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OCR for page 88
Colloquium
Enhanced hematopoietic differentiation of embryonic
stem cells conditionally expressing Stats
Michael Kybat, Rita C. R. Perlingeirot$, Russell R. Hoovert§, Chi-Wei Lut, Jonathan Piercet, and George Q. Dales
iWhitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142; and Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, and Division of Pediatric Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute,
Boston, MA 02115
. . .
The signal transducer Stats plays a key role in the regulation of
hematopoietic differentiation and hematopoietic stem cell func-
tion. To evaluate the effects of Stats signaling in the earliest
hematopoietic progenitors, we have generated an embryonic stem
cell line in which Stats signaling can be induced with doxycycline.
Ectopic Stats activation at the point of origin of the hematopoietic
lineage (from day 4 to day 6 of embryoid body differentiation)
significantly enhances the number of hematopoietic progenitors
with colony-forming potential. It does so without significantly
altering total numbers or apoptosis of hematopoietic cells, sug-
gesting a cell-intrinsic effect of Stats on either the developmental
potential or clonogenicity of this population. From day-6 embryoid
bodies, under the influence of Stats signaling, a population of
semiadherent cells can be expanded on OP9 stromal cells that is
comprised of primitive hematopoietic blast cells with ongoing,
mainly myeloid, differentiation. When these cells are injected into
lethally irradiated mice, they engraft transiently in a doxycycline-
dependent manner. These results demonstrate that the hemato-
poietic commitment of embryonic stem cells may be augmented by
a Stats-mediated signal, and highlight the utility of manipulating
individual components of signaling pathways for engineering
tissue-specific differentiation of stem cells.
hematopoiesis
When differentiated as suspension aggregates (embryoid
bodies, EBs), embryonic stem (ES) cells will readily give
rise to differentiated hematopoietic cells (1) as well as colony-
forming cells (CFCs) that can be assayed in secondary semisolid
hematopoietic cultures (24. However, ES cell differentiation
does not efficiently generate hematopoietic stem cells (HSCs)
capable of repopulating the hematopoietic system of lethally
irradiated adult recipients. In this regard, ES cells recapitulate
the development of the earliest embryonic hematopoietic tissue,
the yolk sac. Analysis of knockout mice has implicated several
genes in the embryonic development of the definitive HSC (3-5~;
however, a detailed understanding of the extracellular signals
that guide development from the pluripotent state to the lineage-
restricted HSC state is lacking.
In an effort to evaluate signals that promote hematopoietic
differentiation of pluripotent cells, as well as the generation of
cells with hematopoietic repopulating potential, we sought to
test the effects of Stats activation during in vitro differentiation
of ES cells. Stats are cytoplasmic signal transducers that are
recruited by ligand-activated receptors via Src homology 2-
mediated interactions with receptor-bound Janus kineses. This
interaction results in Stat protein phosphorylation, disengage-
ment, dimerization, and translocation to the nucleus where these
proteins then function as a transcription factors, binding to and
activating the transcription of target genes (6~. Stats is encoded
by two genes, StatSa and StatSb, with 95% sequence identity (7)
and is activated by engagement of numerous hematopoietic and
11904-11910 1 PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1
nonhematopoietic receptors (8-11~. Stats signaling has been
implicated in cellular proliferation (12, 13), resistance to apo-
ptosis (14-16), and differentiation (17, 18~.
Mice genetically null for both Stat5a and Stat5b display
obvious defects in response to growth hormone and prolactin
(15) and subtler defects in embryonic hematopoietic develop-
ment (144. Although definitive HSCs develop in Stats knockout
mice, they display a profound defect in competitive repopulation
(19-21), suggesting that Stats may be interacting cooperatively
and redundantly with other signal transducers in HSC regula-
tion. In the classic HSC pathology, chronic myeloid leukemia,
regulation of proliferation is disrupted by the oncogene Bcr/Abl,
with concomitant activation of Stats (22-254. Moreover, dom-
inant negative Stats mutants can block transformation by Bcr/
Abl (26), indicating that inappropriate activation of StatS can
have dramatic consequences for HSC regulation.
Because of the pivotal role of Stats signaling in hematopoiesis
and HSC homeostasis, we selected this pathway for study during
the earliest stages of hematopoietic specification in an in vitro
system of ES cell differentiation. For this purpose, we have
generated an ES cell line with a tetracycline-inducible, domi-
nant-active allele of Stats. We report that induction of Stats
signaling during EB development dramatically enhances hema-
topoiesis. Furthermore, on OP9 stromal cell coculture, Stats
promotes the expansion of a blast cell population from day-6
EBs. Cultures expanded in this way are rich in primitive,
undifferentiated cells, with surface marker similarities to HSCs,
and have the capacity to engraft lethally irradiated adult mice in
a transient, Stats-dependent manner.
--rib - r~r~
_ ~
Materials and Methods
Generation of Stat5CA Inducible ES Cells. The cDNA for the
constitutively active mutant of Stats (H299R/S711F, a gift from
T. Kitamura, University of Tokyo, Tokyo) was subcloned on an
EcoRI-NotI fragment from murine stem cell virus (MSCV)-
Stat5CA-iresGFP (16) into pLox. This was then co-electropo-
rated along with pSalk-CRE (a gift from S. O'Gorman, The Salk
Institute, San Diego) into the targeting cell line Ainvl5. The
targeting ES cell line and targeting plasmid, pLox, have been
described (274. The resulting cell line was selected and expanded
This paper results from the Arthur M. Sackier Colloquium of the National Acaclemy of
Sciences, "Regenerative Meclicine," held October 18-22, 2002, at the Arnold and Mabel
Beckman Center of the National Acaclemies of Science and Engineering in Irvine, CA.
