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HEMATOPOIESIS AND STEM CELLS
Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells
and induced pluripotent stem cells
Hyung Joon Joo,1 Honsoul Kim,1 Sang-Wook Park,1 Hyun-Jai Cho,2 Hyo-Soo Kim,2 Do-Sun Lim,3 Hyung-Min Chung,4
Injune Kim,1 Yong-Mahn Han,1 and Gou Young Koh1
1Laboratory
for Vascular Biology and Stem Cell, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology
(KAIST), Daejeon, Korea; 2National Research Laboratory for Cardiovascular Stem Cell, Innovative Research Institute for Cell Therapy, Seoul National University
Hospital, Seoul, Korea; 3Department of Cardiology, College of Medicine, Korea University, Seoul, Korea; and 4Stem Cell Research Laboratory, CHA Stem Cell
Institute, Pochon CHA University, Seoul, Korea
Angiopoietin-1 (Ang1) plays a crucial role
in vascular and hematopoietic development, mainly through its cognate receptor Tie2. However, little is known about
the precise role of Ang1 in embryonic
stem cell (ESC) differentiation. In the
present study, we used COMP-Ang1 (a
soluble and potent variant of Ang1) to
explore the effect of Ang1 on endothelial
and hematopoietic differentiation of
mouse ESCs in an OP9 coculture system
and found that Ang1 promoted endothe-
lial cell (EC) differentiation from Flk-1ⴙ
mesodermal precursors. This effect
mainly occurred through Tie2 signaling
and was altered in the presence of soluble
Tie2-Fc. We accounted for this Ang1induced expansion of ECs as enhanced
proliferation and survival. Ang1 also had
an effect on CD41ⴙ cells, transient precursors that can differentiate into both endothelial and hematopoietic lineages. Intriguingly, Ang1 induced the preferential
differentiation of CD41ⴙ cells toward ECs
instead of hematopoietic cells. This EC
expansion promoted by Ang1 was also
recapitulated in induced pluripotent stem
cells (iPSCs) and human ESCs. We successfully achieved in vivo neovascularization in mice by transplantation of ECs
obtained from Ang1-stimulated ESCs. We
conclude that Ang1/Tie2 signaling has a
pivotal role in ESC-EC differentiation and
that this effect can be exploited to expand
EC populations. (Blood. 2011;118(8):
2094-2104)
Introduction
Embryonic stem cells (ESCs) have been used frequently not only
for research in the field of developmental biology, but also in
cell-based regenerative medicine.1 Many scientists in the field of
vascular biology have studied the endothelial cell (EC) differentiation of ESCs in an attempt to develop treatments for the
associated pathologies, and therefore our knowledge of EC
differentiation has significantly expanded.2,3 However, the pluripotency of ESCs capable of generating various cell types and the
overwhelming complexity of the multiple signaling pathways
involved in the process of differentiation make it extremely
challenging to control EC differentiation in a precise and
efficient manner. Our knowledge of ESC-EC differentiation still
remains far from satisfactory, and the possibility of successfully
applying stem cells in vascular regenerative medicine remains
merely optimistic.
Several recent studies have provided evidence supporting the
presence of an intimate linkage between ECs and hematopoietic
cells (HCs) during early embryonic development.4,5 It has been
suggested that both the endothelial and hematopoietic lineages
arise from a common progenitor, the hemangioblasts, which
express Flk-1 during the early stage of differentiation.6-8 Moreover, several studies have suggested that the Flk-1⫹ cells at this
stage are actually mesodermal precursor cells (MPCs), based on
the fact that they can give rise to not only ECs and HSCs but also
to vascular smooth muscle cells (VSMCs) and even cardiomyocytes.9,10 Recent studies have also demonstrated that some HSCs
are derived from a subset of ECs called the hemogenic
endothelium during the differentiation from ESCs.11-13 Other
studies revealed that the initial definitive hematopoietic cells
could originate from hemogenic endothelium that resides in the
aorta during early embryonic development.14-16 However, the
underlying link between ECs and HSCs still remains obscure
and is the subject of controversy.
During the early stages of life, various growth factors influence
endothelial and hematopoietic development. Among them, Ang1, a
cognate ligand for the Tie2 receptor, is one of the key molecules
known to regulate the development of the vasculature and the
hematopoietic system.17,18 The fact that Ang1-deficient mouse
embryos die between embryonic day 10.5 (E10.5) and E12.5 due to
failure of vascular remodeling, impaired definitive hematopoiesis,
and endocardial defect17 and the fact that Tie2 expression is present
in both ECs and HSCs18,19 attracted our attention. Based on these
results, we decided to pursue the idea that Ang1/Tie2 signaling
might have a pivotal role in endothelial and hematopoietic differentiation from ESCs.
The primary goal of the present study was to investigate the role
of the Ang1/Tie2 signaling pathway and its underlying mechanisms
in ESC-EC differentiation. In addition, we explored whether Ang1
could be used to maximize the expansion of the endothelial
population as a result of controlled ESC-EC differentiation to
investigate the possibility of applying Ang1 in therapeutic protocols that require EC production.
