Endothelial Cells from Embryonic Stem Cells
in a Chemically Defined Medium
Alicia A. Blancas,1Albert J. Shih,2Nicholas E. Lauer,3and Kara E. McCloskey1–3
Endothelial cells (ECs) are desired for their therapeutic potential in a variety of areas including gene therapy,
cardiac regeneration, development of tissue-engineered vascular grafts, and prevascularized tissue transplants.
Pluripotent embryonic stem cells (ESCs) can be induced to differentiate into ECs in vitro using embryoid bodies,
monolayer cultures, or by genetic manipulation and immortalization. However, obtaining homogeneous cul-
tures of proliferating ESC-derived ECs without genetic manipulation is a challenging undertaking and often
requires optimization of protocols and rigorous purification techniques. Moreover, current differentiation
methods that use medium containing fetal calf or bovine serum components introduce additional challenges
because of our limited ability to control the differentiation signals and batch-to-batch variations of serum. We
have explored the development of new medium formulations for deriving ECs from murine embryonic stem
cells (mESCs) using only chemically defined reagents. We present 2 different medium formulations along with
the detailed methodologies, including the optimization of extracellular matrix–derived substrates known to play
a role in cell attachment and proliferation as well as cell differentiation. Characterization of the ESC-derived ECs
indicate that (1) chemically defined medium formulations reproducibly generate superior ECs compared with
previous serum-containing formulations, (2) fibronectin, and not collagen type-IV, is the optimal substrate for
EC induction in our chemically defined medium formulations, (3) without additional activation of Notch-
signaling, ESC-ECs develop predominantly into venous ECs, and (4) using these medium formulations, a second
rigorous selection step is not required to generate proliferating ECs from ESCs, but it does enhance the final
purity of the ECs.
functions including vascular, cardiovascular, as well as the
immune system. ECs regulate blood pressure through con-
trolling vasodilation and vasoconstriction via synthesis of
nitric oxide. ECs also regulate the permeability of the endo-
thelium for recruiting and permitting transmigration of leu-
also help inhibit platelet adhesion and clotting and are key
players in initiating new blood vessel growth and assembly.
Vascular ECs or endothelial progenitor cells derived from
stem cells could potentially lead to a variety of clinically
relevant therapeutic applications . Endothelial progenitor
cell transplantation has been shown to induce new vessel
formation in ischemic myocardium and hind limb [2–4],
supporting enthusiasm that these cells could be used in
strategies for the repair and revascularization of ischemic
tissue in patients exhibiting vascular defects [4,5]. Ad-
ditionally, because ECs inhibit platelet adhesion and clotting,
ndothelial cells (ECs) are highly dynamic cells that
participate in the regulation of a variety of tissue system
lining the lumen of a synthetic or tissue-engineered vascular
graft may aid in patency of vascular grafts [6,7] or in the
development of prevascularized tissue-engineered materials.
Moreover, because ECs line the lumen of blood vessels and
can directly release proteins into the blood stream, they are
ideal candidates to be used as vehicles of gene therapy.
EC differentiation from embryonic stem cells
Human and murine embryonic stem cells (ESCs), isolated
from the inner cell mass of a developing blastocyst, are
pluripotent cells that are also capable of self-renewal as well
as to differentiate into cells from all 3 germ layers . ESCs
are an especially attractive cell culture system because they
can be easily maintained and expanded in culture. Although
it is possible to obtain stem cells from adult sources, such as
bone marrow and adipose tissue, adult cells exhibit limited
pluripotency compared with ESCs or induced-pluripotent
stem cells. Additionally, adult stem cells can be difficult to
identify, isolate, and expand in culture. For these reasons,
1Graduate Program in Quantitative and Systems Biology,2School of Engineering, and3Graduate Program in Biological Engineering and
Small-Scale Technologies, University of California, Merced, California.
STEM CELLS AND DEVELOPMENT
Volume 20, Number 12, 2011
? Mary Ann Liebert, Inc.
ESCs are an ideal cell culture system for studying stem cell
fate and vascular development.
Successful methods for the in vitro differentiation of ECs
from ESCs [9–16] and adult stem cells [17–19] have been pre-
of several cell types from ESCs, including ECs, involves the
formation of a 3-dimensional aggregate called an embryoid
body [9,14]. This structure allows the differentiation of ESCs
it is difficult to control the cells’ microenvironment within the
embryoid body. Conversely, a 2-dimensional monolayer in-
better control over the cells’ microenvironment [12,13,15]. En-
dothelial promoting growth factors, such as vascular endo-
thelial growth factor (VEGF), can be also added to the
differentiation medium to increase cell differentiation and
proliferation of a specific cell phenotype.
