SCIENCE ADVANCES | RESEARCH ARTICLE

CELL BIOLOGY Copyright 2017

The Authors, some

Directed differentiation of human pluripotent stem cells rights reserved;

exclusive licensee
to blood-brain barrier endothelial cells American Association
for the Advancement
of Science. No claim to
Tongcheng Qian, Shaenah E. Maguire, Scott G. Canfield, Xiaoping Bao, William R. Olson, original U.S. Government
Eric V. Shusta,* Sean P. Palecek* Works. Distributed
under a Creative
The blood-brain barrier (BBB) is composed of specialized endothelial cells that are critical to neurological health. A Commons Attribution
key tool for understanding human BBB development and its role in neurological disease is a reliable and scalable NonCommercial
source of functional brain microvascular endothelial cells (BMECs). Human pluripotent stem cells (hPSCs) can the- License 4.0 (CC BY-NC).
oretically generate unlimited quantities of any cell lineage in vitro, including BMECs, for disease modeling, drug
screening, and cell-based therapies. We demonstrate a facile, chemically defined method to differentiate hPSCs
to BMECs in a developmentally relevant progression via small-molecule activation of key signaling pathways.
hPSCs are first induced to mesoderm commitment by activating canonical Wnt signaling. Next, these mesoderm
precursors progress to endothelial progenitors, and treatment with retinoic acid leads to acquisition of BBB-specific
markers and phenotypes. hPSC-derived BMECs generated via this protocol exhibit endothelial properties, includ-

Downloaded from http://advances.sciencemag.org/ on November 12, 2017

ing tube formation and low-density lipoprotein uptake, as well as efflux transporter activities characteristic of
BMECs. Notably, these cells exhibit high transendothelial electrical resistance above 3000 ohmcm2. These hPSC-
derived BMECs serve as a robust human in vitro BBB model that can be used to study brain disease and inform
therapeutic development.

INTRODUCTION
An intact blood-brain barrier (BBB) serves as a key interface between
the blood circulation and the central nervous system (CNS). The pri-
mary anatomical component of the BBB is provided by brain micro-
vascular endothelial cells (BMECs) (1, 2) that work in concert with
supporting cells, such as astrocytes, pericytes, and neurons, to form
the neurovascular unit (1, 3, 4). BMECs are connected by tight junc-
tions and display low levels of vesicular traffic, leading to extremely
low vascular permeability. BMECs also express molecular influx and
efflux transporters, which regulate the delivery of nutrients from the
blood to the brain and removal of compounds from the brain, respec-
tively. A functional BBB prevents most of the small-molecule drugs
and nearly all large-molecule biologics from entering the brain (5).
Thus, the BBB is a highly efficient barrier that protects the brain
and limits CNS drug delivery (6). Moreover, BBB dysfunction has
been associated with many CNS disorders, including stroke (79),
Alzheimers disease (10, 11), multiple sclerosis (12), Parkinsons dis-
ease (13), traumatic brain injury (14, 15), and HIV (1618).
Although the BBB has been extensively studied in animal models
(1921), in vitro models based on primary human BMECs (22, 23),
and immortalized human brain EC lines (2, 24, 25), these models lack
key attributes of the human BBB. Animal models cannot fully repre-
sent the human BBB because of species differences, particularly in
transporter expression and function (26). Human primary BMECs
are difficult to obtain in sufficient quantities for drug screening and
disease models and cannot be readily expanded in high-fidelity cultures.
Immortalized cell lines exhibit a loss of BMEC-specific properties, in-
cluding loss of tight junctions yielding subphysiologic transendothelial
electrical resistance (TEER) (27). These limitations have prevented
our full understanding of human BBB development, function, and dis-
ease (28).
Department of Chemical and Biological Engineering, University of Wisconsin-
Madison, Madison, WI 53706, USA.
*Corresponding author. Email: sppalecek@wisc.edu (S.P.P.); eshusta@wisc.edu
(E.V.S.)
Human pluripotent stem cells (hPSCs) have the potential to generate
large quantities of specialized human cells for studying development
and modeling disease (2931). Previously, we reported the generation
of pure populations of hPSC-derived BMECs via co-differentiation of
hPSCs to neural and endothelial progenitors, followed by selective pu-
rification of the BMECs (32). In addition, we demonstrated that retinoic
acid (RA) addition during BMEC differentiation enhanced barrier
properties to physiologic levels (33). Presumably, the neural progenitors
in this co-differentiation platform induce the endothelial progenitors to
acquire BMEC-specific traits, which are then enhanced by RA treat-
ment. However, the undefined nature of this co-differentiation platform
complicates the investigation of mechanisms that specify BMEC fates in
the hPSC-derived ECs. In addition, this undefined protocol can result in
line-to-line and batch-to-batch variability in BMEC yield and pheno-
types (32, 3436), despite recent efforts that have explored the use of
defined medium to accelerate portions of the differentiation process
(36). Other studies have also shown that human BMEC-like cells can
be generated from alternative stem and progenitor cell sources, includ-
ing hematopoietic stem cells (37), endothelial progenitors (38), and
hPSC-derived ECs cocultured with hPSC-derived pericytes, astrocytes,
and neurons (39) or C6 glioma cells (40). Nevertheless, none of these
previous studies report a chemically defined, robust process for gener-
ating human BMECs exhibiting physiologic BBB phenotypes.
During embryonic development, mesoderm-derived ECs form a
vascular plexus covering the developing neural tube (41, 42). As
nascent blood vessels enter the developing CNS, canonical Wnt
signaling is necessary to induce BMEC barrier properties (4345).
RA has also been shown to regulate BMEC specification. During
BBB development, radial glial cells supply the CNS with RA (46),
and this RA signaling induces barrier formation and BBB-specific
gene expression (33, 46, 47). In addition to Wnt regulation of BBB in-
duction in vivo, previous studies have demonstrated that activation of
canonical Wnt signaling can also direct hPSCs to mesodermal lineages
in vitro (31, 4850). Thus, we hypothesized that appropriate differenti-
ation stagespecific modulation of canonical Wnt signaling would in-
duce mesodermal and endothelial commitment in hPSCs, and combine

Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017 1 of 12

SCIENCE ADVANCES | RESEARCH ARTICLE

with subsequent RA signaling to drive the acquisition of BMEC markers RESULTS

and phenotypes.
Here, we report a chemically defined method to differentiate hPSCs
to BMECs via sequential Wnt and RA pathway activation. During this
differentiation process, hPSCs progress through stages resembling
primitive streak and intermediate mesoderm to vascular endothelial
growth factor receptor 2positive (VEGFR2+) endothelial progenitors
that become virtually pure populations of CD31+ ECs displaying key
BMEC phenotypes including tight junctions, low passive permeability,
and polarized efflux transporters. The resultant, developmentally rele-
vant BMEC differentiation strategy is defined, robust, and facile.
hPSCs progress from intermediate mesoderm to BMECs
Given the roles of canonical Wnt signaling in both mesoderm specifi-
cation and BBB development, we first treated hPSCs with CHIR99021,
a glycogen synthase kinase 3b (GSK-3b) inhibitor and canonical Wnt
pathway agonist, to direct hPSCs to mesoderm-derived endothelial
progenitors. Before treatment, IMR90-4 induced pluripotent stem cells
(iPSCs) were seeded on a Matrigel-coated six-well plate at a density of
35 103 cells/cm2 and expanded in an undifferentiated state for 3 days
in mTeSR1 (Fig. 1A). Previously, we showed that 6 mM CHIR99021
treatment induced hPSC differentiation to a primitive streaklike stage

A
35 103 cells/cm2 6 M
CHIR Replate A
B
D0 D1 D6 D8 D10

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3D mTeSR1 1D DeSR1 5D DeSR2 2D hECSR1 hECSR2

Primitive Intermediate EC
hPSCs mesoderm progenitors BMECs
streak
D0 D1 D4 D5 D10
OCT4 Brachyury PAX2 VEGFR2 CD31
NANOG Claudin-5
Pgp
OCT4/DAPI Brachyury/DAPI
104
B E F 99.6 0.3%
103
Brachyury

102

101 IgG
100
0 200 400 600 800 1.0k

NANOG/DAPI PAX2/DAPI FSC

104
C G H 94.2 1.1%
103
PAX2

102

101 IgG
0
10
0 200 400 600 800 1.0k

TRA-1-60/DAPI VEGFR2/DAPI FSC

J M
4
10
D I 98.9 0.3%
103
VEGFR2

102

101 IgG
100
0 200 400 600 800 1.0k

FSC
Fig. 1. Schematic of BMEC differentiation protocol. (A) Singularized hPSCs are seeded on six-well plates coated with Matrigel, vitronectin, or Synthemax and
expanded for 3 days in mTeSR1. Differentiation to primitive streak is initiated by 24-hour treatment with 6 mM CHIR99021 in DeSR1. Cells progress to intermediate
mesoderm and endothelial progenitors during culture in serum-free defined DeSR2 medium. At day 6, BMEC specification is induced by culture in hESFM supplemented
with 2% B27, 10 mM RA, and bFGF (20 ng/ml) (hECSR1) for 2 days. After replating on Matrigel or fibronectin/collagen IV substrates, BMECs are obtained. (B to D) The
pluripotent state of expanded hPSCs was verified before differentiation by immunofluorescence for OCT4 (B), NANOG (C), and TRA-1-60 (D). DAPI, 4,6-diamidino-2-
phenylindole. (E and F) The expression of the primitive streak marker brachyury was assessed by immunofluorescence (E) and flow cytometry (F) 24 hours after
CHIR99021 treatment. IgG, immunoglobulin G; FSC, forward scatter. (G and H) On day 4 of differentiation, the expression of the intermediate mesoderm marker
PAX2 was quantified. (I and J) On day 5, the expression of the endothelial progenitor marker VEGFR2 was analyzed. Scale bars, 100 mm.

Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017 2 of 12

SCIENCE ADVANCES | RESEARCH ARTICLE
in a serum- and albumin-free medium (51). Hence, at day 0, 6 mM
CHIR99021 was added to DeSR1 [unconditioned medium (UM)
lacking KnockOut Serum Replacement: DMEM/F12, 1% minimum es-
sential medium (MEM)nonessential amino acids (NEAA), 0.5%
GlutaMAX, and 0.1 mM b-mercaptoethanol (32)] to initiate differenti-
ation. After 24 hours, the medium was removed and cells were transi-
tioned to DeSR2 (DeSR1 plus B27 supplement) for another 5 days with
daily medium changes. At day 0, pluripotency was verified by OCT4,
NANOG, and TRA-1-60 immunofluorescence (Fig. 1, B to D). After
24 hours of CHIR99021 treatment, almost 100% of the cells expressed
the primitive streak marker brachyury, as assessed by immunolabeling
(Fig. 1E) and flow cytometry (Fig. 1F). In concert with brachyury ex-
pression, primitive streak genes T and MIXL1 (52) peaked at day 2 and
then markedly decreased (fig. S1). At day 4, more than 90% of the cells
expressed the intermediate mesoderm marker PAX2 (Fig. 1, G and H),
and PAX2 expression peaked at day 6 (fig. S1). Nearly 100% of the cells
expressed the endothelial progenitor marker VEGFR2 at day 5 (Fig. 1,
I and J), whereas the expression level of the endothelial progenitor
marker CD34 gradually increased and then diminished after day 6
(fig. S1).
At day 6, cells were switched to hECSR1 medium [human endo-
thelial serum-free medium (hESFM) supplemented with basic fibro-
blast growth factor (bFGF, 20 ng/ml), 10 mM RA, and B27] to induce
RA signaling in the hPSC-derived endothelial progenitors in an at-
tempt to drive the specification to BMECs. Cells were maintained in
this medium for 2 days. At day 8, cells were replated onto a Matrigel-
coated substrate in hECSR1, and at day 9, the medium was switched
to hECSR2 (hECSR1 lacking RA and bFGF). The expression of CDH5
[vascular endothelial cadherin (VE-cadherin)] was substantially induced
after RA treatment (fig. S1). The expression of the tight junctionrelated
genes TJP1, CLDN5, and OCLN and the efflux transporter ABCB1 also
increased during differentiation (fig. S1). The resultant day 10 BMEC-
like cells were a pure population expressing endothelial markers (CD31
and VE-cadherin), BBB glucose transporter (GLUT-1), tight junction
proteins (ZO-1, claudin-5, and occludin), and efflux transporters (BCRP,
MRP1, and Pgp) (Fig. 2, A to I). Thus, sequential treatment of hPSCs
with CHIR99021 and RA directed hPSCs through endothelial pro-
genitors to ECs that expressed BMEC markers. We next tested whether
the differentiation protocol illustrated in Fig. 1A generated cells ex-
pressing BMEC markers in additional hPSC lines, including H9 hu-
man embryonic stem cells (hESCs) and 19-9-11 iPSCs. These lines
also produced cells expressing endothelial and BMEC markers, in-
cluding CD31, GLUT-1, ZO-1, claudin-5, occludin, MRP1, BCRP1,
and Pgp, at day 10 (fig. S2).
Next, RNA sequencing was used to compare global gene expres-
sion profiles in the hPSC-derived BMECs differentiated, as shown
in Fig. 1A, with BMECs generated from our previously reported co-
differentiation system [UM (32)] and primary human BMECs. As
expected, hPSC-derived BMECs from three independent differentia-
tions clustered closely and were similar to those generated from the
undefined UM platform. Moreover, the hPSC-derived BMECs clustered
with primary human BMECs and were distinct from undifferentiated
hPSCs and hPSC-derived ectoderm, endoderm, and mesoderm (Fig. 2J).
The Pearson correlation coefficient between defined BMECs and
primary human BMECs was 0.77 (P < 0.001), which indicates a strong
positive association between these two groups. We next analyzed the
expression of a subset of genes that regulate key BBB attributes, includ-
ing tight junctions and molecular transporters. The gene set comprises
20 tight junctionrelated genes (1, 5356) and an unbiased list of all
Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017
25 CLDN genes, all 407 solute carrier (SLC) transporters, and all 53
adenosine 5-triphosphate (ATP)binding cassette (ABC) transport-
ers regardless of previous knowledge of BBB association (table S1).
Primary human BMECs expressed 234 of these genes. BMECs differ-
entiated from hPSCs via the defined method expressed many of these
same genes [206 of 234 (88%)], as did BMECs differentiated via the
UM method [208 of 234 (89%); Fig. 2K], indicating a close similarity
between hPSC-derived BMECs and primary human BMECs with re-
spect to transcripts having likely relevance to BBB function.
Initially, differentiation was performed on Matrigel, which has been
shown to support BMEC generation from hPSCs (32). However, to re-
move the lot-to-lot variability inherent to Matrigel and to fully define
the differentiation platform, we explored differentiation on Synthemax
and recombinant human vitronectin coatings. Undifferentiated
IMR90-4 iPSCs were expanded on either Synthemax- or vitronectin-
coated surfaces for 3 days and then subjected to the differentiation
process shown in Fig. 1A. Cells were replated onto a human placenta
Downloaded from http://advances.sciencemag.org/ on November 12, 2017
derived collagen IV/human plasmaderived fibronectin-coated sur-
face at day 8. Immunofluorescence at day 10 demonstrated the
expression of key BMEC proteins in cells differentiated on defined
matrices (fig. S3, A and B).
hPSC-derived BMECs exhibit BBB phenotypes
In addition to the examination of BMEC gene and protein expression,
we also evaluated endothelial and BMEC phenotypes. After 8 days of
differentiation, cells were replated onto a Matrigel-coated surface at
1 million cells/cm2 and maintained in hECSR1 medium. At day 9,
culture medium was switched to hECSR2. Day 10 hPSC-derived
BMECs exhibited EC properties, including the expression of von
Willebrand factor (vWF) (Fig. 3A), formation of tube-like structures
on Matrigel in the presence of VEGF (Fig. 3B), uptake of acetylated
low-density lipoprotein (LDL) (Fig. 3C), and up-regulation of inter-
cellular adhesion molecule 1 (ICAM-1) expression after treatment
with tumor necrosis factora (TNF-a) (Fig. 3, D to F). BMEC efflux
transporter activities were also measured at day 10. Efflux transport-
er accumulation assays were performed by quantifying intracellular
accumulation of fluorescent substrates, including the Pgp substrate
rhodamine 123, the MRP family substrate 2,7-dichlorofluorescein
diacetate (DCFDA), and the BCRP family substrate Hoechst. In the
presence of the transporter-specific inhibitors cyclosporine A (CsA;
Pgp), MK571 (MRP), and Ko143 (BCRP), the intracellular accumu-
lation of fluorescent substrates increased between 150 and 220%, in-
dicating the activity of each class of transporters in hPSC-derived
BMECs (Fig. 3, G to I). In addition, BMECs differentiated on defined
vitronectin or Synthemax substrates also exhibited Pgp, MRP, and
BCRP transporter activities similar to those differentiated on Matrigel
(fig. S3C). Next, polarization of Pgp activity was demonstrated by
measuring rhodamine 123 flux across the BMEC monolayer in the pres-
ence and absence of the Pgp-specific inhibitor CsA and in both the ap-
ical to basolateral (A-B) and basolateral to apical (B-A) directions. As
shown in Fig. 3J, CsA treatment increased rhodamine 123 transport
across the BMEC monolayer by 160% in the A-B direction. In con-
trast, CsA inhibition resulted in a 23% decrease in rhodamine 123
crossing the barrier in the B-A direction (indicated in Fig. 1A), indi-
cating Pgp efflux function polarized in the B-A direction. Finally,
BMECs differentiated via the defined protocol exhibited Pgp accu-
mulation and transport similar to those differentiated via our previ-
ously reported undefined co-differentiation protocol (fig. S4, UM
protocol).
3 of 12
SCIENCE ADVANCES | RESEARCH ARTICLE

A CD31 VE-cadherin GLUT-1

ZO-1 Claudin-5 Occludin

BCRP MRP1 Pgp

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4
B 10 99.2 0.8% C 10 4
99.9 0.1% D 10 4
99.9 0.3% E 104 99.8 0.1%
3
10 103 103 103

Claudin-5
GLUT-1
CD31

ZO-1

102 102 102 102

1 IgG IgG IgG IgG
10 10 1
101 101

100 0 200 400 600 800 1.0k 100 0 200 400 600 800 1.0k 100 0 200 400 600 800 1.0k 100 0 200 400 600 800 1.0k
FSC FSC FSC FSC
F 10 4
99.7 0.2% G 10 4
99.9 0.1% H 10 4
99.8 0.1% I 104
98.6 0.8%

103 103 103 103

Occludin

MRP1
BCRP

Pgp

102 102 102 102

10 1 IgG 10 1 IgG 10 1 IgG 101 IgG

100 0 200 400 600 800 1.0k 100 0 200 400 600 800 1.0k 100 0 200 400 600 800 1.0k 100 0 200 400 600 800 1.0k
FSC FSC FSC FSC