Abbreviations: ES, embryonic stem; ED, embryoicl bocly; HSC, hematopoietic stem cell; CFC,
colony-forming cell; TRE, tetracycline response element; TPO, thrombopoietin; SCF, stem
cell factor; VEGF, vascular enclothelial growth factor; IFS, inactivated fetal serum; MSCV,
murine stem cell virus; IMDM, Iscove's modified Dulbecco's meclium.
"Present aciciress: ViaCell, Inc., 26 LanciscJowne Street 580, Cambricige, MA 02139-4216.
§Present aciciress: Vertex Pharmaceuticals, 130 Waverly Street, Cambricige, MA 02139-4242.
Vito whom correspondence should be aciciressecl. E-mail: claley@wi.mit.eclu.
2003 by The National Acaclemy of Sciences of the USA
www.pnas.org/cgi/cdoi/10. 1 073/pnas. 1734140100
OCR for page 89
a
b
/
pLoxStatSCA
~1 Chromosome 6
+1 ~ X
~ X-chromosome
plasma seq. ~
X
o
O
O4
As ~ ~
~ ·~ · -
~ ˘ ˘
˘
V
.
~~ I,
1 ,
X
o
˘
VO
·_1
~.4
Fig. 1. Generation of the Stats-inducible ES cell line. (a) Integration of pLoxStat5CA into the LoxP site on the X chromosome of Ainv15 ES cells places the cDNA
for Stat5CA underthe control of the tetracycline response element. Recombination between the chromosomal and plasmid LoxP sites, denoted by X, is mediated
by transient expression of Cre recombinase. Reconstitution of neo gene function by the promoter-ATG sequence 5' to the loxP site allows for selection of
successfu I i nteg ration events. rtTA, reverse tetracycl i ne tra nsactivator. (b) Exposu re of iStat5CA ES cel Is to doxycycl i ne, but not the pa renta I cel I I i ne Al nv 1 5, resu Its
in the bandshift of a probe containing Stats binding sites. Arrowhead denotes the Stats-specific bandshift.
in 400 ,ug/ml G418 (Sigma). ES cells were maintained on
irradiated mouse embryonic fibroblasts in DME/15% inacti-
vated fetal serum (IFS)/0. 1 mM nonessential amino acids
(GIBCO/BRL)/2 mM glutamine/50 units/ml penicillin/50
1lg/ml streptomycin (GIBCO/BRL)/0.1 mM 2-mercaptoethanol
(Sigma)/1,000 units/ml leukemia inhibitory factor (PeproTech,
Boston). To induce StatSCA expression in ES cells, 2 ,ug/ml
doxycycline (Sigma) was added to the culture medium.
Bandshift Assay. To generate whole cell extracts, cells were
washed in ice-cold PBS and lysed in EMSA lysis buffer (150 mM
NaCl/20 mM Tris HCl, pH 7.4/1 mM EDTA/10 mM Na3VO4/1
mM MgCl2/1% Nonidet P-40/1 mM phenylmethyl-sulfonyl
fluoride/10% glycerol) on ice for 10 min. Cells and lysate were
scraped with a sterile cell scraper, collected, and spun for 10 min
at max at 4°C on a benchtop centrifuge. Complimentary oligo-
nucleotides that contained a Stats consensus binding site from
the ,B-casein promoter (5'-AGATTTCTAGGAATTCAATCC-
3') were annealed and radiolabeled with ty-32PJATP by using T4
polynucleotide kinase (New England Biolabs). Approximately
20,000 cpm (0.2 ng) of probe was incubated with 20 ,ug of whole
cell extract in 20 ,ul of 10 mM Hepes, pH 7.9/50 mM KCl/0.2
mM DTT/10% glycerol/0.05% Nonidet P-40/1 ,ug poly~dIdC)
(Sigma) for 20 min at room temperature. Resulting DNA/
protein complexes were resolved on a 5% nondenaturing poly-
acrylamide gel.
EB Differentiation. ES cells were trypsinized, collected in EBD
tIscove's modified Dulbecco's medium (IMDM)/15% IFS/200
~g/ml iron-saturated transferrin (Sigma)/4.5 mM monothioglyc-
erol (Sigma)/50 ,u~g/ml ascorbic acid (Sigma)/2 mM glutamine]
and plated onto fresh T25 flasks (Corning) for 45 min to allow
mouse embryonic fibroblasts to adhere. Nonadherent cells were
collected and plated in hanging drops at 100 cells per 10-~l drop
in an inverted bacterial Petri dish, and cultured for 2 days. They
were then collected from the hanging drops and further cultured
in 10 ml of EBD in slowly rotating 10-cm bacterial Petri dishes.
At day 4, EBs were fed by exchanging half of their spent medium
for fresh EBD. In some cultures, doxycycline was added at day
4 at 2 ,ug/ml to induce expression of Stat5CA.
CFC Assay. Day-6 EBs were dissociated by trypsinization, col-
lected, and resuspended in IMDM/10% IFS at a concentration
of 5 x 105 cells per ml. A total of 100 ~l of this cell suspension
Kyba et a/.
was added to 1.5 ml of complete methylcellulose for murine
colonies (StemCell Technologies, Vancouver, catalog no. 3434~.