Our laboratory and others have published methods for the
differentiation of ECs from ESCs [9–13,15]. These methods in-
corporate treatment with VEGF to promote EC specification;
however, they also rely on fetal bovine serum to further pro-
mote differentiation and proliferation of the EC populations.
Unfortunately, the presence of serum in the induction medium
formulations often leads to problems with reproducibility due
to uncontrolled variations from batch-to-batch of serum and
also limits one’s ability to directly control stem cell fate.
For these reasons, serum-free replacements have been
explored and successfully used in the maintenance of ESCs
and stem cell differentiation [20–24], but this has not yet been
accomplished for EC induction from mouse ESCs. Although
most formulations of serum replacements are proprietary,
they are generally free from animal components and do not
demonstrate the batch-to-batch variation seen in serum.
Using a chemically defined medium, one can more accu-
rately control the cell’s microenvironment and, therefore,
better evaluate the response of a particular biochemical or
physical signal. In addition, the final yield and quality of
functionally mature tissue-specific cells derived in vitro from
stem cells may be improved by using chemically defined
medium formulations that allow directed differentiation,
rather than serum-containing formulations.
We set out to explore methodologies for directed differ-
entiation of ESCs toward ECs using only chemically defined
medium formulations. Here, we present our novel medium
formulations for the induction and culture of these cells at
well-defined stages of maturation. Moreover, we explored
optimal time points and matrix substrates required for the
initial generation of high numbers of Flk-1+vascular pro-
genitor cells and Flk-1+outgrowths. The methods presented
in this article allow a more consistent and robust generation
of ECs with appropriate expression of endothelial markers as
well as an improved low-density lipoprotein (LDL) uptake
compared with previous derivation methods that included
the use of serum .
Materials and Methods
R1 and D3 mESCs (ATTC) and E14 mESCs (gift from
Bruce Conklin) were cultured on 0.5% gelatin-coated cell
culture dishes in serum-free medium . This medium
contains knockout Dulbecco’s modified Eagle’s medium
(DMEM; Invitrogen), 15% knockout serum replacer (In-
vitrogen), 1·penicillin–streptomycin (Invitrogen), 1·non-
2,000U/mL of leukemia inhibitory factor (LIF-ESGRO;
Chemicon), and 10ng/mL of bone morphogenetic protein-4
(BMP-4; R&D Systems).
Induction of Flk-1+cells in a chemically
Undifferentiated R1 and E14 mESCs were harvested from
gelatin-coated dishes using 0.25% trypsin/2.21mM EDTA
(Mediatech) and plated on cell culture plates coated with
various commercially available substrates including 0.5%
gelatin, 50mg/mL fibronectin, 50mg/mL collagen type-I,
50mg/mL collagen type-IV, and 50mg/mL laminin as per
manufacturer’s instructions (BD Biosciences). The initial in-
duction medium optimized by our laboratory was named
‘‘NS1D2b.’’ (Table 1). This consists of alpha-MEM (Cellgro),
20% knockout serum replacer (Invitrogen), 1·penicillin–
(Invitrogen), 2mM l-glutamine (Invitrogen), 0.05mM 2-
mercaptoethanol (Calbiochem), 30ng/mL of VEGF (R&D
Systems), and 5ng/mL BMP-4 (R&D Systems). Because the
optimal induction lengths for generating Flk-1+vascular
progenitor cells does vary between cell lines , undifferen-
tiated ESCs were also cultured on plates in NS1D2b medium
for 2, 3, 4, and 5 days to determine the optimal number of
days for initial induction of Flk-1+vascular progenitor cells.
In addition to verifying induction time, we also optimized
the substrate for induction of Flk-1+cells by using culture
plates coated with either gelatin, fibronectin, collagen type-I,
collagen type-IV, or laminin. The adherent cells were disso-
ciated, counted, stained with AlexaFluor 647-conjugated
anti-Flk-1 antibodies (Biolegend), and analyzed for Flk-1
expression using a BD LSRII flow cytometer and FlowJo
Purification and expansion of ESC-derived ECs
in chemically defined conditions
After the initial induction period, the cell population was
enriched for Flk-1+vascular progenitor cells using either
fluorescent-activated cell sorting (FACS) or a MiniMACS
(Miltenyi Biotec). AlexaFluor 647–conjugated anti-mouse
Flk-1 antibodies (Biolegend) and anti-AlexaFluor 647 mag-
netic beads (Miltenyi Biotec) were used to label the Flk-1+
expressing cells. Postenrichment, the Flk-1+cells were
replated on dishes coated with either fibronectin, laminin,
collagen type-I, collage type-IV, or gelatin. These cells were
expanded in another chemically defined medium developed
in our laboratory that we have named ‘‘LDSk.’’ (Table 1). The
medium consists of 70% alpha-MEM (Mediatech) and 30%
DMEM (Invitrogen) plus 100ng/mL VEGF (R&D Systems),
2·Nutridoma CS (Roche), 50ng/mL basic fibroblast growth
factor (Sigma), 2mM l-glutamine (Invitrogen), 1·penicillin–
(Invitrogen), and 0.1mM 2-mercaptoethanol (Calbiochem).