J K
Row max
HBMEC
UM-BMEC
D-BMEC2
D-BMEC3
D-BMEC1
Endo
hPSC
Mes
Ecto
Row min

Fig. 2. hPSC-derived BMECs express key BMEC proteins and have gene expression profiles similar to those of primary human BMECs. (A to I) At day 10, BMECs
differentiated as shown in Fig. 1A were characterized by immunocytofluorescence (A) and flow cytometry (B to I) for key endothelial and BMEC markers. Scale bars,
100 mm. (J) Hierarchical clustering of whole transcripts counted by RNA sequencing was plotted using GENE-E. Hierarchical clustering analysis was performed on log2-
transformed gene counts of RNA sequencing expression data of undifferentiated hPSCs (7678); hPSC-derived endoderm (Endo) (76), ectoderm (Ecto) (77), and mesoderm
(Mes) (78); BMECs differentiated under defined conditions as illustrated in Fig. 1A (D-BMEC1, D-BMEC2, and D-BMEC3: IMR90-4derived BMECs at day 10 generated from three
independent differentiations); BMECs differentiated in UM (UM-BMECs); and human primary BMECs (hBMECs). Distances were computed using one minus Pearson correlation
with average linkage. (K) A set of 506 tight junction and transporter genes (table S1) was used to investigate gene expression similarity between primary human BMECs, hPSC-
derived BMECs differentiated under defined conditions, and hPSC-derived BMECs differentiated in UM (32). The gene set included 20 tight junctionrelated genes (1, 5356), all
25 CLDN genes, all 407 solute carrier (SLC) transporters, and all 53 ATP-binding cassette (ABC) transporters. CLDN, SLC, and ABC genes were included without previous knowl-
edge of BBB association. A threshold of >1 fragment per kilobase million (FPKM) was used to define expressed versus nonexpressed transcripts.

Finally, previous studies have shown that coculturing BMECs, includ- tained as a monoculture or cocultured with primary human pericytes
ing those that are iPSC-derived, with neural progenitor cells, astrocytes, for the first 24 hours, followed by coculture with hPSC EZ sphere
and pericytes can enhance BBB properties such as TEER (33, 5760). derived astrocytes and neurons (1:3) (61) for additional 3 days. Max-
Day 8 iPSC-derived BMECs seeded on Transwells were either main- imum TEER for the monoculture system was ~3000 ohmcm2, and

Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017 4 of 12

SCIENCE ADVANCES | RESEARCH ARTICLE

A vWF/DAPI B C LDL/DIC

D ICAM-1/before E ICAM-1/after F 2.0k IgG

ICAM-1
1.5k

Count
62% increase
1.0k ICAM-1
after TNF-

500

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0
100 101 102 103 104

Fluorescence
Rhodamine 123 accumulation Hoechst accumulation DCFDA accumulation Rhodamine 123 transport

G *** H ** I ** J **
A-B **
Percentage

B-A

*
**
**

Fig. 3. hPSC-derived BMECs exhibit key BBB phenotypes. hPSC-derived BMECs were differentiated as illustrated in Fig. 1A. (A) Immunofluorescence images of vWF
(red) and DAPI staining (blue) in hPSC-derived BMECs at day 10. (B) hPSC-derived BMECs were dissociated with Accutase and replated at 1 105 cells/cm2 on Matrigel.
Images were taken after 24 hours in hECSR2 with VEGF (50 ng/ml). (C) hPSC-derived BMECs at day 10 were analyzed with an LDL Uptake Assay kit. LDL is shown in red on a
merged bright-field image. (D to F) ICAM-1 induction in hPSC-derived BMECs. hPSC-derived BMECs at day 10 were treated with TNF-a (10 ng/ml) for 16 hours. Immuno-
fluorescence images for ICAM-1 were acquired before (D) and after (E) TNF-a treatment. (F) Cells were dissociated with Accutase, and ICAM-1 expression was quantified by
flow cytometry before and after TNF-a treatment. Efflux transporter activities were measured by the intracellular accumulation of (G) rhodamine 123, (H) Hoechst, and
(I) DCFDA, substrates for Pgp, BCRP, and MRP, respectively. CsA, Ko143, and MK571 were used as specific inhibitors of Pgp, BCRP, and MRP, respectively. (J) The polarization of
Pgp was measured by rhodamine 123 transport across the BMEC monolayer from the apical to basolateral side (A-B) and vice versa (B-A). Inhibitor-treated samples were
independently normalized to each respective noninhibitor-treated control sample. (K) TEER was measured in hPSC-derived BMECs cocultured with astrocytes, neurons, and
pericytes. Data were collected from three independent replicates and are means SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 mm.

coculture further elevated TEER by 30% at day 2 and helped maintain
an elevated TEER throughout the duration of the experiment com-
pared to the monoculture control (Fig. 3K).
Cell density is crucial for BMEC differentiation
Cell density has been shown to be crucial for efficient hPSC differen-
tiation to a variety of lineages, including BMECs (35, 6264). Thus, in
addition to the optimal initial day 3 cell seeding density used above
(35 103 cells/cm2), we tested a range of seeding densities, from 8.8
103 to 140 103 cells/cm2, to explore how density affects BMEC yield
and phenotype. As shown in Fig. 4A, TEER was a strong function of
seeding density, with only 35 103 cells/cm2 yielding BMECs with
substantial barrier function at day 2 after transfer onto Transwells.
The BMEC TEER peaked 2 days after replating and plateaued above

Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017 5 of 12

SCIENCE ADVANCES | RESEARCH ARTICLE

A B
***

D Claudin-5 E
C 800
Isotype 8.8k 35k

Geometric mean
140k 100
104 104 10104 4 100

Claudin-5 (%)
15.4 1.1% 99.2 0.3% 600 70k
Claudin-5

Count
103 103 10103 3
35k 80
400 17.5k
102 102 10102 2
50 60
8.8k
101 101 10101 1
200 Isotype 40

Downloaded from http://advances.sciencemag.org/ on November 12, 2017

100 10100 0 20
100
0 200 400 600 800 1.0k 0 200 400 600 800 1.0k 00 200
200 400
400 600
600 800
800 1.0k
1.0k 0 0
100 101 102 103 104 8.8k 17.5k 35k 70k 140k
FSC FSC FSC Fluorescence Density (cells/cm2)
F
Claudin-5/DAPI 8.8k Claudin-5/DAPI 17.5k Claudin-5/DAPI 35k Claudin-5/DAPI 70k Claudin-5/DAPI 140k

H Occludin I
G Isotype 8.8k 35k 800
104 104

Geometric mean
10 4
100 120
23.5 1.3% 95.1 1.1% 140k
Occludin

Occludin (%)
103 103 600 80
103
70k 100
Count

102 102 102 35k 60

400
17.5k 40 80
101 101 101 8.8k
200
0 0 Isotype 20
10 10 100 60
0 200 400 600 800 1.0k 0 200 400 600 800 1.0k 0 200 400 600 800 1.0k
0 0
FSC 8.8k 17.5k 35k 70k 140k
FSC FSC 100 10 1
10 2
10 3
104