Methylcellulose suspension cultures were not supplemented with
doxycycline. EryP colonies were counted on day 6 of methyl-
cellulose culture, all other colonies were counted at day 10.
OP9 Coculture. Day-6 EBs were trypsinized to a single cell
suspension and plated on a semiconfluent monolayer of OP9
cells (a gift of T. Nakano, University of Osaka, Osaka) at a
density of 200,000 cells per well of a six-well dish in IMDM, 10%
IFS supplemented with 2 mM glutamine, 50 units/ml penicillin,
50 ,ug/ml streptomycin (GIBCO/BRL), 0.1 mM 2-mercapto-
ethanol (Sigma), cytokines t40 ng/ml vascular endothelial
growth factor (VEGF), 40 ng/ml thrombopoietin (TPO), 100
ng/ml stem cell factor (SCF), and 100 ng/ml Flt-3 ligandi, and
doxycycline at 1 ~g/ml. When cells became confluent, they were
passaged by trypsinization onto fresh OP9.
Fluorescent Staining and FACS Analysis. Staining of day-6 EB cells. EBs
were disaggregated by washing once with PBS followed by
resuspension in 0.1% trypsin/PBS and pipetting for 30 s. Trypsin
was blocked with IMDM/10% IFS, the cells were strained to
remove clumps and collected by centrifugation. Annexin V-
phycoerythrin staining was done at room temperature for 15 min
according to the manufacturer's specifications (CLONTECH).
After staining, cells were transferred to 4°C, and 1 ~l of
FITC-conjugated CD41 antibody was added. After 20 min,
samples were diluted with annexin V binding buffer containing
propidium iodide and analyzed by FACS.
Staining of OP9 cultures. Cells were collected by trypsinization and
resuspended in PBS containing 5% IFS. Samples of one million
cells in 100 ,ul were blocked with 1 ,ul of Fc block (PharMingen)
and stained with 1 ,ul of phycoerythrin- or FITC-conjugated
antibody for 20 min at 4°C. Samples were washed twice with
PBS/5% IFS, and resuspended in PBS/5% IFS containing
propidium iodide. All antibodies were purchased from Phar-
Mingen. FACS analyses were performed on a Becton Dickinson
FACSCalibur. Dead cells were excluded from phycoerythrin-
stained cells by gating on FL2 vs. FL3.
RT-PCR. The following primers were used: actin~f) 5'-GT-
GGGGCGCCCCAGGCACCA-3'; actintr) 5'-CTCCTTAATGT-
CACGCACGATTTC-3'; ,l3-Hl~f) 5'-AGTCCCCATGGAGT-
CAAAGA-3'; b-Hltr) 5'-CTCAAGGAGACCTTTGCTCA-3';
PNAS | September 30, 2003 | vo~. 100 | supp~. ~ | 11905
OCR for page 90
13-maj(f) 5'-CTGACAGATGCTCTCTTGGG-3'; `(3-maj(r) 5'- a 4o
CACAACCCCAGAAACAGACA-3'. Cycle conditions were as
follows: 2 min at 96°C; 30 cycles of 45 s at 95°C, 1 min at 60°C, and ~ 35
45 s at 72°C; and then 5 min at 72°C. —c' 30.
o 25
To
O 20
a)
15
Q
At) 1 0
Retroviral Labeling with GFP. GFP retroviral supernatants were
produced by FUGENE transfection of 293 cells with pMSC~i-
resGFP (28) and pCL-Eco, a packaging-defective helper plasmid
(294. 293 cells were grown in DME/10% IFS, and medium was
replaced on the day after transfection Forty-eight hours after
transfection, supernatants were collected, filtered, plated onto
iStatSCA blast cells growing on OP9 at 3 ml per well of a six-well
dish, supplemented with 4 ,ug/ml polybrene and cytokines (100
ng/ml SCF, 40 ng/ml VEGF, 40 ng/ml TPO, 100 ng/ml Flt-3
ligand), and spin-infected at 2,500 rpm for 90 min in a Beckman
GS-6R centrifuge. After several days of growth, GFP-positive
cells were separated by FACS and cultured on fresh OP9.
Filtered supernatants from GFP-positive cells expanded on OP9
were tested for lateral transfer to 10T1/2 cells and found to be
negative.
Mouse Transplantation. Two- to four-month-old 12901a/Hsd
(Harlan Breeders, Indianapolis) mice were exposed to 2 x 500
cGy of y-irradiation, separated by 4 h, and injected with 1.75 x
106 cells in 500 ,ul of IMDM/10% IFS via lateral tail vein. Mice
that received doxycycline were provided drinking water supple-
mented with 500 ,ug/ml doxycycline hydrochloride (Sigma) and
5% sucrose.
Results
Stats Signaling During EB Development. To generate an ES cell line
with inducible StatS signaling, we made use of the Tet-On
targeting cell line, Ainvl5 (27~. These cells constitutively express
the reverse tetracycline transactivator from the ROSA26 locus,
and have a tetracycline response element (TRE) integrated into
the transcriptionally open chromatin 5' to the HPRT gene on the
X chromosome. Downstream of the TRE is a single LoxP site,
into which we integrated, by Cre-Lox recombination, the cDNA c
for StatSCA, a constitutively active mutant of Stat5a (a double
mutant of H299R and S711F, also known as 1*6) (12) (Fig. la).