The cells were expanded for 2 weeks until clear cobblestone
morphology became visible.
2154BLANCAS ET AL.
Second purification of ESC-derived ECs
We have previously published that a second purification is
necessary for proliferation of the D3 ESC-derived ECs pro-
[10,25,26]. Although we have rigorously examined a variety
of purification techniques including magnetic activated cell
sorting and FACS, the manual picking of cobblestone colo-
nies consistently results in the most pure EC cultures (>95%
purity ). Moreover, we found that obtaining relatively
homogeneous EC cultures is a critical factor allowing the
expansion of the maturing ECs . Here, we also compare
ECs generated with and without this second purification step
under serum-free conditions.
ECs with cobblestone morphology were manually picked
with flame-pulled microtip Pasteur pipettes in a sterile
laminar flow hood outfitted with a stereoscope (Zeiss). The
9’’ Pasteur pipettes (VWR) were flame-pulled to a thin point
and attached to a mouth aspirator line (Sigma-Aldrich) with
a 0.22-mm filter (Whatman) for performing the sterile manual
selection. The culture plates were washed with phosphate-
buffered saline followed by 10min in cell dissociation buffer
(Invitrogen) to allow gentle cell scraping and aspiration of
the picked cells with the microtip pipette. The manually
picked cells were then plated onto fibronectin-coated dishes
in LDSk medium.
Flow cytometry analysis
Vascular cells, both with and without the second manual
selection, were stained for the following endothelial markers:
Flk-1 (Biolegend), vascular endothelial-cadherin (VE-cad;
eBioscience), Flt-1 (Santa Cruz), EphB4 (Santa Cruz), ephrin-
B2 (Santa Cruz), and Tie-1 (Santa Cruz). Some cells were also
stained for calponin (Santa Cruz), a marker indicating
smooth muscle cells (SMCs). The secondary antibodies in-
clude anti-rat PE (Abcam), donkey anti-rabbit PE (Fitzger-
ald), and donkey anti-goat FITC (Abcam). All samples were
analyzed using a BD LSRII flow cytometer and FlowJo
ESC-derived ECs derived in serum-containing medium
and those derived under chemically defined conditions were
plated on Permanox microscope slides (Nunc). Commer-
cially available Alexa Fluor 488 Acetylated-LDL (Invitrogen)
was diluted to 1:100 in DMEM (Invitrogen) and incubated
with the cells for 4h at 37?C. The slides were then stained
with DAPI and fixed with 4% formaldehyde. The slides were
imaged with a Leica fluorecent scope.
R1-ESCs were induced toward vascular progenitor cells in
chemically defined NS1D2b medium on various substrates
over 5 days and then analyzed for the expression of the
vascular progenitor cell marker, Flk-1, using flow cytometry
analysis (Fig. 1). Unfortunately, the ESCs at day 1 exhibited
very low levels of cell adhesion/proliferation and we were
not able to consistently obtain enough cells for flow cytom-
etry analysis. The percentage of Flk-1+cells from R1-ESCs
(ESCs). Histograms of the Flk-1+ expression of R1 mouse ESC on day 0 (prior to induction) and those induced in chemically
defined medium on days 2, 3, 4, and 5. The optimal expression of Flk-1+ cells occurs at day 2 for this cell line and
The largest numbers of Flk-1+ vascular progenitor cells are seen at day 2 of induction from embryonic stem cells
Table 1. Chemically Defined Medium Formulations for the Derivation of Endothelial Cell
from Murine Embryonic Stem Cell
l· Nonessential amino acids
20% Knockout serum replacement
70% Alpha-MEM, 30% DMEM
l· Nonessential amino acids
0.l mM 2-Mercaptoethanol
The first induction medium optimized for the generation of Flk-1+ vascular progenitor cells is called ‘‘NS1D2b.’’ The second medium
formulation called ‘‘LDSk’’ is optimized for EC specification and expansion.
bFGF, basic fibroblast growth factor; BMP-4, bone morphogenetic protein-4; DMEM, Dulbecco’s modified Eagle’s medium; EC, endothelial
cells; VEGF, vascular endothelial growth factor.