Density (cells/cm2)
Fluorescence
J
Occludin/DAPI 8.8k Occludin/DAPI 17.5k Occludin/DAPI 35k Occludin/DAPI 70k Occludin/DAPI 140k

Fig. 4. Initial seeding density is critical for BMEC differentiation. (A) hPSCs were seeded at the indicated densities (from 8800 to 140,000 cells/cm2) and differ-
entiated to BMECs, as illustrated in Fig. 1A. TEER was measured 2 days after replating on Transwell membranes at 106 cells/cm2. (B) TEERs of hPSC-derived BMECs were
measured daily for 7 days after replating on Transwell membranes. Data were collected from three independent replicates and are means SEM. ***P < 0.001. (C to E) The
percentage of claudin-5positive cells and expression levels of claudin-5 were quantified by flow cytometry at day 8 for cells differentiated at the indicated seeding density
(cells/cm2). (F) The localization of claudin-5 in cells differentiated at different seeding densities was investigated by immunofluorescence. White arrows indicate sample
areas lacking claudin-5 expression, and red arrows indicate discontinuous claudin-5. (G to I) The percentage of occludin-positive cells and expression levels of occludin
were quantified by flow cytometry at day 8 for cells differentiated at the indicated seeding density (cells/cm2). (J) The localization of occludin in cells differentiated at
different seeding densities was investigated by immunofluorescence. White arrows indicate sample areas lacking occludin expression, and red arrows indicate dis-
continuous occludin. Flow cytometry plots are representative of three independent experiments. Insets indicate the mean percentage of cells in the immunopositive
gated region SEM. Scale bars, 100 mm.

2000 ohmcm2 through day 7 (Fig. 4B). At non-optimum seeding den- cess. Cells differentiated at all densities tested yielded EC populations
sities, TEER gradually increased through 6 days after replating, reach- with nearly 100% VEGFR2+ cells at day 5 and more than 90% CD31+
ing approximately 1000 ohmcm2. We next assessed the expression of cells at day 10 (fig. S5). This suggested that deficits in barrier function
endothelial markers to investigate the endothelial specification pro- may be a result of poor BMEC specification. Thus, we assessed BMEC

Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017 6 of 12

SCIENCE ADVANCES | RESEARCH ARTICLE
marker expression in populations differentiated at different seeding
densities. Nearly 100% of cells expressed Pgp after the differentiation
process at either day 8 or day 10 (fig. S6). However, only cells differ-
entiated at the optimal seeding density of 35 103 cells/cm2 yielded a
pure claudin-5expressing population with maximal claudin-5 expres-
sion (Fig. 4, C to E). In addition, a subpopulation of cells differentiated
from non-optimal starting densities also lacked junctional claudin-5
(Fig. 4F, white arrows) or exhibited discontinuous claudin-5 localization
at cell junctions (Fig. 4F, red arrows). Similarly, only cells differen-
tiated from seeding densities of at least 35 103 cells/cm2 yielded a
nearly pure population of occludin-expressing cells (Fig. 4, G to I).
Immunofluorescence analysis of occludin also showed that a large
fraction of cells differentiated from initial densities less than 35
103 cells/cm2 lacked occludin expression (Fig. 4J, white arrows). Un-
like claudin-5 and occludin, seeding density did not have a significant
effect on ZO-1 expression, but cells differentiated at initial densities of
8.8 103 cells/cm2 showed discontinuous ZO-1 localization (fig. S7, red
arrows). Immunofluorescence for additional BMEC markers also indi-
cated poor localization of CD31, MRP1, and BCRP in cells differen-
tiated at non-optimum initial cell densities (fig. S7, compare 35k to
other densities). H9 hESCs and 19-9-11 iPSCs yielded BMECs exhibit-
ing TEER at or above 2000 ohmcm2 at an optimal initial seeding den-
sity of 35 103 cells/cm2 (fig. S8, A and B). We also compared the
differentiation reproducibility with that of the previously reported
UM protocol (33). Although both methods produce BMECs capable
of substantial barrier formation from multiple hPSC lines, BMECs
differentiated from H9 hESCs and 19-9-11 iPSCs using the defined
method exhibited higher TEERs and lower batch-to-batch variation
(fig. S8A). Finally, 35 103 cells/cm2 was also found to be the optimal
seeding density for differentiation on Synthemax and vitronectin
substrates, with vitronectin substrates performing more closely to
Matrigel than Synthemax substrates in TEER assays (fig. S9).
RA enhances BMEC phenotypes
Previously, we have shown that RA induces BBB properties in hPSC-
derived BMECs (33). Other studies have also demonstrated that RA
signaling regulates BBB formation and induces BBB phenotypes
(46, 47). To determine the role of RA in specifying BMEC differen-
tiation and enhancing the BMEC phenotypes described in Figs. 2
and 3, we compared differentiation in the presence and absence of
RA using the protocol illustrated in Fig. 1A. From day 6 to day 8, cells
were maintained either in hECSR1 or in hECSR1 lacking RA. Quan-
titative polymerase chain reaction (qPCR) showed that the expres-
sion of the tight junctionrelated genes TJP1, CLDN5, and OCLN
and the efflux transporters ABCG2, ABCC1, and ABCB1 was greater
(3- to 20-fold) in cells exposed to RA (Fig. 5A). Nearly 100% of cells
expressed CD31 at day 6, and this expression was preserved in the
presence of RA induction at day 8 (Fig. 5B). Immunofluorescence for
CD31 and other BMEC markers for cells differentiated in the absence
of RA, including VE-cadherin, GLUT-1, and MRP1, is shown in fig.
S10A. Nearly 100% of cells differentiated in the absence and presence
of RA expressed Pgp, but RA-treated cells expressed more Pgp than
nontreated cells (Fig. 5C). To evaluate the barrier formation potential
of the differentiated BMECs and to assess the effects of RA treatment,
we replated day 8 BMECs onto Transwells and measured TEER at day
10. As shown in Fig. 5D, cells differentiated in the presence of RA ex-
hibited physiologically relevant TEER (~4000 ohmcm2), whereas
those differentiated in the absence of RA exhibited significantly re-
duced barrier properties. Similarly, BMECs differentiated on vitronec-
Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017
tin or Synthemax required RA supplementation to achieve substantial
barrier properties (fig. S11). We then investigated the expression and
localization of tight junction proteins. Both occludin and ZO-1 were
expressed in nearly all cells at day 10 regardless of RA treatment; how-
ever, RA treatment significantly increased the expression levels of
occludin and ZO-1 (Fig. 5, E and F). Although occludin and ZO-1 ex-
pression was lower in the absence of RA, immunofluorescence results
indicate that nearly all the cells differentiated in the absence of RA still
expressed occludin and ZO-1; however, the junctional distribution
was nonuniform (Fig. 5G, indicated with red arrows and compare
to Fig. 2A). In contrast to the results with occludin and ZO-1, in the
absence of RA, only around 60% of the ECs expressed claudin-5
compared to 100% of RA-treated cells expressing claudin-5 (Fig. 5,
H and I). In addition, claudin-5 expression was also substantially
greater in RA-treated cells (Fig. 5I). Immunofluorescence indicated
that in the absence of RA, many of the cells did not express claudin-5
(Fig. 5J, white arrows), and those that did exhibited nonuniform junc-
Downloaded from http://advances.sciencemag.org/ on November 12, 2017
tional distribution of claudin-5 similar to that observed with occludin
and ZO-1 (Fig. 5J, red arrows). Together, these results suggest that RA
is not necessary for hPSC differentiation to ECs but enhances key
BMEC phenotypes in the hPSC-derived ECs, including the expression
and localization of tight junction proteins that promote barrier func-
tion as measured by TEER.
DISCUSSION
Here, we demonstrate a robust and efficient process to differentiate
hPSCs to BMECs in a defined manner. The cells progress as a homo-
geneous population from a pluripotent state to primitive streaklike
and intermediate mesodermlike stages, to endothelial progenitors,
and eventually to ECs that express BMEC markers and exhibit BBB
barrier and efflux transporter properties. This differentiation method
uses a completely defined platform, including culture medium and
substrates. Defined reagents exhibit less lot-to-lot variability, leading
to more robust and efficient differentiation and allowing differentia-
tion results to be more reliable and reproducible. We have tested three
different hPSC lines with this differentiation protocol, and all of these
lines were able to differentiate into pure populations of BMEC, with
definitive BMEC properties at the same optimum cell density.
In vivo, ECs that form the BBB originate from mesoderm progeni-
tors located outside the CNS (65). In contrast to previous BMEC dif-
ferentiation protocols (32, 37) that rely on coculture of endothelial
progenitors with pericytes, astrocytes, or differentiating neural cells,
this differentiation strategy instead relies on sequential Wnt and RA
signaling activation to first specify ECs and then enhance BMEC prop-
erties, respectively. First, activation of canonical Wnt signaling by
CHIR99021 addition directs hPSCs to brachyury-positive primitive
streaklike cells that then differentiate to PAX2-positive intermediate
mesoderm and EC progenitors when differentiated at the appropriate
density in DeSR2 medium. Next, RA treatment for 2 days drives these
endothelial progenitor cells to express key BMEC markers and exhibit
BMEC-specific properties, including high TEER and efflux transporter
activity. Our experiments showed that although RA was not necessary
to obtain ECs, RA treatment significantly increased BBB properties such
as TEER. The TEER enhancement correlated with increased expression
and improved localization of the tight junction proteins occludin and
claudin-5. These findings are similar to those results observed after RA
treatment of hPSC-derived BMECs generated by co-differentiation with
neural cells using our previously reported undefined protocol (33), in
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SCIENCE ADVANCES | RESEARCH ARTICLE