The resulting ES cell line, named iStatSCA, as well as its
differentiated progeny, express this mutant cDNA when exposed
to doxycycline. Expression results in binding of Stat5CA to DNA
as measured by bandshifting activity against an oligonucleotide
probe encoding a StatS DNA-binding consensus sequence, in the
lysate of doxycycline-treated iStat5CA ES cells (Fig. lb).
We used this cell line to generate EBs and applied doxycycline
to the cultures for 48 h, from day 4 to day 6 of differentiation.
This time window was chosen based on the kinetics of colony
formation in EBs: the bipotent precursor to the hematopoietic
and endothelial lineages, the hemangioblast, peaks at day 3.75
and is no longer present by day 5 (30), whereas hematopoietic
CFC, particularly m~xed erythroid-myeloid colonies, first be-
come detectable between days 5 and 6. Thus, StatS signaling was
induced at the time of specification of the hematopoietic lineage.
At day 6, the EBs were disaggregated into single cells and
assayed for hematopoietic CFC content by plating in methylcel-
lulose suspension medium with hematopoietic cytokines. As
shown in Fig. 2a, exposure of EBs to doxycycline increased the
numbers of all types of hematopoietic colonies assayed between
2- and 4-fold.
To investigate the mechanism of StatS-mediated hematopoi-
etic enhancement, we analyzed cells from doxycycline-treated or
untreated day-6 EBs for apoptosis. Preliminary results (not
shown) demonstrated that StatS activation modestly decreased
the number of apoptotic (annexin V-positive) cells from day-6
EBs; however, a general reduction in apoptosis would increase
CFC frequency only if the hematopoietic lineage were subject to
11906 1 www.pnas.org/cgi/doi/10.1073/pnas.1734140100
|ONO dox 1
1~ Dox d4-6
CD41
C No dox
CD41
1 '
._
y
C~
. . . . . . . .
CD41 CD41
Dox d4-6
Dox d4-6
1 11 0.5
Fig. 2. Effects of Stats signaling during EB differentiation. EBs were grown
for 6 days, either exposed or not exposed to doxycycline from day 4 to day 6.
(a) CFC assay: day-6 EB cells were disaggregated and plated into methylcellu-
lose suspension culture with hematopoietic cytokines. Filled bars denote
colony number from doxycycline-treated EBs; open bars denote colony num-
ber from untreated EBs. Standard errors for three independent experiments
are shown. (n = 3 for each bar; P < 0.05 for combined CFCs.) (b) Apoptosis
assay: day-6 EB cells were disaggregated and stained with annexin V (yaxis) to
label apoptotic cells and anti-CD41 (x axis) to label hematopoietic cells. The
percentage of cells falling into single- and double-positive quadrants is
shown. (c) Hematopoietic compartment quantitation: day-6 EB cells were
disaggregated and stained with antibodies to c-Kit (y axis) and CD41 (x axis).
The percentage of cells falling within the double-positive rectangular gate is
shown.
higher levels of apoptosis than the other nonhematopoietic
lineages that arise in a day-6 EB. To assay the levels of apoptosis
in hematopoietic vs. nonhematopoietic cells, we stained EB cells
with both annexin V and a pan-hematopoietic antibody. Studies
of adult hematopoiesis commonly use the CD45 antigen as a
pan-hematopoietic marker; however, this marker is not univer-
sally expressed by the earliest embryonic hematopoietic progen-
itors. The recent discovery that the adult megakaryocytic anti-
gen CD41 is actually pan-hematopoietic in very early embryos as
well as in day-6 EBs (31, 32) prompted us to use this marker
rather than CD45 for this purpose. To our surprise, this assay
revealed that apoptosis in day-6 EBs is almost completely
restricted to the nonhematopoietic population (Fig. 2b).
Kyba et a/.
OCR for page 91
a
Q 10
He
=
~ O
a) ~
at_
~ 6
in Vitro Growth
Air
9
8
.
h ~ ~
O O
A ~
·_ ·_I
BOX - +
IL-3
a"
C
Day
I:
ii
I
O. + DOX
.,
...
Fig. 3. Stats signaling promotes blast cell outgrowth. (a) Cumulative cell number from OP9 stromal cell cocultures initiated by 2 x 105 cells. (b) Stats
DNA-binding activity was seen in OP9 cocultures of iStat5CA day-6 EB cells grown in the presence of doxycycline, but not in the residual growth that appeared
in the absence of doxycycline. For comparison, the Stats bandshift from BaF/3 cells growing exponentially in the presence of IL-3, or BaF/3 cells infected with
a MSCV retrovirus expressing Stat5CA are shown. Arrowhead denotes Stats-specific bandshift product. (c) Cytospin of iStat5CA day-6 EB cells expanded on OP9.
Cells were spun onto glass slides and stained with Wright-Giemsa.
An alternative to a reduction in apoptosis would be overpro-
liferation of hematopoietic cells in response to StatS. Because
hematopoietic CFCs from the day-6 EB are CD41 and c-Kit
double positive (31, 32), we assayed the frequency of this
population in the presence vs. absence of Stats induction (Fig.
2c). We observed only a very modest increase with doxycycline
treatment, which was not sufficient to account for the increase
in CFCs. We therefore conclude that StatS activation is either
influencing development within the hematopoietic compart-
ment, such that it contains a higher ratio of CFCs to more
differentiated cells, or enhancing the clonogenicity of the CFCs
that are present.
