA-1) known to play a role in leukocyte extravasation
from the vasculature by binding to ICAMs on endothelial
cells (Hogg et al., 2011). In a related study, we show that
CD11A is upregulated as HSCs lose self-renewal potential
and may be involved in HSC mobilization out of the
bone marrow (J.W.F., M.A.I., Nathaniel B. Fernhoff, J.S.,
and I.L.W., unpublished data). In this study, we show
that CD11A upregulation is also associated with the loss
of multilineage potential and marks a subset of lineage-
committed progenitors that are downstream of embryonic
multipotent cells. These changes in adhesion molecule
expression may play an important role in the migration
of progenitors from their tissues of origin (YS, PL, and
AGM) to secondary sites of hematopoiesis (FL and BM).
For example, the downregulation of VE-cadherin may
allow cells to detach from the endothelium and enter circu-
lation, whereas the upregulation of CD11A may allow cells
to exit circulation and seed new sites.
In this study, we created an assay to look for clonal multi-
lineage potential and used it to identify emerging multipo-
tent hematopoietic progenitors within the embryo. Other
markers we focused on were also critical for distinguishing
multipotent cells from downstream progenitors. Due to
space limitations, we include an in-depth technical discus-
sion of these markers and their use in the Supplemental
Information. Our data suggest that a multipotent, self-
renewing hematopoietic wave arises in the YS blood islands
and appears simultaneously in multiple sites at e9.5. We
confirmed the multipotency of this population in vivo.
We hypothesize that this population represents a critical
intermediate in the origins of definitive hematopoiesis,
and armed with a panel of markers that can identify these
cells with high resolution, we can now begin to dissect the
critical steps in the emergence of and maturation of the
first HSCs in embryonic development.
EXPERIMENTAL PROCEDURES
Antibodies
A detailed list of all antibodies used in this study is shown in
Table S1.
Clonal Multipotency Assay
ODT stroma was cultured as described (Vodyanik and Slukvin,
2007). Briefly, ODT was plated onto gelatin-coated dishes and
cultured in the presence of OP9 media (aMEM [made from powder,
Invitrogen catalog No. 12000-022] in 20% serum [Omega Scientific
FB-11]). ODT was passaged around 1:10 every 5 days. OP9 cells
differentiate rapidly to adipocytes when confluent, so great care
was taken to avoid confluence. For clonal assays, ODT was plated
Figure 7. In Vivo Competitive Comparison of Engraftment and Lineage Potential of CD11A
and CD11A
+
KLS
Newborn NSG mice were transplanted with four embryo equivalents of CD11A
KLS and CD11A
+
KLS cells attained from different tissues at
e11.5 along with 100 adult BM KLS cells. Blood was analyzed at 4, 8, 12, and 15 weeks to examine donor chimerism and lineages produced.
(A) Comparison of donor chimerism between BM KLS (green), CD11A
KLS (red), and CD11A
+
KLS (blue). Stacked graphs show the dis-
tribution of donor cells in the blood at 4, 8, 12, and 15 weeks posttransplant, listed as a percentage of total CD45
+
cells (including those of
the recipient). The tissue of origin for e11.5 KLS cells is indicated above each graph. Note that the scale of the y axis is different in each
graph. The number of CD11A
and CD11A
+
KLS cells for each transplant is shown on the table on the right.
(B) Head-to-head comparison of e11.5 donor cells at 4 weeks. Only donor contributions of e11.5 CD11A
KLS (red) and CD11A
+
KLS (blue)
were compared.
(C) FACS analysis of donor lineages from e11.5 FL KLS cells (‘‘FL #1’’) at 15 weeks posttransplant. The percentage displayed for each lineage
is out of that donor’s total cells. For example, the percentage of NK cells shown for CD11A
KLS cells is out of total donor CD11A
KLS-
derived cells.
(D) Time course of the distribution of donor lineages derived from adult BM KLS (top row), CD11A
+
KLS (middle row), and CD11A
KLS
(bottom row) in the blood of recipient mice at 4, 8, 12, and 15 weeks posttransplant. Percentages shown are out of total CD45
+
cells and
shown for NK cells (orange), T cells (purple), B cells (red), macrophages (green), and granulocytes (blue). The gates used to identify each
lineage are shown in (C). Note that the y axis scale is different with each graph.
Stem Cell Reports j Vol. 2 j 457–472 j April 8, 2014 j ª2014 The Authors 469
Stem Cell Reports
Mouse Embryonic Multipotent Hematopoietic Cells
the day prior to use onto gelatin-coated 96-well plates (Falcon
3072), around 1,000–2,000 cells per well, in OP9 media. The day
of use, the media was replaced with 100 ml of differentiation media
(described in Vodyanik and Slukvin, 2007), aMEM, 10% serum,
100 mM monothioglycerol (catalog No. M6145; Sigma), 50 mg/ml
ascorbic acid (catalog No. A-0278; Sigma), 100 U/ml penicillin/
100 mg/ml streptomycin (catalog No. 15140; Invitrogen), and 13
GlutaMAX (catalog No. 35050; Invitrogen) and the cytokines
SCF (rmSCF; Peprotech 250-03; 10 ng/ml), TPO (rmTPO; Peprotech
315-14; 10 ng/ml), EPO (rhEPO; Invitrogen PHC2054; 0.5 U/ml),
Flt3L (mFlt3L; Peprotech 250-31L; 10 ng/ml), IL-7 (mIL-7; Pepro-
tech 217-17; 10 ng/ml), and IL-15 (IL15/IL15R complex; Ebio-
science 14-8152; 5 ng/ml) were added. Colonies were visibly
confirmed at 3 days after plating. Around 4 to 5 days after plating,
wells with colonies were fed by adding 100 ml of differentiation
media with IL-7 (10 ng/ml), IL-15 (5 ng/ml), and DOX (1 mg/ml).