EMBRYONIC STEM CELL–DERIVED ENDOTHELIAL CELLS2155
cultured for 0, 2, 3, 4, and 5 days on fibronectin under the
chemically defined conditions peaked on day 2 (over 70%),
continually decreasing over the next 5 days. We also exam-
ined the effect of cell-matrix signaling during the 2-day in-
duction of Flk-1+progenitors. We plated these cells on
collagen type-I, collagen type-IV, laminin, fibronectin, and
gelatin. Figure 2A shows that, contrary to currently accepted
reports that collagen type-IV is the optimal substrate for the
induction of Flk-1+vascular cells [12,13,15], the actual per-
centages of Flk-1+cells induced from R1-ESCs under chem-
ically defined conditions did not vary significantly between
substrates. We subsequently counted the adherent cells on
the various substrates at the end of the 2-day induction pe-
riod to quantify their ability to promote adhesion and pro-
liferation of the differentiating ESCs (Fig. 2B). Combining
Flk-1+cell percentages with total cell numbers, the optimal
substrates yielding the greatest total number of Flk-1 cells are
gelatin and fibronectin (Fig. 2C). We suspect that these re-
sults might not necessarily be due to directed differentiation,
but enhanced proliferation of the adherent cells on gelatin
atin yields the largest numbers of Flk-1+
cells at day 2. (A) The levels of Flk-1 ex-
pression at various initial induction peri-
ods. Lack of adequate cell adhesion
forbids the testing on day 1. Flk-1+ ex-
pression decreases as the induction period
is lengthened. Shown here is the expres-
sion profile for induction on fibronectin-
coated dishes at days 2–5. Error bars
represent SEM. (B) Total number of ad-
herent cells at day 2 of induction for the
various substrates. The dashed line rep-
resents the initial seeding number of
50,000 cells. Note that the fibronectin and
gelatin substrates encourage the largest
proliferation of adherent cells. Error bars
represent SEM. (C) Graph depicts the
number of Flk-1+ adherent cells at day 2
of induction. Note that fibronectin results
in the best combination of cell yield (B)
and Flk-1 expression (C). One-way AN-
OVA and Tukey tests were used to
analyze statistically significant differences
between substrates. The number of Flk-
1+ cells generated on gelatin and fibro-
nectin were both statistically greater than
that on collagen IV (*) or laminin (#).
Culture on fibronectin and gel-
2156 BLANCAS ET AL.
and fibronectin. The results of this study shown in Fig. 2 are
particularly important to note, as many previous studies,
including ours [10,25], use collagen type-IV for differentia-
tion of vascular cells in serum-containing medium. The cul-
ture of ESCs on collagen type-IV in a chemically defined
medium without serum actually yields the lowest number of
Flk-1+cells of all the substrates tested. We hypothesize that
the superior results on fibronectin, and not collagen IV, is
due to the fact that important adhesion-related proteins, such
as fibronectin, found in serum would not be available in our
The Flk-1+cells were then enriched and plated onto either
collagen type-I, collagen type-IV, laminin, gelatin, or fibro-
nectin-coated dishes in LDSk medium. After 2 weeks, the
outgrowths from the Flk-1–expressing cells were analyzed for
markers of venous endothelium (EphB4), arterial endothelium
(ephrin-B2), endothelial VE-cad, and vascular smooth muscle
smooth muscle cell (SMC) contamination. (A) Expression of endothelial VE-cadherin in outgrowths of Flk-1+ vascular
progenitors cultured on collagen type-I, collagen type-IV, laminin, fibronectin, and gelatin. (B) Expression of calponin, a
marker expressed early on SMCs, which were cultured on the same 5 material substrates. (C) The cell yield (total cell numbers
per dish) on the various substrates. (D) Table summarizing the quantitative differences in vascular cell specification between
groups. Note that although collagen type-IV and fibronectin allow induction of equivalent percentages of ECs, collagen type-
IV also allows the differentiation/proliferation of more calponin-expressing SMCs, whereas fibronectin does not lead to
cultures with as many contaminating SMCs. Student’s t-tests were used to analyze statistically significant differences between
substrates. Statistical significance (P<0.05) between cells cultured on the various substrates are indicated by various symbols:
collagen I (*), collagen IV (#), laminin (>), and fibronectin (##).
Flk-1+ outgrowths cultured on fibronectin generate the largest number of endothelial cells (ECs) with minimal
fibronectin for 2 days of induction with bone morphogenetic protein-4 (BMP-4) and vascular endothelial growth factor
(VEGF) treatment. The Flk-1+ cells are then isolated and replated on fibronectin in medium containing VEGF and basic
fibroblast growth factor (bFGF). The Flk-1+ outgrowths generate cobblestone-like cell sheets. These sheets are manually
selected for optimal EC purification.