Stats Signaling During OP9 Stromal Cell Coculture of Day-6 EB Cells in
Vitro. Cells from day-6 EBs that had been exposed to doxycycline
for 48 h were also plated on OP9 stromal cells with a cytokine
mixture tailored for HSC expansion, consisting of TPO, SCF,
Flt-3 ligand, and VEGF. In the absence of doxycycline, there was
minimal growth, whereas in the presence of doxycycline, there
was a dramatic expansion of a semiadherent cell type growing
attached to the OP9 feeder layer. These semiadherent cells could
be passaged by trypsinization and expanded exponentially
(Fig. 3a).
Whole cell protein extracts from cells growing on OP9 in the
presence of doxycycline, but not in its absence, contained
StatS-specific DNA-binding activity (Fig. 3b). The intensity of
the StatS bandshift was similar to that observed in the pro-B cell
line BaF/3 growing in the presence of IL-3, but much less than
the bandshift observed in BaF/3 cells infected with a retrovirus
expressing StatSCA (Fig. 3b). This demonstrates that the level of
StatS activation achieved by exposure to doxycycline approxi-
mates the physiological level that hematopoietic cells experience
when growing in the presence of cytokines.
The dominant cell type in the OP9 expansion cultures was a
primitive hematopoietic blast; however, other cell types could
also be detected, particularly differentiated myeloid cells (Fig.
3c). We analyzed these cells for surface antigen expression by
flow cytometry (Fig. 4 a and b). Consistent with the blast cell
morphology, the majority of cells were negative for markers of
hematopoietic differentiation. Of those cells that were positive
Kyba et a/.
for lineage markers, the majority expressed the myeloid markers,
Gr-1 and Mac-1, but a small number expressed markers of
lymphoid (B220) and erythroid (Ter-119) differentiation. We
observed strong positivity for the hematopoietic stem cell mark-
ers c-Kit and Sca-1. The cells were negative for CD45 but positive
for CD41, consistent with an early embryonic hematopoietic
character, and the majority of both the c-Kit- and Sca-1-positive
cells were double positive for CD41. The cells were also strongly
positive for CD31, a marker displayed by hematopoietic stem
cells, some differentiated hematopoietic cells, as well as endo-
thelial cells. This expression is likely hematopoietic in origin as
opposed to endothelial given the compression of CD41. This
profile is suggestive of the expansion of an undifferentiated
embryonic hematopoietic progenitor with many characteristics
of the HSC, which undergoes concomitant differentiation mainly
along the myeloid lineage in vitro.
We assayed globin gene expression in these cells and com-
pared it to that seen in a similar cell population obtained by
expression of HoxB4, which we have previously described (27~.
Whereas embryonic (,B-H1) globin is almost undetectable in the
HoxB4-expanded cells, it is clearly present in those expanded by
Stats. The embryonic globin signal is weak compared with adult
(,B-major) globin; however, its presence suggests that StatS
signaling does not efficiently drive primitive to definitive hema-
topoietic switching in the same way that HoxB4 appears to.
Stats-Dependent Engraftment in Vivo. To determine the capacity of
these cells to undergo differentiation in salvo, they were marked
with GFP by retroviral infection with the virus MSCViresGFP
(28~. Cells were then injected into 10 irradiated isogenic recip-
ient adult mice, in two independent experiments, and the
peripheral blood was sampled periodically for GFP positivity.
We observed no engraftment in mice not treated with doxycy-
cline, even at time points as early as 2 weeks. However, when
mice were fed drinking water supplemented with doxycycline, we
observed transient donor cell contribution to the peripheral
blood, liver, spleen, and marrow, which was exhausted by 8 weeks
after transplantation. Although we observed contribution to the
spleen, we did not observe donor-derived CFU-S. Engraftment
was best when OP9 cocultures were injected as soon as sufficient
PNAS I September30, 2003 1 vol. 100 1 supple. ~ I 11907
OCR for page 92
CD4 0.5%
.`,,.,,, I, ......
Ter-119 2.1%
66.71
._
By
~ '
~ .'
~ .
:
In
m ~
PA
O
~ 4 -
M actor
CD41 CD41
m U m ~
~ ~ TIC
0 ~ O
p-H1 p-maj
CD31 50 7%1
Fig. 4. Characterization of Stats-induced blast cells growing on OP9. (a) Surface antigen expression. Day-6 iStat5CA EB cells expanded on OP9 were labeled
with the antibodies indicated and analyzed by flow cytometry. Cell number is plotted on the y axis; fluorescence intensity is plotted on the x axis. The first plot,
labeled "iso," is staining by an isotype control, nonspecific antibody. Percentages of cells positive for each marker are indicated. (b) Double staining. The same
cells were costained with antibodies against CD41 (x axis) vs. c-Kit, Sca-1, or CD31 (y axis). The percentage of cells falling into each single- and double-positive
quadrant is shown. (c) Globin gene expression. RNA was derived from cells expanded on OP9 with Stat5CA induction or with HoxB4-induction. RT-PCR for actin,
f-H1 globin, and p-major globin was performed. M represents the molecular mass marker lane.
cells were available, and was gradually lost with extensive
passage in vitro.
FACS analysis of the bone marrow of a typical recipient 1
month after transplantation is shown in Fig. 5. Although we
observed low levels of GFP positive cells overall, they counter-
stained with markers representing differentiation into all three
hematopoietic lineages: lymphoid (B220 and CD4), myeloid
(Gr-1 and Mac-1), and erythroid (Ter-119), demonstrating that
these cells have a broad differentiation potential in vivo. In one
case, we observed an animal succumb to a GFP-positive myeloid
leukemia 2 months after transplant, suggesting that at some
frequency, continual activation of Stats signaling can result in
the eventual outgrowth of a malignant population, as has been
seen after retroviral transduction of Stat5CA in bone marrow
transplant models (334. However, because donor cells were
eventually lost by the majority of recipients, our results demon-
strate that this cell population has limited self-renewal potential
in viva, even with maintenance of induction of Stats signaling.
