(2020) 17:36
Yamashita et al. Fluids Barriers CNS
https://doi.org/10.1186/s12987-020-00197-1

Fluids and Barriers of the CNS
Open Access

RESEARCH

Inhibition of transforming growth factor
beta signaling pathway promotes differentiationof human induced pluripotent stem cell‑derived
brain microvascular endothelial‑like cells
Misaki Yamashita, Hiromasa Aoki, Tadahiro Hashita, Takahiro Iwao and Tamihide Matsunaga*

Abstract
Background: The blood–brain barrier (BBB) plays an important role as a biological barrier by regulating moleculartransport between circulating blood and the brain parenchyma. In drug development, the accurate evaluation of
BBB permeability is essential to predict not only the efficacy but also the safety of drugs. Recently, brain microvascular endothelial-like cells derived from human induced pluripotent stem cells (iPSCs) have attracted much attention.
However, the differentiation protocol has not been optimized, and the enhancement of iPSC-derived brain microvascular endothelial-like cells (iBMELCs) function is required to develop highly functional BBB models for pharmaceuticalresearch. Thus, we attempted to improve the functions of differentiated iBMELCs and develop a versatile BBB modelby modulating TGF-β signaling pathway without implementing complex techniques such as co-culture systems.
Methods: iPSCs were differentiated into iBMELCs, and TGF-β inhibitor was used in the late stage of differentiation. To
investigate the effect of TGF-β on freezing–thawing, iBMELCs were frozen for 60–90 min or 1 month. The barrier integrity of iBMELCs was evaluated by transendothelial electrical resistance (TEER) values and permeability of Lucifer yellow.
Characterization of iBMELCs was conducted by RT-qPCR, immunofluorescence analysis, vascular tube formation assay,
and acetylated LDL uptake assay. Functions of efflux transporters were defined by intracellular accumulation of thesubstrates.
Results: When we added a TGF-β inhibitor during iBMELCs differentiation, expression of the vascular endothelial
cell marker was increased and blood vessel-like structure formation was enhanced. Furthermore, TEER values wereremarkably increased in three iPSC lines. Additionally, it was revealed that TGF-β pathway inhibition suppressed thedamage caused by the freezing–thawing of iBMELCs.
Conclusion: We succeeded in significantly enhancing the function and endothelial characteristics of iBMELCs by
adding a small molecular compound, a TGF-β inhibitor. Moreover, the iBMELCs could maintain high barrier function
even after freezing–thawing. Taken together, these results suggest that TGF-β pathway inhibition may be useful fordeveloping iPSC-derived in vitro BBB models for further pharmaceutical research.
Keywords: Blood–brain barrier, Cell differentiation, Endothelial cells, Induced pluripotent stem cells, Transforming
growth factor beta

*Correspondence: tmatsu@phar.nagoya‑cu.ac.jp
Department of Clinical Pharmacy, Graduate School of Pharmaceutical
Sciences, Nagoya City University, 3‑1 Tanabe‑dori, Mizuho‑ku,
Nagoya 467‑8603, Japan© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds thepermitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​
mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Yamashita et al. Fluids Barriers CNS(2020) 17:36

BackgroundThe blood–brain barrier (BBB) plays a very importantbiological barrier role by regulating molecular transport
between the circulating blood and the brain parenchyma[1]. The BBB consists of brain microvascular endothelial
cells (BMECs), which are characterized by specialized
tight junctions [2, 3] and high expression of multidrug
efflux transporters [4].
In drug development, accurate evaluation of BBB permeability is required to predict not only the efficacy but
also the safety of drugs [5]. To evaluate BBB permeability, in vitro models are often used; however, there is no
ideal in vitro BBB model available currently. Although
BBB permeability has been evaluated by primary BMECs
obtained from animals [6–8], species differences between
human and animals have made it difficult to apply accurately animal findings in humans [9]. In addition, it isvery difficult to obtain primary human BMECs for pharmacokinetic studies. Human immortalized BMECs havethe advantage of promoting reproducibility of experimental data. However, it is difficult to predict BBB permeability accurately due to low barrier functions in mosthuman immortalized BMECs [10] compared with physiological transendothelial electrical resistance (TEER)
values. The reported physiological TEER values are morethan 1000 Ω × cm2 in capillaries of rat or frog brain [11,12]. Thus, non-endothelial cells with high-TEER values,
such as Caco-2 cells and Madin–Darby canine kidneycells, have been used for pharmacokinetic studies [13].
However, the functions of transporters in these cell lines
differ from those in human BMECs. Therefore, there isa focus on generating functional BMECs from humaninduced pluripotent stem cells (iPSCs), which, because ofpluripotency, can differentiate into various cell types inour organs and tissues [14].
Recently, some human iPSC-derived BBB models
have been developed [15–23]. However, expression levels of endothelial cell markers were low [18], and thebarrier functions were unstable depending on the iPSC
line used [17, 23]. Therefore, these reports suggested
that the differentiation protocol has not been optimized
and enhancement of iPSC-derived brain microvascular endothelial-like cells (iBMELCs) function is required
to develop highly functional BBB models for pharmaceutical research. Previously, co-culture systems with
pericytes, astrocytes, or neurons—all components of
BBB—have been tried to promote differentiation into
highly functional iBMELCs [15–21]. However, the systems are not reproducible and suitable for drug screening
due to complex manipulation. Additionally, although cryopreservation of iBMELCs using Rho-associated coiledcoil forming kinase inhibitor Y-27632 has been reported,
iBMELCs were not able to maintain the barrier function