Flowchart of EC differentiation procedure. Undifferentiated ESCs are expanded on gelatin and then transferred to
EMBRYONIC STEM CELL–DERIVED ENDOTHELIAL CELLS2157
of vascular cells on different substrates (Fig. 3). Laminin gen-
erates the largest number of venous ECs, whereas both colla-
gen IV and fibronectin generate the most number of arterial
ECs (Fig. 3D). However, all of these percentages are still quite
low at this early stage of endothelial maturation. Looking at an
earlier endothelial marker, VE-cad, we see that collagen I, col-
lagen IV, and fibronectin yield the largest percentages of VE-
cad+cells (Fig. 3A, D). We then looked closely at the SMC
outgrowths and observed that the cells cultured on fibronectin
also contained the lowest percentage of contaminating SMCs
(Fig. 3B) and that the greatest cell yield was again observed in
cells cultured on fibronectin (Fig. 3C). Based on these data, we
continued toexpandESC-derived ECs onfibronectintoreduce
the percentage of SMCs in the cultures while promoting opti-
mal adhesion and proliferation of our ECs.
The expanded Flk-1+ cells subcultured in LDSk medium
on fibronectin robustly supported the differentiation and
proliferation of ECs, and some of the Flk-1+ exhibited dis-
of endothelial markers Flk-1, VE-cadherin, Flt-1, Tie-1, EphB4 (venous), and ephrin-B2 (arterial). The histograms include the
ECs derived using R1 ESCs under chemically defined conditions (green). Also included are ECs derived from R1-ESCs (aqua)
and D3-ESCs (red) using our previous medium formulation that contained FBS. The last 2 columns include comparisons of
the EC marker expression for the ECs from R1-ESCs (gold) and E14-ESCs (purple) derived under the new chemically defined
conditions, but these did not include the second EC purification.
ECs derived in chemically defined conditions express appropriate endothelial makers. We examined the expression
Table 2. Quantitative Expression of Various
Endothelial Cell Markers
The percentage of positive cells expressing the listed EC markers
for the ECs derived using our chemically defined medium compared
with old serum formulations (quantitative data from Fig. 5). Also
included are data comparing the percentage of positive cells with
and without a second rigorous manual selection for optimal
purification of the ECs.
VE-cad, vascular endothelial-cadherin.
2158BLANCAS ET AL.
tinct cobblestone-like morphologies (Fig. 4), allowing manual
selection to be used for further enrichment of the ECs. The
expanded ESC-derived ECs were then analyzed for EC
markers Flk-1, VE-cad, Flt-1, Tie-1, venous-specific marker,
EphB4, and the arterial-specific marker, ephrin-B2, using
FACS analysis (Fig. 5 and Table 2). Here, the differentiation
and expansion procedures of the ESC-R1 cells derived in our
chemically defined medium formulations, as described here,
expressed very high levels of most of the EC markers that
were examined, but did not exhibit a high level of ephrin-
B2 arterial surface marker, indicating a largely venous
phenotype (EphB4) for these cells (Fig. 5, green). We also
included characterization of our ESC-ECs derived from
serum-containing medium for 2 different ESC lines. Com-
paring columns II (aqua) and III (red), the serum-containing
induction protocol worked well for the ESC-D3 cell line (Fig.
5, red) as previously described [10,25,27], but the same se-
rum-containing medium was not able to generate the same
level of quality ECs from the ESC-R1 cell line (Fig. 5, aqua).
We expect that this difference is due to the inherent hetero-
geneity between ESC lines.
All 3 of the ESC-ECs presented in the first 3 columns (green,
aqua, and red) followed a rigorous differentiation and
expansion methodology including 2 distinct isolations. These
include an early isolation of vascular progenitor cells plus a
late isolation that purifies the progenitor cells further into
relatively pure (>95%) ESC-ECs . In addition, the second
manual selection (or another method of post-Flk-1+ sorting
enrichment) of late developing ECs was required for EC
proliferation of the ESCs derived using serum-containing
medium ; we expect, because of the high levels of con-
taminating SMCs in serum-containing cultures. However, the
EC induction methodology presented in this article uses
chemically defined mediums that allow the generation of
proliferative ECs with minimal contaminating SMCs. There-
fore, we sought to characterize the outgrowths of the none-
nriched Flk-1+ to explore whether the second late-stage
selection is a necessary manipulation under these chemically
defined conditions (Fig 5, gold). The data indicate that al-
though these cells do exhibit equivalent expression of EC
molecules Flk-1 and VE-cad, the expression levels of Flt-1, Tie-
1, and EphB4 were lower compared with the ESC-ECs that
did undergo the second isolation, indicating that this second
selection process does generate better quality and, potentially,
more mature ECs. Figure 6 examined the coexpression of Flk-
1 with endothelial markers, EphB4 and Ephrin-B2, as well as
chemically defined conditions
take up low-density lipopro-
tein (LDL). The 2 (A and B)
distinct isolations of ECs de-
rived from R1-ESCs both take
up LDL. We also examined the
LDL uptake of the R1s derived
in the same chemically defined
manually selected for addi-
tional purification. (C) Some of
the cells did take up LDL, but
(D) some of the cells in the
same culture did not take up
LDL. These results indicate
that the nonpurified cultures
contained some heterogeneity
(LDL, green; DAPI, blue).