DIscusslon
ES cells are competent to differentiate into cells of all embryonic
and adult lineages, as evidenced by the derivation of chimeric
animals from blastocysts injected with ES cells (344. This potency
11908 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 1734140100
makes ES cells promising source material for regenerative
medicine. However, putting this potential into practice in adults,
as opposed to embryos, will require the derivation of adult-
repopulating stem cells from ES cells in vitro. In the case of the
hematopoietic system, this has proven to be more difficult than
expected, given that ES cells will readily generate blood in vitro
when differentiated as EBs (1~. ES cells seem predisposed to an
embryonic mode of blood differentiation akin to that of the early
extraembryonic yolk sac, producing mainly primitive erythro-
cytes and myeloid progenitors (2, 35), but lacking adult-
repopulating cells that are thought to arise in the embryo proper
(36, 37~.
The HSC for this primitive (embryonic) mode of hematopoi-
esis seems to have the latent potential to generate definitive
(adult) lineages. When adult engraftment is enabled by trans-
formation with Bcr/Abl, an oncogene with specific growth-
promoting effects on the HSC, contribution to these lineages can
be observed (38~. Adult engraftment and multilineage hemato-
poiesis has also been observed when EB-derived cells are made
to overexpress the transcription factor HoxB4 (27~. This tran-
scription factor has growth-promoting effects on the HSC similar
to but less transforming than Bcr/Abl. HoxB4 also induces a
switch in the expression pattern of several markers that distin-
Kyba et a/.
OCR for page 93
uninjected, iso
n on
to
C) ~ ~ . , . , . .
10 10 1o2 103 1 4
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CON
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, \ so
o
~ ISO
1 _ _ 1 ~
No
100 1o1 1o2 103 104
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— > GFP
B220
At' I . . T I . ~ 1 . .... '.~1 . . ~ ~ 8 ,
10 10 10 10- 10
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o.5 onto
.. ..
.. ~ ~
~ At ~ ~ -
1~ 1o1 1o2 103 104
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10 10 10 10 10
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Ter-119
~ .~
~ 0.6 GO
100 1 . , .l . . 4
100 1 1 1 o2 l o3 1 ( 14
Fig. 5. FACS analysis of bone marrow of a doxycycline-treated recipient mouse. Bone marrow cells were harvested 1 month after transplantation, stained with
the indicated antibodies, and subjected to flow cytometry. GFP fluorescence is plotted on the x axis; antibody staining is plotted on the y axis. "iso" represents
an isotype control nonspecific antibody. The upper left plot is from a control, uninfected mouse; all others are from the experimental mouse. In each plot, the
percentage of double-positive cells is shown in the upper right quadrant. Double positives represent donor cells expressing a given antigen.
guish primitive from definitive hematopoiesis, suggesting the
possibility that it may promote a primitive-definitive switch at
the level of the embryonic HSC.
Under conditions that favor expansion of undifferentiated
hematopoietic progenitors, namely coculture with OP9 stromal
cells, and the cytokines SCF, TPO, Flt-3 ligand, and VEGF,
maintenance of Stats signaling in day-6 EB cells enables the
outgrowth of a very primitive cell population expressing many of
the markers of the HSC. The levels of Stats activation in these
cells are typical of those seen in hematopoietic cells activating
Stats for physiological reasons, such as growing in the presence
of IL-3. This finding implies that the effects we have observed are
not caused by superphysiological levels of StatS activating spu-
rious target genes. Cells expanded by Stats activation have
similarities to both the Bcr/Abl-expanded cells and the HoxB4-
expanded cells that we have previously described. Morphologi-
cally, all three populations consist mainly of undifferentiated
blast cells. By analysis of surface marker expression, the StatS-
expanded cells described here are closer in type to those
expanded by HoxB4, in particular in their expression of CD31,
CD41, and c-Kit, and in the presence of differentiating cells
expressing myeloid lineage markers. The Bcr/Abl-expanded
cells were negative for all lineage markers tested, although we
did observe lineage marker expression on some cells in the
earliest phase of expansion. It may be the case that Bcr/Abl
drives expansion of the most primitive cells more efficiently, and
is better able to suppress their differentiation in vitro. In contrast
to the HoxB4-expanded cells, the in viva engraftment potential
of the Stats-expanded cells is quite distinct. Their engraftment
is strictly doxycycline-dependent, and temporally limited,
whereas HoxB4-expanded cells engraft without the need for
maintenance of HoxB4 expression in vivo, and readily contribute
to long-term hematopoiesis. It may be the case that Stats is
driving self-renewal of lineage-committed progenitors or possi-
bly short-term-repopulating HSCs in vitro, as opposed to long-
Kyba et a/.
term-repopulating HSCs. Given their maintenance of embryonic
globin expression, it is also likely that the Stats-expanded cells
are not undergoing a primitive-definitive hematopoietic switch,
and that their inability to reconstitute adult hematopoiesis is
reflective of their similarity to the earliest hematopoietic pro-
genitors of the yolk sac, which suffer a similar defect in adult
repopulation.