Page 2 of 12

for a prolonged period after freezing–thawing [24]. Thus,
we attempted to improve the functions of differentiated
iBMELCs and develop a versatile BBB model without
implementing complex techniques such as co-culture
systems.
Transforming growth factor beta (TGF-β) signaling is
involved in embryonic development including cell differentiation, apoptosis, cellular homeostasis, and othercellular functions [25, 26]. Furthermore, TGF-β is associated with BBB dysfunction [27–29]. However, the effectof TGF-β inhibition on iBMELCs differentiation has not
be analyzed. In this study, we show that TGF-β inhibitors
critically enhance the BBB properties of human iBMELCs
without complex manipulations such as the co-culture
with pericytes or astrocytes. This approach may provide
a powerful tool in pharmaceutical research.

Methods
Culture of human iPSCs

Human iPSCs (610B1, 606A1 and 648A1) were purchased from RIKEN (Saitama, Japan) and were maintained on a feeder layer of mitomycin C-treated mouseembryonic fibroblasts in iPSC medium [Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (Wako Pure Chemical
Industries (Wako), Osaka, Japan) containing 20% KnockOut Serum Replacement (Invitrogen, Carlsbad, CA,USA), 2 mM l-glutamine (Wako), 1% minimal essential
medium with non-essential amino acids (Invitrogen),
0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis,
MO, USA), and 5 ng/mL human fibroblast growth factor-2 (FGF-2) (GenScript, Nanjing, China)] at 37 °C in 5%­CO2.
Differentiation of human iPSCs into BMELCs

Prior to differentiation, human iPSCs were seeded onto
Growth Factor Reduced Matrigel (Matrigel) (Corning, Corning, NY, USA)-coated plates and cultured
with StemSure hPSC medium (Wako) supplemented
with 35 ng/mL FGF2 for 3–4 days. Differentiation
into human iPSC-derived BMECs was performed aspreviously described [15, 16]. The protocol has been
described in Fig. 1a. Briefly, after reaching 70% confluence, cells were cultured in standard unconditioned
medium (UM; iPSC medium without FGF2) for 6 days.
The medium was changed every day. Then, the culture medium was switched to EC medium [Human
Endothelial-SFM (Thermo Fisher Scientific, Waltham,
MA, USA) supplemented with 1% platelet-poor plasma
derived bovine serum (PDS) (Alfa Aesar, Haverhill, MA, USA), 20 ng/mL FGF2, and 10 μM all-transretinoic acid (RA) (Tocris Bioscience, Bristol, UK)].
After 2 days, the cells were detached using Accutase(Nacalai Tesque, Kyoto, Japan) (20 min, 37 °C) andYamashita et al. Fluids Barriers CNS(2020) 17:36

Page 3 of 12

a Day 0Day 8 Passage Day 8

Day 10

1 day

1 day

UM

EM

EM

EM
- RA
- bFGF

Fibronectin
CollagencdControl

2.0

**

1.5
1.0
0.5
0

A-83-01

A-83-01

Control

Mean fluorescenceintensity

VE-cadherin

Control
A-83-01

Ac-LDL

Positive cell ratio (%)