Submitted December 8, 2010; accepted June 3, 2011. Prepublished online as
Blood First Edition paper, June 16, 2011; DOI 10.1182/blood-2010-12-323907.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
2094
BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
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BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Methods
Cell culture
A mouse ESC line (R1) was obtained from the American Type Culture
Collection. OP9 cells were gifted from J.K. Yamashita (Kyoto University,
Japan). Mouse induced pluripotent stem cells (iPSCs) were prepared as
described previously.20 Flk-1⫹ MPC induction and OP9 coculture for
endothelial and hematopoietic differentiation were performed as described previously.10,21 Briefly, for Flk-1⫹ MPC induction, ESCs were
cultured on 0.1% gelatin-coated cell-culture plates at a density of
1-2 ⫻ 103 cells/cm2 for 96-108 hours in the differentiation medium
(␣-MEM [Invitrogen] supplemented with 10% selected FBS [Welgene])
without leukemia inhibitory factor. For endothelial differentiation,
Flk-1⫹ MPCs were purified at E4.5 with a FACSAria II (BD Biosciences) or an AutoMACS Pro Separator (Miltenyi Biotec) combined with
allophycocyanin (APC)–conjugated anti–mouse Flk-1 antibody (clone
AVAS12a1; eBioscience) and/or anti-APC MicroBeads (Miltenyi Biotec), plated onto the confluent mitomycin-C (Sigma-Aldrich)–treated
OP9 cells at a density of 1-2 ⫻ 104 cells/cm2, and cultured in the
differentiation medium. Endothelial differentiation of human ESCs
(CHA4-hES)22,23 was also performed as described previously.23 Briefly,
undifferentiated hESCs were cultured for 3 days in ESC medium
(ESCM; DMEM/F12 medium supplemented with 20% knockout serum
replacement, 1% nonessential amino acids, 0.1mM ␤-mercaptoethanol;
all from Invitrogen) with MEK/ERK inhibitor (50␮M, PD98059;
Calbiochem) and BMP4 (20 ng/mL; Peprotech), and cultured for
another 6 days in ESCM with VEGF-A (100 ng/mL) and b-FGF (100
ng/mL; R&D Systems). CD34⫹ progenitor cells were purified by MACS
using anti–human CD34 microbeads, replated, and cultured in EC basal
medium-2 with growth supplements (EGM-2; Clonetics). hESC-derived
ECs were induced from CD34⫹ progenitor cells and analyzed upon EC
differentiation 9 days later. Various recombinant proteins and other
reagents, including COMP-Ang124 (hereafter abbreviated as Ang1),
Ang3, Ang4, sTie2-Fc, VEGF-A, and VEGF-Trap, were prepared as
described previously.25,26 RGD peptides (GRADSP and GRGDSP) were
purchased from Calbiochem.
Immunofluorescence staining
Cells were fixed with 2% paraformaldehyde and blocked with 5% goat (or
donkey) serum in PBST (0.3% Triton X-100 in PBS) for 1 hour at room
temperature. The cells were incubated overnight at 4°C with the following
primary antibodies: anti–mouse CD144 antibody (clone 11D4.1; BD
Pharmingen), anti–mouse CD31 antibody (clone 2H8; Chemicon), anti–
mouse phospho-histone H3 (Ser10) antibody (rabbit polyclonal; Upstate
Biotechnology), anti–mouse Flk-1 antibody (rabbit polyclonal; gifted from
Dr Rolf Brekken, University of Texas-Southwestern Medical Center,
Dallas,, TX), FITC-conjugated anti-CD41 antibody (clone MWReg30;
eBioscience). After washing in PBST 6 times, the cells were incubated for
3 hours at room temperature with the following secondary antibodies:
Cy3-conjugated anti–hamster IgG antibody (Jackson ImmunoResearch),
Cy3-conjugated anti–rabbit IgG antibody (Jackson ImmunoResearch),
and FITC-conjugated anti–rat antibody (Jackson ImmunoResearch).
The cells were then mounted in fluorescent mounting medium (DAKO).
Nuclei were stained with Hoechst 33258 (Invitrogen). Immunofluorescent images were acquired using an LSM510 confocal fluorescence
microscope (Carl Zeiss).
Ang1 PROMOTES ENDOTHELIAL DIFFERENTIATION
2095
phycoerythrin (PE) or APC antibody (clone MEC13.3; eBioscience),
anti–mouse CD144-Alexa Fluor 647 antibody (clone BV13; eBioscience).
anti–mouse CD45-APC antibody (clone 30-F11; eBioscience), anti–mouse
CD41-PE or APC antibody (clone MWReg30; BD Biosciences), antimouse CD117-APC antibody (clone 2B8; eBioscience), anti–mouse Sca-1–
APC antibody (clone D7; eBioscience), anti–mouse Tie2-PE antibody
(clone TEK4; eBioscience), anti–mouse Ter119-APC antibody (clone
Ter119; eBioscience), and anti–mouse CD34-FITC antibody (clone RAM34;
eBioscience). Dead cells were excluded by 7-aminoactinomycin D (Invitrogen). Data were analyzed using FlowJo Version 7.2.5 software (TreeStar).
Cell purity of ⬎ 95% was confirmed after cell sorting.
Apoptosis assay
After 3 days of differentiation of purified Flk-1⫹ MPCs on OP9 cells, the
cells were quickly trypsinized with 0.25% trypsin-EDTA and stained
with anti–mouse Flk-1–APC antibody. Annexin V and propidium iodide
staining was performed using the annexin V-FLUO staining kit (Roche)
for 10 minutes at room temperature according to the manufacturer’s
instructions, and then flow cytometric analysis was performed.
In vitro tube-forming assay
For cell preparation, Flk-1⫹ MPCs differentiated from ESCs were purified
and cultured for 3 days on OP9 cells. Ang1 (200 ng/mL) was given on days
0 and 2. CD31⫹/CD144⫹ cells were sorted by FACS on day 3 and used as
Ang1-induced ESC-derived ECs (Ang1-EC-ESCs) for the in vitro tubeforming assay. Matrigel (300 ␮L, growth factor reduced; BD Biosciences)
was spread on a 24-well polystyrene plate and allowed to solidify at 37°C.
Ang1-EC-ESCs were plated on Matrigel-covered plates in EGM-2 at a
density of ⬃ 20 000-40 000 cells/cm2. After 24 hours, the tube structure of
the plated cells was observed using an Axiovert25 microscope (Carl Zeiss).
Phase-contrast images were taken using an Infinity X digital camera and
DpxView LE software (DeltaPix). HUVECs were prepared as described
previously27 and were used as a positive control.