ECs derived in
chemically defined conditions
coexpress Flk-1 and EphB4
(venous). These cultures con-
tain very few ephrin-B2+ ar-
terial cells and do not contain
EMBRYONIC STEM CELL–DERIVED ENDOTHELIAL CELLS2159
the smooth muscle marker, calponin. Interestingly, 45% of the
Flk-1+ cells coexpress the marker for venous endothelium,
EphB4, but only 8% of these cells express Ephrin-B2. This
indicated that the remaining 37%–45% of the Flk-1+ cells
have not yet specified an endothelial subphenotype (previous
studies indicate that none of the cells are lymphatic; data not
shown) and may retain some level of plasticity.
Lastly, the entire derivation procedure was repeated in a
third ESC line (E14) to verify that the chemically defined
medium formulations presented in this article can be applied
toward other mouse ESCs. These ESCs were also able to
generate ECs, but require a second manual selection for
purification of the ECs (Fig. 5, purple).
Functional assays are also an important indicator of EC
quality. The uptake of LDL is considered an important EC
function that was not seen in our ESC-ECs derived from D3-
ESCs in serum-containing medium[10,25]; however,as seen in
Fig. 7A and B, our ESC-ECs derived in chemically defined
conditions were able to take up LDL. We also observed that
some of the ESC-ECs that did not undergo the second isolation
(Fig. 5, gold) could take up LDL (Fig. 7C). However, not all of
the cells in the culture could take up LDL (Fig. 7D), indicating
that these unpurified cells were also remaining somewhat
heterogeneous, even in the chemically defined medium.
Our initial stage of induction focused on promoting the
differentiation of ESCs into Flk-1+ cells similar to studies
using serum-containing medium formulations [10–13,15,16].
Our rationale is supported by the fact that the Flk-1 surface
molecule is currently considered to be the first lineage-
commitment marker expressed on vascular and hematopoi-
etic progenitors [28–41]. BMP-4 and VEGF are also known to
promote ventral mesoderm and hematopoietic development
while inhibiting neuronal development [42–44]; therefore,
these were also incorporated into our serum-free differenti-
ation medium. Conversely, basic fibroblast growth factor,
considered to be a pro-angiogenic factor, was not required in
our early stage of induction (i.e., generation of Flk-1+ cells)
 but incorporated with VEGF at the later, more mature,
stages of development for enhancing EC proliferation.
We also examined the effect of ECM substrate on the EC
inductions. Our results using a chemically defined medium
indicate that early-stage EC inductions focused on generating
Flk-1+ mesodermal cells can take place on any of the sub-
strates examined, including gelatin. However, the substrates’
role in directing the specification of the Flk-1+ cells toward
various cell phenotypes was much more enlightening. It
seems that fibronectin directs the most EC differentiation,
leading to the greatest number of VE-cad+ ECs, whereas
culture on gelatin, laminin, and collagen I result in more
SMCs. If one wanted to study the codevelopment of ECs and
SMCs, collagen type-IV would remain the optimal substrate
that allows the simultaneous proliferation and differentiation
of both cell types. These data from ESC-EC derivations in a
chemically defined medium also challenges the currently ac-
cepted belief that collagen IV substrate generates largest
number of vascular progenitors and ECs [13,15].
Lastly, it has been proposed that the venous lineage is the
default pathway during EC development , presumably
because of insufficient Notch activation . Based on the
high EphB4 and limited ephrin-B2 expression levels of our
cells, we found that our ESC-derived ECs also resembled
venous endothelium compared with ephrin-B2–expressing
arterial endothelium. One study using cells cultured in serum
and VEGF found that >90% of the cells became venous
ECs, but when Notch signaling was activated by stimulating
the cyclic adenosine monophosphate (cAMP) pathway (by
adding either 8bromo-cAMP or adrenomedullin—a cAMP-
elevating factor—to the serum- and VEGF-containing medi-
um), up to 70% of the cells expressed ephrin-B2 arterial
marker . On the basis of this study, one would expect that
specifically activating Notch signaling in our chemically de-
fined cultures may also lead to the generation of arterial ECs.