A B C Pgp D
104 800 ***
NO RA-D10
** ** ** ** ** * D6 97.2 0.4% 99.6 0.3%
Isotype
103 D8 97.4 0.3% 600 RA-D10
99.8 0.1%

Count
79.2% increase

CD31
102 400

CD31
200
101

0
100 0 200 400 600 800 1.0k 100 101 102 103 104

FSC Fluorescence

E Occludin F ZO-1
NO RA-D10 1.2k NO RA-D10
1.2k
Isotype 95.1 0.6% Isotype 99.1 0.3%
RA-D10 RA-D10 G
99.1 0.5% 900 99.4 0.1%
900
191% increase 328% increase Occludin/DAPI ZO-1/DAPI
Count

Count

600 600

Downloaded from http://advances.sciencemag.org/ on November 12, 2017

300 300

0 0
100 101 102 103 104 100 101 102 103 104

Fluorescence Fluorescence
Claudin-5
H 104 Isotype I 800 Isotype
NO RA-D10 60.0 2.8% NO RA-D10
RA-D10 99.8 0.1% RA-D10 J
103 600 436% increase Claudin-5/DAPI
Claudin-5

Count

2
10 400

101 200

100 0
0 200 400 600 800 1.0k 100 101 102 103 104
FSC Fluorescence
Fig. 5. RA induces acquisition of key BMEC phenotypes in EC progenitors. BMECs were differentiated as shown in Fig. 1A in the presence or absence of RA, as
indicated. (A) At day 8, the expression of tight junction and transporter genes was assessed by qPCR. (B) Flow cytometry for CD31 expression was performed at days 6 (D6)
and 8 (D8). (C) Pgp expression was quantified by flow cytometry at day 10. At day 6, the medium was switched to hESFM containing or lacking RA, as indicated. (D) At day 8,
cells were replated onto Matrigel-coated Transwell membranes at 106 cells/cm2 in the presence or absence of 10 mM Y-27632. TEER was measured at day 10, 2 days after
replating. (E to G) Occludin (E and G) and ZO-1 (F and G) expression and localization were assessed by flow cytometry and immunofluorescence at day 10. Red arrows indicate
discontinuous occludin or ZO-1 localization. (H and I) At day 10, the expression levels of claudin-5 in BMECs differentiated in the presence or absence of RA were assessed by
flow cytometry. (J) The localization and expression of claudin-5 of cells differentiated in the absence of RA were determined via immunofluorescence (white arrows indicate
cells lacking claudin-5, and red arrows indicate discontinuous claudin-5). Images, bar graphs, and flow cytometry plots are representative of three independent experiments.
Data from three independent replicates are means SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Insets denote either percentage of positively labeled cells or percentage increase
in marker expression upon RA treatment. Scale bars, 100 mm.

addition to those studies that have explored the barrier-enhancing
effects of astrocyte or neuron coculture with hPSC-derived BMECs
(33, 60). However, even in the absence of RA treatment, the previously
reported undefined protocol still yielded pure populations of claudin-5
expressing BMECs, whereas RA was critical in the defined protocol for
generating pure claudin-5expressing populations. Thus, the effects of
defined differentiation do not exactly mimic those provided by cocul-
tured neural cells, yet the resultant BMECs were quite similar on a
whole-transcriptome level to BMECs generated using the undefined co-
culture approach and to primary human BMECs.
Previously, we have shown that cell seeding density can affect
BMEC differentiation from hPSCs using the neural co-differentiation
protocol (35). Other studies have also demonstrated that cell seeding
density is crucial for efficient hPSC differentiation to various lineages
(6668). An initial cell seeding density of 35 103 cells/cm2 at day 3
is necessary to yield homogeneous populations of BMECs with high
expression and proper localization of key BBB proteins, in turn
leading to optimal barrier properties. In addition, this optimum
seeding density translated to multiple hPSC lines and to differentia-
tion on defined matrices. Cells differentiated at non-optimal seeding
densities expressed BMEC markers but exhibited a reduced TEER,
likely resulting from diminished claudin-5 and occludin expression
and improper junctional localization. Thus, RA signaling and cell
density similarly regulate the capability for the endothelial progenitors
to acquire BMEC properties. Although we determined that seeding
density is a crucial regulator of hPSC differentiation to BMECs, we
do not yet know the mechanism by which density affects differentia-
tion efficiency.

Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017 8 of 12

SCIENCE ADVANCES | RESEARCH ARTICLE
Coculturing hPSC-derived BMECs with pericytes, astrocytes, and
neurons further elevated TEER, consistent with previous studies that
showed that coculturing BMECs with these neural cells can enhance
BBB properties (57, 58, 6971). These data suggest that it will be possible
to integrate these defined hPSC-derived BMECs with other cells of the
neurovascular unit to create an isogenic patient-derived model that can
be used to study the role of neurovascular unit in human neurological
diseases (60). In addition, this defined BMEC differentiation method
has the potential to be a powerful and robust tool for preclinical studies
of pharmaceutical transport through the BBB.
MATERIALS AND METHODS
hPSC culture and differentiation
Human iPSCs [iPS(IMR90)-4 (72), iPS-DF 19-9-11T (73), and hESCs
(H9) (29)] were maintained on Matrigel (Corning)coated surfaces in
mTeSR1 (STEMCELL Technologies), as previously described (74).
Before differentiation, hPSCs were singularized with Accutase (Inno-
vative Cell Technologies) and plated onto Matrigel-coated plates at a
density between 25 103 and 50 103 cells/cm2 in mTeSR1 supple-
mented with 10 mM Rho-associated protein kinase (ROCK) inhibitor
Y-27632 (Selleckchem). hPSCs were expanded in mTeSR1 for 3 days.
For differentiation on defined substrates, singularized hPSCs were
plated onto the vitronectin-coated (Thermo Fisher Scientific) or
Synthemax-coated (Corning) surface at a density between 25 103
and 50 103 cells/cm2 in mTeSR1 supplemented with 10 mM ROCK
inhibitor Y-27632 (Selleckchem). To initiate differentiation at day 0,
cells were treated with 6 mM CHIR99021 (Selleckchem) in DeSR1:
DMEM/Hams F12 (Thermo Fisher Scientific), 1 MEM-NEAA
(Thermo Fisher Scientific), 0.5 GlutaMAX (Thermo Fisher Scientific),
and 0.1 mM b-mercaptoethanol (Sigma). After 24 hours, the medium
was changed to DeSR2: DeSR1 plus 1 B27 (Thermo Fisher Scientific)
every day for another 5 days. At day 6, the medium was switched to
hECSR1: hESFM (Thermo Fisher Scientific) supplemented with bFGF
(20 ng/ml), 10 mM RA, and 1 B27. After 2 days of culture in hECSR1
medium, day 8 cells were dissociated with Accutase and plated at
1 106 cells/cm2 in hECSR1 onto 48-well tissue culture plates or
1.12-cm2 Transwell-Clear permeable inserts (0.4 mm pore size) coated
with Matrigel (100 mg/ml). For cells differentiated on vitronectin or
Synthemax substrates, cells were dissociated with Accutase and plated
at 1 106 cells/cm2 in hECSR1 onto 48-well tissue culture plates or
1.12-cm2 Transwell-Clear permeable inserts (0.4 mm pore size) coated
with human placentaderived collagen IV (400 mg/ml)/human plasma
derived fibronectin (100 mg/ml). At day 9, the medium was changed to
hECSR2 (hECSR1 without RA or bFGF) for longer-term mainte-
nance. Y-27632 was added to increase the attachment (75) of cells dif-
ferentiated in the absence of RA and was permitted to ensure confluent
monolayer formation and confirm that the reduction in TEER was not
only a result of incomplete monolayer formation.
Immunochemistry
Cells were rinsed with ice-cold phosphate-buffered saline (PBS) once
and fixed in either ice-cold methanol or 4% paraformaldehyde (PFA)
for 15 min. Cells were then blocked with 10% goat serum in PBS
containing 0.3% Triton X-100 for 30 min (10% PBSGT). Primary anti-
bodies were diluted in 10% PBSGT, and cells were incubated in the
antibody solutions at 4C overnight or at room temperature for 2 hours.
After three PBS washes, cells were incubated with secondary antibodies
in 10% PBSGT (goat anti-rabbit Alexa Fluor 594 and goat anti-mouse
Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017
Alexa Fluor 488; 1:200) for 1 hour at room temperature. Cells were
then washed with PBS three times, followed by treatment with DAPI
Fluoromount-G (Southern Biotech), and were visualized. A detailed
list of antibodies is shown in table S2.
Flow cytometry
Cells were dissociated with Accutase, fixed in 1% PFA for 15 min at
room temperature, and then washed with 0.5% bovine serum albumin
(BSA) (Bio-Rad) plus 0.1% Triton X-100 three times. Cells were stained
with primary and secondary antibodies diluted in 0.5% BSA plus
0.1% Triton X-100, as previously described (31). Data were collected
on a FACSCalibur flow cytometer (Becton Dickinson) and analyzed
using FlowJo. Corresponding isotype antibodies were used as FACS
(fluorescence-activated cell sorting) gating control. Details about anti-
body source and usage are provided in table S2.
Quantitative real-time PCR
Downloaded from http://advances.sciencemag.org/ on November 12, 2017
Total RNA was extracted with the RNeasy Mini Kit (Qiagen) and treated
with deoxyribonuclease (Qiagen). One microgram of total RNA was
reverse-transcribed into complementary DNA using an oligo(dT)
primer with SuperScript III Reverse Transcriptase (Invitrogen).
Quantitative real-time PCR was done in triplicate with iQ SYBR Green
SuperMix (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as an endogenous housekeeping control. Primer
sequences are provided in table S3.
LDL uptake assay
Differentiated BMECs at day 10 were analyzed with the LDL Uptake
Assay Kit (Abcam). Culture medium was aspirated and replaced with
LDL-DyLight 550 working solution. Cells were then incubated for
3 hours at 37C, followed by three washes with PBS, and then visualized
via fluorescent microscopy with excitation and emission wavelengths of
540 and 570 nm, respectively. After visualization, cells were fixed with a
cell-based fixative solution for 10 min. Cells were then washed with tris-
buffered saline plus 0.1% Triton X-100 for 5 min, followed by 30-min
blocking with Cell-Based Assay Blocking Solution. Cells were then
stained with rabbit anti-LDL receptor primary antibody and DyLight
488conjugated secondary antibody. Images were taken with a fluores-