Matrigel plug assay
Six- to 8-week-old CD31-knockout mice were gifted from Dr Tak W. Mak
(University of Toronto, Toronto, Ontario, Canada) and used for the
Ang1-EC-ESC transplantation experiment. The mice were immunosuppressed with cyclosporine A. From 2 days before to 3 days after
transplantation, 30 mg/kg/d of cyclosporine A was administered intraperitoneally and then the dosage was reduced to 15 mg/kg/d. Animal care and
experimental procedures were performed with the approval of the animal
care committee of KAIST. Ang1-EC-ESCs (0.5 ⫻ 106 cells) were mixed
with 500 ng/mL of VEGF165 and 200 ng/mL of Ang1 in a total volume of
100 ␮L. Matrigel was implanted subcutaneously into the dorsal side of the
immunosuppressed CD31-deficient mice. After 2 weeks, 100 ␮g of
FITC-lectin (Sigma-Aldrich) was injected into the tail vein 20 minutes
before the mice were killed and examined to assess blood perfusion. The
implanted Matrigel was fixed by systemic vascular perfusion with 1%
paraformaldehyde in PBS, harvested, and whole-mounted for immunofluorescence staining.
Statistics
Data are presented as means ⫾ SD. Significant differences between means
were determined by ANOVA followed by the Student-Newman-Keuls test.
Significance was set at P ⬍ .05.
Flow cytometry and cell sorting
Cells were harvested with 0.25% trypsin-EDTA or dissociation buffer
(Invitrogen) and resuspended in Hank buffered salt solution/2% FBS at
1 ⫻ 106 cells per 100 ␮L. The cells were incubated with antibodies
described below for 20 minutes, washed twice, and resuspended. Analyses
and sorting were performed with a FACS LSRII (BD Biosciences) or
FACSAria II (BD Biosciences). The antibodies were anti–mouse Flk-1APC antibody (clone AVAS12a1; eBioscience), anti–mouse CD31-
Results
Ang1 promotes endothelial differentiation via Tie2 signaling
According to previous studies,11,28-30 the formation of CD31⫹/
CD144⫹ EC colonies originating from ESC-derived Flk-1⫹ MPCs
is regarded as EC differentiation. In agreement with previous
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BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Figure 1. Ang1 promotes EC differentiation via Tie2
signaling. Flk-1⫹ MPCs were purified on E4.5 and
cultured on OP9 cells. Control buffer (Control), Ang1
(200 ng/mL), sTie2-Fc (sT2, 25␮g/mL), RGD peptide
(GRGDSP, 25␮g/mL), and control peptide (GRADSP,
25␮g/mL) were administered alone or together on days 0
and 2, and analyses were performed on day 3. (A) Treatment scheme of Ang1 or inhibitor. (B) Comparison of cell
number of each EC colony on day 3 (n ⫽ 4). *P ⬍ .05
compared with control. (C) Images showing CD144⫹ EC
colonies on day 3. Nuclei were stained with Hoechst.
Scale bar represents 200 ␮m. (D) Dose dependency of
Ang1 on the percentage of CD31⫹/CD144⫹ ECs from
purified Flk-1⫹ cells on day 3 (n ⫽ 3). *P ⬍ .05 compared
with control. (E,G) Representative FACS analyses of the
population of CD31⫹/CD144⫹ ECs. Numbers indicate the
percentage of CD31⫹/CD144⫹ ECs. (F,H) Comparison
percentages of CD31⫹/CD144⫹ ECs (n ⫽ 4). *P ⬍ .05
compared with control, GRGDSP, or GRADSP; #P ⬍ .01
compared with Ang1.
studies,11,21,28,30 the Flk-1⫹ MPCs in our OP9 coculture system
successfully gave rise to EC colonies (Figure 1A). In this setting,
we examined the effect of various growth factors, including
VEGF-A, VEGF-C, Ang1, Ang2, Ang3, Ang4, bFGF, EGF, TGF-␤,
and PDGF-␣, on EC differentiation. Of these, Ang1 exerted the
strongest effect on the expansion of the EC colony (supplemental Figure 1A, available on the Blood Web site; see the
Supplemental Materials link at the top of the online article), so
our study was focused on the role of Ang1 in ESC-EC
differentiation. We used COMP-Ang1, a soluble and potent
Ang1 variant protein, for Ang1 supplementation.24 Compared
with controls, additional Ang1 (200 ng/mL on days 0 and 2)
increased the number and size of the CD144⫹ EC colony and
subsequently promoted the formation of larger EC colonies by
fusion on day 3 (Figure 1B-C and supplemental Videos 1 and 2).
Flow cytometric analyses revealed a positive correlation between
the frequency of generated ECs and the concentration of Ang1 treatment
within the range of ⬃ 25-200 ng/mL; however, the effect of Ang1
reached a plateau beyond 200 ng/mL (Figure 1D). We chose
200 ng/mL of Ang1 for the following experiments based on its
maximum effect on EC differentiation.
Pretreatment with an excess amount (25 ␮g/mL) of soluble
Tie2-Fc (sT2), a selective blocker of Ang1, completely abrogated
the Ang1-induced promotion of CD31⫹/CD144⫹ EC frequency on
day 3 (Figure 1E-F). Treatment of sTie2 also significantly suppressed (⬃ 2.0-fold) the percentage of CD31⫹/CD144⫹ ECs in the
controls, suggesting the possibility that angiopoietins secreted from
either differentiating ESCs or OP9 feeder cells may have served to
constitutionally maintain endothelial differentiation. In fact, both
CD31⫹/CD144⫹ ECs and non-ECs (including OP9 cells) expressed Ang1, whereas only CD31⫹/CD144⫹ ECs expressed
Ang2 (supplemental Figure 1B). However, the addition of Ang2
(25-800 ng/mL) did not alter the profile of CD31⫹/CD144⫹ EC
production (supplemental Figure 1C). In addition, because our
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BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Ang1 PROMOTES ENDOTHELIAL DIFFERENTIATION
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Figure 2. Ang1 promotes the EC population but
reduces the HSC and VSMC populations during Flk-1ⴙ
MPC differentiation. Flk-1⫹ MPCs were purified on
E4.5 and cultured on OP9 cells. Control or Ang1 was
administered on days 0, 2, and 4, and analyses were
performed on days 1.5, 3, and 6. (A,C,E) Representative
FACS analyses of the populations of CD31⫹/CD144⫹
ECs, CD45⫹ HSCs, and ␣-SMA⫹ VSMCs. Numbers
indicate percentages of ECs, HSCs and VSMCs.