The generation of ECs from ESCs in chemically defined
conditions is valuable for potential use of these cells in a
variety of therapeutic applications. By eliminating various
unknown animal contaminants and removing unspecified
and uncontrollable elements found in serum, chemically
defined culture conditions allow better controlled studies of
the effects of various biological and mechanical signaling
variables. Although we can see some developmental varia-
tions between different ESC lines, the reproducibility and
quality of ESC-derived ECs were significantly increased us-
ing the chemically defined conditions described.
This work was funded, in part, by a National Institutes of
Health-National Service Award (NIH-NRSA) from the Na-
tional Heart Lung and Blood Institute (NHLBI) (No.
Author Disclosure Statement
No competing financial interests exist.
1. Nishikawa S, LM Jakt and T Era. (2007). Embryonic stem-cell
culture as a tool for developmental cell biology. Nat Rev Mol
Cell Biol 8:502–507.
2. Kocher AA, et al. (2001). Neovascularization of ischemic
myocardium by human bone-marrow-derived angioblasts
prevents cardiomyocyte apoptosis, reduces remodeling and
improves cardiac function. Nat Med 7:430–436.
3. Kawamoto A, et al. (2001). Therapeutic potential of ex vivo
expanded endothelial progenitor cells for myocardial ische-
mia. Circulation 103:634–637.
4. Kalka C, et al. (2000). Transplantation of ex vivo expanded
endothelial progenitor cells for therapeutic neovasculariza-
tion. Proc Natl Acad Sci U S A 97:3422–3427.
5. Soker S, M Machado and A Atala. (2000). Systems for ther-
apeutic angiogenesis in tissue engineering. World J Urol
6. Kaushal S, et al. (2001). Functional small-diameter neo-
vessels created using endothelial progenitor cells expanded
ex vivo. Nat Med 7:1035–1040.
7. Griese DP, et al. (2003). Isolation and transplantation of
autologous circulating endothelial cells into denuded vessels
and prosthetic grafts: implications for cell-based vascular
therapy. Circulation 108:2710–2715.
8. Amit M, et al. (2000). Clonally derived human embryonic
stem cell lines maintain pluripotency and proliferative po-
tential for prolonged periods of culture. Dev Biol 227:271–278.
2160 BLANCAS ET AL.
9. Levenberg S, et al. (2002). Endothelial cells derived from
human embryonic stem cells. Proc Natl Acad Sci U S A
10. McCloskey KE, et al. (2003). Purified and proliferating en-
dothelial cells derived and expanded in vitro from embry-
onic stem cells. Endothelium 10:329–336.
11. McCloskey KE, SL Stice and RM Nerem. (2006). In vitro
derivation and expansion of endothelial cells from embry-
onic stem cells. Methods Mol Biol 330:287–301.
12. Nishikawa SI, et al. (2001). Cell biology of vascular endo-
thelial cells. Ann N Y Acad Sci 947:35–40; discussion 41.
13. Nishikawa SI, et al. (1998). Progressive lineage analysis by
cell sorting and culture identifies FLK1+ VE-cadherin+ cells
at a diverging point of endothelial and hemopoietic lineages.
14. Vittet D, et al. (1996). Embryonic stem cells differentiate in
vitro to endothelial cells through successive maturation
steps. Blood 88:3424–3431.
15. Yamashita J, et al. (2000). Flk1-positive cells derived from
embryonic stem cells serve as vascular progenitors. Nature
16. Yamashita JK. (2007). Differentiation of arterial, venous, and
lymphatic endothelial cells from vascular progenitors.
Trends Cardiovasc Med 17:59–63.
17. Goodell MA, et al. (2001). Stem cell plasticity in muscle and
bone marrow. Ann N Y Acad Sci 938:208–220.
18. Majka SM, et al. (2003). Distinct progenitor populations in
skeletal muscle are bone marrow derived and exhibit dif-
ferent cell fates during vascular regeneration. J Clin Invest
19. Planat-Benard V, et al. (2004). Plasticity of human adipose
lineage cells toward endothelial cells physiological and
therapeutic perspectives. Circulation 190:656–663.
20. Adelman CA, S Chattopadhyay and JJ Bieker. (2002). The
BMP/BMPR/Smad pathway directs expression of the ery-
throid-specific EKLF and GATA1 transcription factors dur-
ing embryoid body differentiation in serum-free media.