Although genetic modification can enable engraftment, it
should in principle be possible to guide the differentiation of
unmodified ES cells into definitive HSCs. To achieve this, it will
be necessary to recapitulate, in a temporally appropriate man-
ner, all of the extracellular signals that an ES cell and its
lineage-restricted progeny experience from the point of blasto-
cyst injection to the point of differentiation to fetal liver-stage
HSC. This is a daunting task, not only because of the large
number of known extracellular signaling molecules and combi-
natorial possibilities, but also because the relevant molecules
may not be known at present. Given that many extracellular
signaling molecules share common intracellular mediators, the
endeavor may be simplified by focusing on these signal trans-
ducers. Focusing on individual mediators has the additional
advantage of bypassing or deconvoluting the complexity of cell
surface receptor-mediated signaling, in which multiple pathways
are commonly activated by binding of a single ligand. For
example, signaling by the leukemia inhibitory factor receptor
promotes ES cell self-renewal through activation of Stat3 (39~;
however, it also activates the mitogen-activated protein kineses,
ERK1 and ERK2, resulting a counteractive signal that attenu-
ates self-renewal (40~. By interfering with or activating individual
pathways, unique outcomes can be selected from the manifold of
effects initiated by receptor activation.
We chose to study the effects of StatS activation on the process
of hematopoietic differentiation of ES cells, in part because
StatS is a major downstream mediator of Bcr/Abl signaling,
which we have previously shown induces the expansion of an
PNAS | September 30, 2003 | vol. 100 | supple. ~ | 11909
OCR for page 94
~ -I'
embryonic hematopoietic stem cell population from EBs (38),
and in part because of the critical role that Stats plays in normal
hematopoiesis by acting to transduce signals from a variety of
cytokine receptors. Our results demonstrate that at the point of
origin of the hematopoietic lineage, day 4 to day 6 of EB
differentiation, the precursors of this lineage are competent to
respond to Stats activation. We have determined that the
enhanced hematopoiesis driven by Stats is not the result of the
rescue of hematopoietic precursors that were destined for apo-
ptosis, nor is it the result of over proliferation of the hemato-
poietic compartment. An attractive possibility is that Stats
modulates the rate of progression from stem cell to committed
progenitor (CFC) to differentiated progeny within the early
embryonic hematopoietic compartment such that stem cells and
progenitors accumulate to greater numbers in the presence of
. .
slgna 1ng.
The fact that Stats null embryos generate hematopoietic tissue
means that Stats is not essential for hematopoietic development
(15~. However, the fetal anemia seen in these embryos, which has
been attributed to a defect in erythropoietin signaling (14) might
also be due in part to reduction of the stem cell pool in the
absence of Stats. It is noteworthy that StatS functions in a
partially redundant manner in the regulation of the definitive
HSC: Stats null HSCs are capable of repopulating irradiated
recipients, but they are at a tremendous disadvantage when
placed into competition with wild-type HSCs. If Stats has a
1. Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. & Kemler, R. (1985)
J. Embryol. Exp. Morphol. 87, 27-45.
2. Keller, G., Kennedy, M., Papayannopoulou, T. & Wiles, M. V. (1993) Mol. Cell.
Biol. 13, 473-486.
3. Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F. W. & Orkin, S. H.
(1996) Cell 86, 47-57.
4. North, T., Gu, T. L., Stacy, T., Wang, Q., Howard, L., Binder, M., Marin-
Padilla, M. & Speck, N. A. (1999) Development (Cambridge, U.K) 126, 23.
2563-2575. 24.
5. Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., 25.
Breitman, M. L. & Schun, A. C. (1995) Nature 376, 62-66. 26.
6. Ihle, J. N. (1996) Cell 84, 331-334.
7. Liu, X., Robinson, G. W., Gouilleux, F., Groner, B. & Hennighausen, L. (1995)
Proc. Natl. Acad. Sci. USA 92, 8831-8835.
8. Wakao, H., Harada, N., Kitamura, T., Mui, A. L.-F. & Miyajima, A. (1995)
EMBO J. 14, 2527-2535.
9. Pallard, C., Gouilleux, F., Benit, L., Cocault, L., Souyi, M., Levy, D., Groner,
B., Gisselbrecht, S. & Dusanter-Fourt, I. (1995) EMBO J. 14, 2847-2856.
10. Hou, J., Schindler, U., Henzel, W. J., Wong, S. C. & McKnight, S. L. (1995)
Immunity 2, 321-329.
11. Wood, T. J., Sliva, D., Lobie, P. E., Pircher, T. J., Gouilleux, F., Wakao, H.,
Gustafsson, J. A., Groner, B., Norstedt, G. & Haldosen, L. A. (1995) J. Biol.
Chem. 270, 9448-9453.
12. Onishi, M., Nosaka, T., Misawa, K., Mui, A. L.-F., Gorman, D., McMahon, M.,
Miyajima, A. & Kitamura, T. (1998) Mol. Cell. Biol. 18, 3871-3879.
13. Moriggl, R., Topham, D. J., Teglund, S., Sexl, V., McKay, C., Wang, D.,
Hoffmeyer, A., van Deursen, J., Sangster, M. Y., Bunting, K. D., et al. (1999)
Immunity 2, 249-259.
14. Socolovsky, M., Fallon, A. E. J., Wang, S., Brugnara, C. & Lodish, H. F. (1999)
Cell 98, 181-191.
15. Teglund, S., McKay, C., Schuetz, E., van Deursen, J. M., Stravopodis, D., Wang,
D., Brown, M., Bodner, S., Grosveld, G. & Ihle, J. N. (1998) Cell 93, 841-850.