(B,D,F) Comparison of percentages of CD31⫹/CD144⫹
ECs, CD45⫹ HSCs, and ␣-SMA⫹ VSMCs over time
(n ⫽ 5). *P ⬍ .01 compared with control.
RT-PCR and FACS analyses revealed that the OP9 cells did not
express Tie2 (supplemental Figure 1B and D), Ang1 would not
have had a significant effect on the OP9 cells. Therefore, we
believe that Ang1, but not Ang2, acted to increase the EC
population in a paracrine manner during differentiation. Because recent studies have indicated that Ang1 can also exert its
cellular actions through integrin,31,32 we attempted to exclude
the possibility of the increased endothelial populations being the
result of Ang1 stimulation via integrin. The pan-integrin inhibitor GRGDSP and its control GRADSP did not alter the basaland Ang1-induced promotion of CD31⫹/CD144⫹ EC percentages (Figure 1G-H). Moreover, GRGDSP and GRADSP had no
measurable effect on EC adhesion (supplemental Figure 2).
These data indicate that Tie2 is the corresponding receptor that
mediated signal transduction of Ang1 to increase endothelial
differentiation.
We also analyzed the expression pattern of Tie2 during
differentiation. Although the Flk-1⫹ cells derived from ESCs at
E4.5 did not express Tie2 immediately after isolation (supplemen-
tal Figure 3A), 12.4% of the cells began to express Tie2 as early
as 12 hours after being replated on OP9 cells (supplemental
Figure 3B). At days 1 and 3, approximately 20%-25% of the
cells derived from the Flk-1⫹ cells expressed Tie2 (supplemental Figure 3B-D). The addition of Ang1 further increased the
Tie2⫹ population up to 38.5% on day 3 (supplemental Figure
3C-D). FACS analysis revealed that most Tie2⫹ cells were
CD31⫹/CD144⫹ ECs not CD45⫹ HSCs (supplemental Figure
3E); therefore, Ang1 increased the percentage of the Tie2⫹ EC
population by ⬃ 2 fold compared with control. In addition,
Ang1 successfully activated Tie2 in the differentiating cells
derived from ESCs (supplemental Figure 4A-B), and pretreatment with the PI3K inhibitor (LY294002, 20␮M) almost
completely inhibited Ang1-induced CD31⫹/CD144⫹ EC expansion, whereas the MEK/ERK inhibitor (PD98059, 50␮M) did
not. Moreover, Ang1 significantly increased phosphorylation of
Akt at serine 473 in the CD144⫹ ECs (supplemental Figure 4C).
These data imply that Ang1 promotes EC differentiation through
the activation of Tie2-PI3K-Akt in Flk-1⫹ MPCs.
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BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Figure 3. Ang1 increases EC proliferation but suppresses EC apoptosis during Flk-1ⴙ MPC differentiation. Control or Ang1 was administered into Flk-1⫹ MPCs
on days 0, 2, and 4 during OP9 coculture. (A) Images
showing PHH3⫹ cells in CD144⫹ EC colonies on days
1.5, 2, 2.5, 3, and 3.5. Nuclei were stained with Hoechst.
Scale bar represents 100 ␮m. (B) Comparison of number
of PHH3⫹ ECs over time (n ⫽ 5). *P ⬍ .05 compared with
control. (C) Representative FACS analyses of the populations of annexin⫹ apoptotic Flk-1⫹ cells and Flk-1⫺ cells
on day 3. Numbers indicate percentages of each population. (D) Comparison of percentage of annexin⫹ apoptotic
Flk-1⫹ cells and Flk-1⫺ cells on day 3 (n ⫽ 3). *P ⬍ .05
compared with control.
Ang1 induces differentiation of ESCs toward ECs
Flk-1⫹ MPCs are known to give rise to 3 major cell types: ECs,
HSCs, and VSMCs.10,33 To assess EC, HSC, and VSMC differentiation from the Flk-1⫹ MPCs, we analyzed the frequencies of
CD31⫹/CD144⫹ ECs, CD45⫹ HSCs, and ␣-SMA⫹ VSMCs on
days 1.5, 3, and 6 (Figure 2). In the absence of additional Ang1, the
percentage of CD31⫹/CD144⫹ ECs was observed to gradually
decrease, whereas the percentage of CD45⫹ HSCs rapidly increased over time (Figure 2). FACS analyses revealed that the
CD45⫹ cells were mainly CD11b⫹ myeloid cells (supplemental
Figure 5A). However, when Ang1 was administered, the percentage of ECs continued to increase but the increase rate of CD45⫹
HSCs was significantly blunted (Figure 2). The suppressive effect
of Ang1 on hematopoietic expansion was also observed at the
hematopoietic progenitor level, as shown by the reduced hematopoietic colony formation (supplemental Figure 5B-C). The population
of ␣-SMA⫹ VSMCs was found to be 10%-15% at day 1.5 and
dramatically increased over time in the absence of Ang1. However,
the rate of increase in VSMCs was markedly slowed down in the
presence of Ang1 (Figure 2). These data indicate that Ang1
preferentially drives EC differentiation from Flk-1⫹ MPCs during
differentiation of ESCs.