21. Cheng J, et al. (2004). Improved generation of C57BL/6J
mouse embryonic stem cells in a defined serum-free media.
22. Furue M, et al. (2005). Leukemia inhibitory factor as an anti-
apoptotic mitogen for pluripotent mouse embryonic stem
cells in a serum-free medium without feeder cells. In Vitro
Cell Dev Biol Anim 41:19–28.
23. Park C, et al. (2004). A hierarchical order of factors in the
generation of FLK1-and SCL-expressing hematopoietic and
endothelial progenitors from embryonic stem cells. Devel-
24. Smith AG. (1991). Culture and differentiation of embryonic
stem cells. Methods Cell Sci 13:89–94.
25. McCloskey KE, et al. (2006). Embryonic stem cell-derived
endothelial cells may lack complete functional maturation in
vitro. J Vasc Res 43:411–421.
26. Blancas AA, NE Lauer and KE McCloskey. (2008). En-
dothelial differentiation of embryonic stem cells. Curr Protoc
Stem Cell Biol 1: Unit 1F 5.
27. McCloskey KE, ME Gilroy and RM Nerem. (2005). Use of
embryonic stem cell-derived endothelial cells as a cell source
to generate vessel structures in vitro. Tissue Eng 11:497–505.
28. Bautch VL. (2006). Flk1 expression: promiscuity revealed.
29. Carmeliet P. (2000). Mechanisms of angiogenesis and arter-
iogenesis. Nat Med 6:389–395.
30. Choi K. (1998). A common precursor for hematopoietic and
endothelial cells. Development 125:725–732.
31. Drake CJ, JE Hungerford, CD Little. (1998). Morphogenesis
of the first blood vessels. Ann N Y Acad Sci 857:155–179.
32. Dumont DJ, et al. (1995). Vascularization of the mouse em-
bryo: a study offlk-1, tek, tie, and vascular endothelial
growth factor expression during development. Am J Anat
33. Ema M, S Takahashi and J Rossant. (2006). Deletion of the
selection cassette, but not cis-acting elements, in targeted
Flk1-lacZ allele reveals Flk1 expression in multipotent me-
sodermal progenitors. Blood 107:111–117.
34. Fehling HJ, et al. (2003). Tracking mesoderm induction and
its specification to the hemangioblast during embryonic
stem cell differentiation. Development 130:4217–4227.
35. Hatzopoulos AK. (1998). Isolation and characterization of
endothelial progenitor cells from mouse embryos. Devel-
36. Heil M, et al. (2006). Arteriogenesis versus angiogenesis:
similarities and differences. J Cell Mol Med 10:45–55.
37. Jain RK, et al. (2005). Molecular regulation of vessel matu-
ration. Nat Biotechnol 23:821–823.
38. Keller GM. (1995). In vitro differentiation of embryonic stem
cells. Curr Opin Cell Biol 7:862–869.
39. Lacaud G, G Keller and V Kouskoff. (2004). Tracking me-
soderm formation and specification to the hemangioblast in
vitro. Trends Cardiovasc Med 14:314–317.
40. Simons M. (2004). Integrative signaling in angiogenesis. Mol
Cell Biochem 264:99–102.
41. Yancopoulos GD, et al. (2000). Vascular-specific growth
factors and blood vessel formation. Nature 407:242–248.
42. Johansson BM and MV Wiles. (1995). Evidence for involve-
ment of activin A and bone morphogenetic protein 4 in
mammalian mesoderm and hematopoietic development.
Mol Cell Biol 15:141–151.
43. Karlsson G, et al. (2007). Smad4 is critical for self-renewal of
hematopoietic stem cells. J Exp Med 204:467.
44. Stavridis MP and AG Smith. (2003). Neural differentiation
of mouse embryonic stem cells. Biochem Soc Trans 31(Pt 1):
45. Thurston G and GD Yancopoulos. (2001). Gridlock in the
blood. Nature 414:163–164.
46. Yurugi-Kobayashi T, et al. (2006). Adrenomedullin/cyclic
AMP pathway induces Notch activation and differentiation
of arterial endothelial cells from vascular progenitors. Ar-
terioscler Thromb Vasc Biol 26:1977–1984.
Address correspondence to:
Dr. Kara E. McCloskey
School of Engineering
University of California, Merced
P.O. Box 2039
Merced, CA 95344
Received for publication September 27, 2010
Accepted after revision March 29, 2011
Prepublished on Liebert Instant Online March 29, 2011
EMBRYONIC STEM CELL–DERIVED ENDOTHELIAL CELLS2161