16. Hoover, R. R., Gerlach, M. J., Koh, E. Y. & Daley, G. Q. (2001) Oncogene 20,
5826-5835.
17. Matsumura, I., Ishikawa, J., Nakajima, K., Oritani, K., Tomiyama, Y., Miya-
gawa, J., Kato, T., Miyazaki, H., Matsuzawa, Y. & Kanakura, Y. (1997) Mol.
Cell. Biol. 17, 2933-2943.
18. Buitenhuis, M., Baltus, B., Lammers, J.-W. J., Coffer, P. J. & Koenderman, L.
(2003) Blood 101, 134-142.
11910 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 1734140100
physiological function in the primitive HSC it is must be similarly
redundant. Physiologically relevant or not, the sensitivity of
embryonic hematopoietic precursors to Stats signaling enables
enhanced hematopoietic development from ES cells exposed to
activation of StatS during differentiation. In our hands this was
achieved through inducible expression of a constitutively active
mutant; however, one could envision a small molecule agonist or
transducible protein having the same effect.
We have demonstrated that expression of several transgenes
(BcrAbl, HoxB4, and now StatSCA) can lead to the outgrowth
of primitive hematopoietic blast cell populations from differen-
tiating ES cells. Each of these populations has its own distinct
characteristics, and none yet equals the definitive HSC in terms
of its efficiency at engrafting adult recipients and contributing to
long-term multilineage hematopoiesis. Realizing that obtaining
such efficiency from ES cells will likely involve more than simple
genetic modifications, we would benefit enormously from a
better understanding of the factors that govern the origin of the
definitive HSC in embryogenesis.
We thank M. William Lensch for comments on the manuscript. This
work was supported by National Institutes of Health Grants CA86991
and DK59279, the Alberta Heritage Foundation for Medical Research,
the Canadian Institutes of Health Research, the State of Sao Paulo
Research Foundation (FAPESP), the MIT Biotechnology Process En-
gineering Center, and the Burroughs Wellcome Fund. G.Q.D. is the
Birnbaum Scholar of the Leukemia and Lymphoma Society of America.
19. Snow, J. W., Abraham, N., Ma, M. C., Abbey, N. W., Herndier, B. & Goldsmith,
M. A. (2002) Blood 99, 95-101.
20. Bunting, K. D., Bradley, H. L., Hawley, T. S., Moriggi, R., Sorrentino, B. P. &
Ihle, J. N. (2002) Blood 99, 479-487.
21. Bradley, H. L., Hawley, T. S. & Bunting, K. D. (2002) Blood 100, 3983-3989.
22. Shuai, K., Halpern, J., Hoeve, J. T., Rao, X. & Sawyers, C. L. (1996) Oncogene
13, 247-254.
Carlesso, N., Frank, D. A. & Griffin, J. D. (1996) J. Exp. Med. 183, 811-820.
Frank, D. A. & Varticovski, L. (1996) Leukemia 10, 1724-1730.
Ilaria, R. L. J. & Etten, R. A. V. (1996) J. Biol. Chem. 271, 31704-31710.
. Nieborowska-Skorska, M., Wasik, M. A., Slupianek, A., Salomoni, P., Kita-
mura, T., Calabretta, B. & Skorski, T. (1999) J. Exp. Med. 189, 1229-1242.
27. Kyba, M., Perlingeiro, R. C. R. & Daley, G. Q. (2002) Cell 109, 29-37.
28. Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D. & Jaenisch, R.
(2000) Mol. Cell. Biol. 20, 7419-7426.
29. Naviaux, R. K., Costanzi, E., Haas, M. & Verma, I. M. (1996) J. Virol. 70,
5701-5705.
30. Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J. C. & Keller, G. (1998)
Development (Cambridge, U.K) 125, 725-732.
31. Mitjavila-Garcia, M. T., Cailleret, M., Godin, I., Nogueira, M. M., Cohen-Solal,
K., Schiavon, V., Lecluse, Y., Pesteur, F. L., Lagrue, A. H. & Vainchenker, W.
(2002) Development (Cambridge, U.K.) 129, 2003-2013.
32. Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D. & Orkin, S. H. (2003)
Blood 101, 508-516.
33. Schwaller, J., Parganas, E., Wang, D., Cain, D., Aster, J. C., Williams, I. R., Lee,
C. K., Gerthner, R., Kitamura, T., Frantsve, J., et al. (2000) Mol. Cell 6, 693-704.
34. Bradley, A., Evans, M., Kaufman, M. H. & Robertson, E. (1984) Nature 309,
255-256.
35. Palis, J., Robertson, S., Kennedy, M., Wall, C. & Keller, G. (1999) Development
(Cambridge, U.K) 126, 5073-5084.
36. Muller, A. M., Medvinsky, A., Strouboulis, J., Grosveld, F. & Dzierzak, E.
(1994) Immunity 1, 291-301.
37. Cumano, A., Dieterlen-Lievre, F. & Godin, I. (1996) Cell 86.
38. Perlingeiro, R. C. R., Kyba, M. & Daley, G. Q. (2001) Development (Cambridge,
U.K) 128, 4597-4604.
39. Niwa, H., Burdon, T., Chambers, I. & Smith, A. (1998) Genes Dev. 12,
1248-2060.
40. Burdon, T., Stracey, C., Chambers, I., Nichols, J. & Smith, A. (1999) Dev. Biol.
210, 30-43.
Kyba et al.
Representative terms from entire chapter:
cell line