Ang1 promotes proliferation and suppresses apoptosis of ECs
during Flk-1ⴙ MPC differentiation
We next examined the underlying mechanism through which Ang1
promotes endothelial differentiation. The proliferative activity
measured by phospho-histone H3 immunostaining revealed that
Ang1 enhanced the proliferative activities of CD144⫹ ECs by
1.5-fold and 1.3-fold at days 2 and 2.5, respectively, compared with
controls (Figure 3A-B). Furthermore, Ang1 inhibited cell apoptosis
in the Flk-1⫹ cells but not in Flk-1⫺ cells, as measured by
costaining with annexin V and propidium iodide (Figure 3C-D).
These data indicate that Ang1 enhanced the proliferation and
extended the survival of ECs, which could be the cause of the
increases in ESC-derived EC populations.
Ang1 drives CD41ⴙ cells preferentially toward ECs rather
than HSCs
CD41 is expressed in cells that are involved in the early stage of
hematopoiesis, and its expression is associated with hematopoietic
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Figure 4. Ang1 drives CD41ⴙ cells to ECs rather than
HSCs. Expression pattern and differentiation potential of
CD41⫹ cells derived from Flk-1⫹ MPCs were characterized. (A) Representative FACS analyses showing surface
expression patterns of CD144, CD31, Tie2, Sca-1, and
c-Kit, each compared with CD41 on Flk-1⫹ MPC–derived
cells on day 1.5. (B) Images showing CD144⫹/CD41⫹
cells in endothelial colonies on day 1.5. Nuclei were
stained with Hoechst. Scale bar represents 20 ␮m.
(C) Experimental scheme for the differentiation potential
of CD41⫹ cells. Sorted CD41⫹ cells on day 1.5 were
replated on the freshly prepared OP9 cells, treated with
Control, Ang1, sT2, or Ang1 ⫹ sT2 on days 1.5, and 3.5, and
analyses were performed on day 6. (D,F) Representative
FACS analyses of the populations of CD31⫹/CD144⫹ ECs
and CD45⫹ HSCs from the sorted CD41⫹ cells. Numbers
indicate percentages of each population. (E,G) Comparison
of percentages of CD31⫹/CD144⫹ ECs and CD45⫹ HSCs
from the sorted CD41⫹ cells (n ⫽ 3). *P ⬍ .05 compared with
control; #P ⬍ .05 compared with Ang1.
commitment of hemogenic endothelium in the early murine yolk
sac.34-36 It was recently reported that certain hemogenic ECs
transiently express CD41 at an early stage of ESC endothelial and
hematopoietic differentiation.12 Consistent with these previous
findings, our data revealed that ⬃ 10%-20% of Flk-1⫹ MPC–
derived cells were CD41⫹ on day 1.5, and this number gradually
decreased over time (Figure 2C). Interestingly, flow cytometric
analyses revealed that CD41⫹ cells on day 1.5 concurrently
expressed both endothelial and primitive hematopoietic markers
(CD144, CD31, Tie2, Sca-1, and c-Kit; Figure 4A). Immunostaining revealed that there were CD144⫹/CD41⫹ cells in the EC
colonies (Figure 4B).
To determine the differentiation potential of these CD41⫹ cells,
we sorted CD41⫹/CD45⫺ cells on day 1.5, replated them on the
freshly prepared OP9 cells, and cultured them for another 4.5 days
(Figure 4C). On day 6 after plating, ⬃ 25% of the cells were
CD144⫹/CD31⫹ ECs and ⬃ 30% were CD45⫹ HSCs. Compared
with controls, Ang1 increased the CD144⫹/CD31⫹ EC population
by ⬃ 2-fold, whereas Ang1 suppressed the CD45⫹ HSC population
by ⬃ 3-fold. We also performed a limiting dilution analysis with
CD41⫹/CD45⫺ cells to determine whether they gave rise to the EC
colony (supplemental Figure 6A). Even after dilution to 10 cells,
the CD41⫹/CD45⫺ cells were still able to generate the CD144⫹ EC
colony, but after dilution to ⬍ 10 CD41⫹/CD45⫺ cells, they were
not (supplemental Figure 6B), suggesting that ⬎ 10 CD41⫹ cells is
necessary for the generation of EC colony by an as-yet-unidentified
mechanism. To confirm the effect of Ang1 on the suppression of
HSC differentiation from the CD41⫹/CD45⫺ cells, we performed a
CFU assay of CD41⫹/CD45⫺ cells that were obtained from Flk1⫹
MPCs on OP9 culture at day 1.5 according to the original
experimental scheme shown in Figure 4C and supplemental Figure
6C. The CFU assay revealed that the CD41⫹/CD45⫺ cells gave rise
to hematopoietic colonies including B/CFU-E, CFU-M/GM, and,
rarely, CFU-GEMM at day 10. FACS analyses revealed that the
Ter119⫹ erythroid cell population in hematopoietic colonies peaked
to 30% at day 5 and decreased thereafter, whereas the CD45⫹
myeloid cell population gradually increased up to 30% at day 10
(supplemental Figure 6D). The addition of Ang1 (1 ␮g/mL) to the
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BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Figure 5. Ang1 increases EC differentiation from
mouse iPSCs and human ESCs. (A-C) iPSC-derived
Flk-1⫹ MPCs were purified on E4.5 and cultured on OP9
cells with control buffer (Control) or Ang1. Analyses were
performed on day 3. (A) Images showing CD144⫹ EC
colonies. Nuclei were stained with Hoechst. Scale bar
represents 200 ␮m. (B) Representative FACS analyses
of the population of CD31⫹/CD144⫹ ECs. Numbers
indicate percentages of ECs from Flk-1⫹ MPCs during
differentiation. (C) Comparison percentages of CD31⫹/
CD144⫹ ECs from purified Flk-1⫹ MPCs (n ⫽ 3). *P ⬍ .05
compared with control. (D-E) hESC-derived CD34⫹ cells
were purified and cultured with Control or Ang1 and
analyses were performed 9 days later. (D) Images showing CD144⫹ EC colonies. Nuclei were stained with
Hoechst. Scale bars represent 200 ␮m. (E) Representative FACS analyses of the population of CD31⫹/CD105⫹
ECs. Numbers indicate percentages of ECs from CD34⫹
cells during differentiation. (F) Comparison percentages
of CD31⫹/CD105⫹ ECs from purified CD34⫹ cells (n ⫽ 3).