cent microscope with excitation and emission wavelengths of 485 and
535 nm, respectively.
Efflux transporter accumulation and transport assay
Pgp, BCRP, and MRP functionality was assessed by intracellular accu-
mulation of fluorescent transporter substrates and transport of fluo-
rescent substrates across BMEC monolayers. Rhodamine 123 (10 mM;
Sigma), Hoechst (20 mM; Thermo Fisher Scientific), and DCFDA
(10 mM; Life Technologies) were used as the specific substrates for
Pgp, BCRP1, and MRP1, respectively. BMECs at day 10 were pretreated
for 1 hour with or without specific transporter inhibitors [10 mM CsA
(Pgp inhibitor), 10 mM Ko143 (BCRP inhibitor) (Sigma), and 1 mM
MK571 (MRP inhibitor) (Sigma) in Hanks balanced salt solution
(HBSS)]. Cells were then treated with transporter substrates in HBSS
and incubated for 1 hour at 37C on an orbital shaker. Cells were
washed with PBS three times and then lysed with radioimmunoprecip-
itation assay buffer (Pierce Biotechnology). Fluorescence intensity was
measured on a plate reader (485-nm excitation and 530-nm emission
for rhodamine 123 and DCFDA and 360-nm excitation and 497-nm
emission for Hoechst). Fluorescence intensity was subsequently nor-
malized to cell number determined using a hemocytometer.
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SCIENCE ADVANCES | RESEARCH ARTICLE
Endothelial cell tube formation
Each well of a 24-well tissue culture plate was coated with 300 ml of
Matrigel (10 mg/liter). BMECs at day 10 were dissociated with Ac-
cutase and plated in hECSM1 plus VEGF (50 ng/ml) without RA or
bFGF at 2 105 cells per well. Phase-contrast images were acquired
after 24 hours.
RNA sequencing and data analysis
Total RNA of day 10 IMR90-4 iPSCderived BMECs with either
defined protocol or UM protocol and primary human brain micro-
vascular ECs (ACBRI 376, Cell Systems) were prepared with the
Direct-zol RNA MiniPrep Plus Kit (Zymo Research) according to
the manufacturers instructions. Samples were sequenced on Illumina
HiSeq 2500 at the University of Wisconsin-Madison Biotechnology
Center. The resulting sequence reads were mapped to the human ge-
nome (hg19) using HISAT49, and the RefSeq transcript levels (FPKMs)
were quantified using the Python script rpkmforgenes.py50. A hierar-
chical clustering of whole transcripts was performed using GENE-E
on the log2-transformed gene counts. Distances were computed using
one minus Pearson correlation with average linkage. FASTQ files of
hPSCs (7678) and hPSC-derived ectoderm (77), endoderm (76), and
mesoderm (78) were downloaded from the Gene Expression Omnibus
(GEO) or ArrayExpress (www.ebi.ac.uk/arrayexpress/) database. The
expression of a subset of genes that regulate key BBB attributes, in-
cluding tight junctions and molecular transporters, was analyzed. The
gene set comprises 20 tight junctionrelated genes (1, 5356) and an
unbiased list of all 25 CLDN genes, all 407 solute carrier (SLC) trans-
porters, and all 53 ATP-binding cassette (ABC) transporters regardless
of previous knowledge of BBB association (table S1). Transcript levels
(FPKMs) were set at a threshold of >1 FPKM, which indicates moderate
expression (79). Primary human BMECs were used to screen out the
BBB-related genes from the gene list with the threshold of >1 FPKM.
Coculture of hPSC-derived BMECs with pericytes, neurons,
and astrocytes
hPSC-derived BMECs at day 8 were replated onto 12-well Transwell
inserts at a density of 1 106 cells/cm2 and cocultured with primary
human pericytes residing on the bottom of the 12-well plates (1 105 cells
per well) for 24 hours in hECSR1 medium. Following coculture with peri-
cytes, BMECs were cocultured with EZ spherederived neurons and as-
trocytes (a total of 1 105 cells per well; 1:3) in hECSR2 for the remainder
of the experiment. TEER was measured as a function of time following
initiation of coculture.
Statistics
Data are presented as means SEM. Statistical significance was de-
termined by Students t test (two-tailed) between two groups. Three
or more groups were analyzed by one-way analysis of variance
(ANOVA) followed by Tukeys post hoc tests. P < 0.05 was considered
statistically significant.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/3/11/e1701679/DC1
fig. S1. Gene expression during hPSC differentiation to BMECs.
fig. S2. BMECs differentiated from H9 hESCs and 19-9-11 iPSCs express EC- and BMEC-related
proteins.
fig. S3. BMECs differentiated on Synthemax and vitronectin express EC- and BMEC-related
proteins and have efflux transporter activities.
Qian et al., Sci. Adv. 2017; 3 : e1701679 8 November 2017
fig. S4. BMECs differentiated from hPSCs in defined and undefined protocols exhibit similar
Pgp activities.
fig. S5. BMECs differentiated at different seeding densities express VEGFR2 and CD31.
fig. S6. BMECs differentiated at different seeding densities express Pgp.
fig. S7. BMECs differentiated at different densities express BMEC proteins but can exhibit
aberrant protein localization.
fig. S8. TEER of BMECs differentiated from different hPSC lines.
fig. S9. TEER in BMECs differentiated from hPSCs at different seeding densities.
fig. S10. BMECs differentiated in the absence of RA exhibit low expression and mislocalization
of EC and BMEC proteins.
fig. S11. RA enhances TEER for cells differentiated on Matrigel-, vitronectin-, and Synthemax-
coated surfaces.
table S1. Expression of tight junction and transporter genes in iPSC-derived and primary
human BMECs.
table S2. Antibodies used in this study.
table S3. qPCR primers used in this study.
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Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells
Tongcheng Qian, Shaenah E. Maguire, Scott G. Canfield, Xiaoping Bao, William R. Olson, Eric V. Shusta and Sean P.
Palecek

Sci Adv 3 (11), e1701679.

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