All wells with colonies at day 3 were harvested, stained, and
analyzed on a BDFortessa FACS analyzer with a high-throughput
sampler on FACS Diva software (BD Biosciences). Colonies were
scored for presence of erythrocytes (TER119
+
), platelets (CD41
+
),
granulocytes (MAC-1
+
, GR1
+
), NK cells (NK1.1
+
), T cells (CD25
+
,
LY6D
+
), and B cells (CD19
+
, LY6D
+
). LY6D is expressed on devel-
oping thymocytes and B cells and was useful as a second marker
to identify T/B lymphocytes (Inlay et al., 2009). Colonies were
scored as MegE if erythrocytes and/or platelets were detected,
GM if granulocytes were detected, and L if NK, T, and/or B cells
were detected. Colonies with MegE, GM, and L potential were
scored as multipotent.
Mouse Embryo Harvest and Cell Sorting
All animal procedures were approved by the International Animal
Care and Use Committee and the Stanford Administrative Panel
on Laboratory Animal Care. Matings of C57B6 mice were estab-
lished and plugs checked in the mornings. Pregnant females
were harvested in the mornings and embryos dissected immedi-
ately. Vitelline and umbilical vessels were typically harvested
with the yolk sac, except where indicated. For the AGM harvest,
the head, tails, feet, and fetal liver/heart were removed, and the
remaining tissue was listed as AGM. Tissues were dissociated in
10 mg/ml Collagenase Type IV (Invitrogen 17104-019) for approx-
imately 30 min to 1 hr, pipetted up and down and filtered in 70
micron mesh. Tissues were typically stained for 15 min on ice in
staining media. See Table S1 for antibodies used. Cells were
analyzed and/or sorted on a BD FACSAria using FACS Diva soft-
ware. For clone sorting, a 100-micron nozzle was used, and cells
were single sorted on ‘‘single cell’’ mode directly onto 96-well
plates with ODT stroma. The authors highly recommend doing a
single sort (as opposed to a double sort) due to substantial loss of
rare cells upon the second sort. We also recommend avoiding
the use of ACK lysis buffer prior to Ab staining, as it dramatically
reduces the VE-cadherin signal.
In Vivo Transplantation and Analysis
CD45.1
+
CFP
+/
males were crossed with CD45.2
+
females to pro-
duce CD45.1
+
CD45.2
+
embryos, half of which were CFP
+
and half
CFP
. CD11A
KLS and CD11A
+
KLS cells from e11.5 tissues were
sorted and pooled such that CFP
+
CD11A
KLS cells were pooled
with CFP
CD11A
+
KLS cells and vice versa. One hundred adult
BM KLS cells (CD45.2
+
CFP
) were also sorted from the mother
and pooled with the sorted e11.5 KLS populations and then trans-
planted via the superficial facial vein into neonatal (days 1–3) NSG
mice (CD45.1
+
) conditioned with 100 rads irradiation. Mice were
bled at 4, 8, 12, and 15 weeks posttransplantation for analysis.
Erythrocytes were lysed in ACK lysis buffer (150 mM NH
4
Cl,
10 mM KHCO
3
, 0.1 mM EDTA), and the remaining cells were
stained with antibodies and analyzed on a BD FACSAria flow
cytometer.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Discussion,
Supplemental Experimental Procedures, seven figures, and one
table and can be found with this article online at http://dx.doi.
org/10.1016/j.stemcr.2014.02.001.
AUTHOR CONTRIBUTIONS
M.A.I., T.S., A.M., and I.L.W. designed experiments. M.A.I., T.S.,
and A.M. performed experiments. J.W.F., I.K.D., and J.S. contrib-
uted unpublished data, efforts, and tools. M.A.I. and T.S. wrote
the manuscript, and I.L.W. edited the manuscript. All experiments
were performed in the laboratory of I.L.W.
ACKNOWLEDGMENTS
The authors wish to thank Adriel Cha, Charles Chan, Nate Fernh-
off, Katharina Seiler, Shah Ali, Bevin Brady, Debashis Sahoo, Kyle
Loh, Seth Karten, Pamela Wenzel, and Roger Pedersen for sharing
reagents and expertise; Libuse Jerabek and Terry Storm for lab man-
agement; Teja Naik and Chrissy Muscat for antibody conjugation;
and Charlene Wang, Aaron McCarty, Humberto Contreas-Trujillo,
and Joel Dollaga for mouse management. The research reported in
this article was supported by the National Institutes of Health (5
T32 AI07290 to M.A.I. and J.W.F.; R01HL058770, R01CA86085,
and U01HL09999 to I.L.W.), the California Institute for Stem
Cell Research (T1-00001 to M.A.I. and J.S.; RT2-02060 to I.L.W.),
the Harvard Stem Cell Institute (to T.S.), the Siebel Stem Cell Insti-
tute and the Thomas and Stacey Siebel Foundation (to I.K.D.), and
the Virginia and D.K. Ludwig Fund for Cancer Research (to I.L.W.).
Received: July 31, 2013
Revised: February 5, 2014
Accepted: February 5, 2014
Published: March 20, 2014
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