*P ⬍ .05 compared with control.
medium suppressed the populations of CD45⫹ myeloid cells, but
had little effect on the population of Ter119⫹ erythroid cells
(supplemental Figure 6D). Likewise, Ang1 significantly reduced
the number of CFU-G/GM cells, whereas Ang1 had no significant
effect on B/CFU-E and CFU-GEMM cells (supplemental Figure
6E). Based on these findings, we conclude that Ang1 suppressed
HSC differentiation from CD41⫹/CD45⫺ cells.
sTie2 markedly suppressed the CD144⫹/CD31⫹ EC population
by 5-fold, but it also significantly increased the CD45⫹ HSC
population compared with control. The addition of Ang1 failed to
reverse the sTie2-induced changes in EC and HSC differentiation
(Figure 4D-G). These data imply that the endogenous Ang1
secreted from differentiating ESC cells and/or OP9 feeder cells
may contribute to EC differentiation. It can also be inferred that
Ang1 drives the differentiation of CD41⫹ cells preferentially
toward ECs rather than HSCs.
Ang1 promotes endothelial differentiation from mouse iPSCs
and human ESCs
To determine the effect of Ang1 on EC differentiation in another
type of pluripotent cell, we applied the same differentiation
protocol to mouse iPSCs.37 Similar to mouse ESCs, the presence of
Ang1 promoted the size of each colony and the number of CD144⫹
ECs that it contained (Figure 5A). FACS analyses revealed that
Ang1 significantly increased the percentage of CD31⫹/CD144⫹
ECs on day 3 compared with controls (Figure 5B-C). We also
tested the effect of Ang1 in human ESC differentiation. Purified
human CD34⫹ cells were cultured in EGM-2 medium with or
without Ang1 (500 ng/mL) after CD34⫹ cells were induced from
human ESCs. Ang1 promoted the CD144⫹ EC number and size
in each colony (Figure 5D). FACS analyses revealed that Ang1
significantly increased the percentage of CD31⫹/CD105⫹ ECs
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BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Ang1 PROMOTES ENDOTHELIAL DIFFERENTIATION
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compared with controls (Figure 5E). In summary, our data
indicated that Ang1 commonly promoted EC differentiation in
both iPSCs and ESCs.
Ang1 promotes EC differentiation in the OP9 cell-free system
To examine whether Ang1 also promotes EC differentiation in the
OP9 cell-free system, Flk-1⫹ MPCs were purified at E4.5, plated
onto Matrigel-coated plates, and cultured in the differentiation
medium containing Ang1 (supplemental Figure 7A). Ang1 also
profoundly promoted EC differentiation in the Matrigel-coated
system (supplemental Figure 7B-D), indicating that it has a direct
effect on EC differentiation from the Flk-1⫹ MPCs.
Ang1-induced ECs have a neovasculogenic potential in vitro
and in vivo
To explore whether the ECs induced by Ang1 have vasculogenic
potential, we purified CD31⫹/CD144⫹ ECs cultured with Ang1
supplements using FACS on day 3 and refer to them as “Ang1-ECESCs.” FACS analysis revealed that some mesenchymal stem cell
(MSC) markers (CD105, Sca-1, and c-Kit) were positive in
Ang1-EC-ESCs, whereas another MSC marker (CD106) was
negative (supplemental Figure 8A). Recently, Kopher et al demonstrated that hESC-derived CD34⫹ endothelial precursors have
phenotypic and functional features similar to MSCs.38 Therefore,
Ang1-EC-ESCs may contain MSC components. When Ang1-ECESCs were plated on a 0.1% gelatin-coated dish in EGM-2
medium, they showed a typical cobblestone-like appearance as
early as 6 hours from the start of incubation (Figure 6A). Typical
endothelial adhesion molecules such as CD144 and CD31 were
abundantly localized on the cell-cell junctions of the Ang1-ECESCs (Figure 6B). Therefore, Ang1-EC-ESCs were morphologically compatible with typical ECs. An in vitro tube-forming assay
was performed to characterize the functional properties of Ang1-ECESCs. When Ang1-EC-ESCs were seeded on Matrigel-covered
plates and incubated in EGM-2 medium, although relatively
abundant cell clusters were observed, obvious vessel-like network
structures similar to those of HUVECs were formed as early as
12 hours from the start of incubation (Figure 6C), indicating that
Ang1-EC-ESCs are capable of achieving in vitro neovascularization. Our additional immunostaining analysis revealed that the
vascular network structure formed with Ang1-EC-ESCs in the in
vitro tube-forming assay consisted of mostly CD144⫹ cells and
some CD144⫺ adjacent cells (supplemental Figure 8B), suggesting
that the Ang1-EC-ESCs in our differentiation system could also
have functionally and phenotypically different characteristics from
mature ECs.
To determine the in vivo vasculogenic potential of Ang1-ECESCs, the cells were mixed with 500 ng/mL of VEGF165 and 200
ng/mL of Ang1 in 100 ␮L of Matrigel and implanted subcutaneously into the dorsal flank of CD31-deficient mice. Before
implantation of the Ang1-EC-ESC mixture, the CD31-deficient
mice were pretreated with cyclosporine A to prevent allograft
immune rejection (Figure 6D). At 14 days after implantation,
FITC-lectin was injected 20 minutes before the implanted
Matrigel was harvested to assess blood perfusion. Immunohistochemistry of the implants revealed the presence of CD31⫹
vessel structures that colocalized with FITC-lectin signals,
showing that Ang1-EC-ESCs are capable of establishing bloodperfused neovasculatures in vivo (Figure 6E). We conclude that
CD31⫹/CD144⫹ ECs stimulated by Ang1 have neovasculogenic
potential not only in vitro but also in vivo.
Figure 6. Ang1-induced CD31ⴙ/CD144ⴙ ECs have a neovasculogenic potential
in vitro and in vivo. Purified Flk-1⫹ MPCs differentiated from mouse ESCs were
cultured for 3 days on OP9 cells. Ang1 was administered on days 0 and 2.
CD31⫹/CD144⫹ cells were sorted by FACS on day 3 and used as Ang1-EC-ESCs for
further experiments. (A) Phase contrast images showing Ang1-EC-ESCs. Scale bar
represents 100 ␮m. (B) Immunostaining images showing Ang1-EC-ESCs. Nuclei
were stained with Hoechst. Scale bar represents 100 ␮m. (C) Images showing
network formations of Ang1-EC-ESCs and HUVECs. Ang1-EC-ESCs and
HUVECs were plated on Matrigel-covered plates and incubated in EGM-2. The
tube structures were observed after 24 hours. Scale bar represents 200 ␮m.
(D-E) Ang1-mESC-ECs were injected subcutaneously into the dorsal skin of
cyclosporine A–treated CD31-deficient mice in Matrigel. The implanted Matrigel
was harvested 14 days after injection of FITC-lectin 20 minutes before the mice
were killed; The presence of CD31⫹ vessel-like structures containing FITC-lectin
can be identified in regions 1 and 2, indicating that effective perfusion had taken
place in the implanted Ang1-EC-ESCs. The bottom 2 panels are the magnified
views of indicated regions. Scale bars represent 50 ␮m.
Discussion
The Ang1/Tie2 axis is well known for its general effect on
adulthood vessels, causing vascular remodeling and maturation.39
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2102
JOO et al
BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Figure 7. Schematic diagram of the effect of Ang1 on
endothelial and hematopoietic differentiation. CD41⫹
cells derived from Flk-1⫹ MPCs differentiate further into
either the endothelial or the hematopoietic lineage. Ang1
potentiates EC differentiation via Ang1/Tie2 signaling,
and its mechanisms are: (1) modulating CD41⫹ cells to
differentiate into the endothelial lineage rather than the
hematopoietic lineage, and (2) promoting EC proliferation
and survival during differentiation.
During the quiescent phase of adult vessels, the Ang1/Tie2 pathway
acts not only to retain vascular integrity but also to induce the
circumferential proliferation of ECs and consequently achieve
vascular enlargement.40 During more active periods of adult
angiogenesis, Ang1/Tie2 signaling is involved in promoting nonleaky and inflammation-free angiogenesis.39,40 In contrast, during
embryonic development, the Ang1/Tie2 axis is thought to induce
EC differentiation and pericyte recruitment, and therefore is
involved in vessel maturation and stabilization.17 Little is known
about the role of the Ang1/Tie2 pathway during early development,
mainly due to the lack of a technically suitable system that can
platform a valid experimental design for ECs of this period. Several
established methods are used to achieve EC differentiation from
Flk-1⫹ MPCs.10,11,28,30,41 Some studies have reported that ECs and
VSMCs are selectively induced by culturing purified Flk-1⫹ MPCs
on type-IV collagen-coated dishes with VEGF-A stimulation.10,11,28
Flk-1⫹ MPCs cultivated on OP9 feeder cells (the OP9 coculture
system) also successfully induced ECs, along with HSCs, VSMCs,
and cardiomyocytes.11,28,30,41 It has been reported that hemogenic
endothelial colonies that are capable of hematopoietic differentiation can be produced using the OP9 coculture system.12 In the
present study, we adopted the OP9 coculture system for the
comprehensive analysis of ESC-EC differentiation.
Our results show that during the early developmental periods of
ESCs, Tie2 is highly expressed in differentiating ECs, whereas
Ang1 is expressed not only in differentiating ECs, but also in
non-ECs—including OP9 cells. We believe that the proper expression and stimulation of the Ang1/Tie2/PI3-kinase/Akt signaling
pathway is essential for EC differentiation because signal interference by treatment with an sTie2 or PI3K inhibitor interfered with
this process. We conclude that Ang1 basically acts in a paracrine
manner and has a pivotal role in EC differentiation during early
development. Conversely, we could not measure any significant
effect of Ang2 on EC differentiation and expansion. The interpretation of Ang2 is more complex, because its effect is context
dependent and can serve either as an antagonist or agonist of Tie2.
However, our preliminary assumption is that Ang2 probably
requires additional components that are not provided in differentiating ESCs.
Previous studies have reported that the activation of the Tie2
pathway has diverse effects on hematopoiesis.18,19,42,43 Considering
the complexity and combined effects of the signal circuit of Ang1,
Ang2 ligands, and their common receptor Tie2,12,14-16,34 we assume
that the diverse effect of Tie2 activation on hematopoietic development would be similar to the temporal and spatial profile of
emergence of embryonal hematopoiesis; the primitive hematopoiesis begins at blood islands in the yolk sac, but is later reformed
into definitive hematopoiesis in several tissues such as the aortagonad-mesonephros.44 Recent accumulating evidence supports the
presence of hemogenic ECs that generate definitive hematopoiesis12,13; however, the identity of these cells remains unclear. In the
present study, we demonstrate that CD41⫹ ECs that are transiently
observed during the process of mesodermal precursor cell
differentiation have the capacity to give rise to both HSCs and
ECs. Therefore, in vitro CD41⫹ ECs are equivalent to in vivo
hemogenic ECs. Our data showed that Tie2 is expressed in these
CD41⫹ ECs, and that stimulation with Ang1 shifted the differentiation toward ECs and away from HSCs. In a similar context, we
hypothesize that Ang1/Tie2 signaling might be involved in the
emergence of definitive hematopoiesis during embryonic development by controlling the spatiotemporal expression pattern of Tie2
in hemogenic ECs.
The notion that ECs can be obtained and expanded by ex vivo
differentiation of stem cells suggested ESCs and/or iPSCs as
promising candidate supplies for vascular regenerative therapies.
In theory, their pluripotency and proliferative capacity identifies
stem cells as ideal resources for therapeutic approaches; however,
in reality, the main obstacle is that it is extremely challenging to
precisely control differentiation into ECs while at the same time
producing sufficient quantities. In this study, we show that Ang1
has a profound role in ESC-EC differentiation mainly through
Tie2 signaling (Figure 7) and that it can maximize the efficiency
of EC production during this process. Moreover, given that Ang1
also promotes EC expansion in the OP9 cell-free system, it could
be a potential supplemental factor to maximize the efficiency of
EC production in the cell-free system. Recently, iPSCs have
become a conceptual but promising source of pluripotent cells.23,45,46
Therefore, it would be useful to establish a method to generate
specific target cells from iPSCs. We have demonstrated herein that
the effect of Ang1 supplementation was recapitulated in iPSCs and
even in human ESCs. In addition, in vivo transplantation of the
ECs derived from ESCs under Ang1 stimulation successfully
established vessel structures, indicating that their functionality
was retained.
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BLOOD, 25 AUGUST 2011 䡠 VOLUME 118, NUMBER 8
Ang1 PROMOTES ENDOTHELIAL DIFFERENTIATION
Because, in the present study, Ang1 supplementation increased
the production of ECs during ESC differentiation, we questioned
how this effect was achieved. Our data show that Ang1 induced
enhanced EC proliferation and extended survival. In fact, Ang1 has
been reported as a crucial factor for EC proliferation and survival in
fully differentiated ECs in vitro and in vivo, with the proliferation
and survival said to be mainly regulated through the ERK1/2 and
PI3K-Akt signaling pathways, respectively.24,40,47 Our current
results showed that Tie2-PI3K-Akt activation could be the major
downstream signaling pathway responsible for Ang1-induced
CD31⫹/CD144⫹ EC expansion. The spatiotemporal expression
profile in our study revealed that the effect of Ang1 supplementation was strongest on proliferation mainly during the earlier
periods, whereas the anti-apoptosis effect was continuously observed throughout the entire experimental period. This suggests
that continuous supplementation of Ang1 is necessary to maximize
the size of the EC population during ESC-EC differentiation.
Another explanation of the surplus EC expansion is that CD41⫹
cells were induced by Ang1/Tie2 signaling to preferentially differentiate into ECs instead of HSCs. On E4.5, the Flk-1⫹ MPCs,
which are considered transient precursors of both ECs and HSCs,
expressed neither endothelial (CD31 and CD34) nor hematopoietic
(CD41 and CD45) markers. Under normal conditions, Tie2⫹ cells
rarely appeared before E4.5 of mouse ESC differentiation. However, we found that purified Flk-1⫹ MPCs began to express Tie2 as
early as 0.5 days after reseeding ESCs in the OP9 coculture system.
On day 1.5 of OP9 coculture, Tie2 was expressed on ⬃ 20%-25%
of all Flk-1⫹ MPC–derived cells. Interestingly, ⬎ 40% of those
Tie2⫹ cells coexpressed CD41, a surface marker that is temporarily
observed in hemogenic ECs undergoing differentiation.12,13,16 Indeed, the fate of these CD41⫹ cells after differentiation turned out
to be bidirectional, producing both ECs and HSCs. Our study
demonstrated that Ang1 supplementation increased the production
of ECs from CD41⫹ cells, suggesting that Ang1 tilted the balance
of the bidirectional Tie2-expressing CD41⫹ cells, guiding these
2103
indecisive cells toward the endothelial lineage. The downstream
signaling pathways responsible for Ang1/Tie2-mediated fate determination in CD41⫹ cells remain to be further elucidated in
subsequent studies.
In conclusion, Ang1 promotes endothelial and suppresses
hematopoietic differentiation of CD41⫹ cells during ESC-EC
differentiation. Ang1 also has strong proliferative and antiapoptotic
effects on ECs during early development. We believe that these
novel effects of Ang1 can be applied to establishing EC protocols
that will solidify stem cell–based therapies to introduce new
directions in vascular regeneration medicine.
Acknowledgments
The authors thank Jin Sun Hong, Jongho Jin, and Eun Soon Lee for
their technical assistance.
This research was supported by grants from the Stem Cell
Research Center of the 21st Century Frontier Research Program
(SC-5120 to G.Y.K.) and from the Korea Healthcare Technology
R&D Project, Ministry for Health, Welfare & Family Affairs,
Republic of Korea (A091345 to H.J.J.).
Authorship
Contribution: H.J.J., H.K., S.-W.P., H.-J.C., and G.Y.K. designed
and performed the experiments, analyzed the data, generated the
figures, and wrote the manuscript; and H.-S.K., D.-S.L., H.-M.C,
I.K., and Y.-M.H. designed the experiments and analyzed the data.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Gou Young Koh, Graduate School of Medical
Science and Engineering, KAIST, 373-1, Guseong-dong, Daejeon,
305-701, Republic of Korea; e-mail:[email protected].
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2011 118: 2094-2104
doi:10.1182/blood-2010-12-323907 originally published
online June 16, 2011
Angiopoietin-1 promotes endothelial differentiation from embryonic
stem cells and induced pluripotent stem cells
Hyung Joon Joo, Honsoul Kim, Sang-Wook Park, Hyun-Jai Cho, Hyo-Soo Kim, Do-Sun Lim,
Hyung-Min Chung, Injune Kim, Yong-Mahn Han and Gou Young Koh
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