2 days

iPS cells Matrigelb± TGF-

6 daysiBMECs

100

***

80
60
40
20
0
Control
A-83-01

Tube formation

6 10 cells

5

8 10 cells

5

10 10 cells

5

8 10 cells

5

10 10 cells

Control

55

A-83-01

6 10 cells

Fig. 1 The effect of TGF-β inhibitor on iBMELCs differentiation. a A schematic diagram of the protocol of differentiation of human iPSCs to BMECs.
b Immunofluorescence for the endothelial cell adhesion molecule (VE-cadherin: red). Blue: DAPI. Scale bar, 100 μm. Statistical significance wascalculated using the unpaired Student’s t-test (**p < 0.01), control = 1. Data are represented as the mean ± SD (n = 3). c Ac-LDL (red) uptake assay.
Blue: Hoechst 33342. Scale bar, 100 μm. Positive cell ratio was defined as the number of cells with Ac-LDL uptake to the total number of cells.
Statistical significance was calculated using the unpaired Student’s t-test (***p < 0.001). Data are represented as the mean ± SD (n = 3). d Tube
formation assay. The cells were stained with calcein (green). Scale bar, 500 μm

plated onto tissue culture polystyrene plates or 0.3cm2 Transwell-Clear permeable inserts (0.4 μm poresize, Corning) coated with a mixture of fibronectin(100 μg/mL; Wako) and collagen IV (400 μg/mL; Nitta
geratin, Osaka, Japan). The cells were seeded at a density of 3.0 × 105 cells/insert and cultured for 24 h with
EC medium. Thereafter, culture medium was replacedwith EC medium lacking FGF2 and RA for 24 h. The
cells were treated with 1 μM TGF-β inhibitors, A-83-01(Wako), SB-431542 (Wako), and RepSox (Wako), fromday 8 to day 10. As shown in Additional file 1: Fig. S5,
the cells were treated with A-83-01 from day 8 to day
10, from day 8 to day 12, or from day 10 to day 12.

Quantitative reverse transcription‑polymerase chain
reaction (RT‑qPCR)

Total RNA was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Brea, CA, USA)according to the manufacturer’s instructions. cDNA wassynthesized from 0.5 ng total RNA using ReverTra Ace
qPCR RT Master Mix (Toyobo, Osaka, Japan). KAPASYBR FAST qPCR Kit Master mix (2×) ABI Prism (Kapa
Biosystems, Wilmington, MA, USA) was used as thereaction solution for RT-qPCR. The samples were amplified using a LightCycler 96 System (Roche Diagnostics,
Basel, Switzerland). PCR primer sequences are shown in
Additional file 1: Table S1. All values were normalized byYamashita et al. Fluids Barriers CNS(2020) 17:36

the value of the hypoxanthine phosphoribosyltransferase(HPRT) gene.
Immunofluorescence staining

Cells were differentiated after seeding on Cell carrier-96(PerkinElmer, Waltham, MA, USA) on day 8. On day 10,
differentiated cells were washed with phosphate buffered saline (PBS) and fixed for 20 min in 4% paraformaldehyde. Next, the cells were washed three times with
PBS containing 10 mM glycine and then permeabilized
in PBS containing 0.1% Triton X-100 for 25 min. After
blocking with 5% donkey serum for 20 min, cells wereincubated with the primary antibody for 120 min, followed by incubation with both secondary antibody and a
1:1000 dilution of 4′,6-diamidino-2-phenylindole (DAPI)(Dojindo, Kumamoto, Japan) for 60 min. The cells werethen washed three times with PBS. The list of antibodies
used in this study is shown in Additional file 1: Table S2.
For vascular endothelial cadherin (VE-cadherin), vonWillebrand factor (vWF), claudin-5, P-glycoprotein(P-gp), breast cancer resistant protein (BCRP), and glucose transporter 1 (GLUT1) staining, differentiatedcells were washed three times with PBS containing 0.1%
bovine serum albumin (BSA), fixed with 4% paraformaldehyde for 15 min, washed three times with PBS containing 0.1% BSA, and permeabilized with 0.1% Triton X-100
for 5 min. After washing three times with PBS containing 0.1% BSA, cells were incubated overnight at 4 °C
with primary antibodies diluted in PBS containing 0.1%
BSA. The cells were washed three times with PBS containing 0.1% BSA and incubated with a 1:200 dilution of
Alexa Fluro 564-labeled secondary antibody for 60 min.
After washing three times with PBS containing 0.1%
BSA, the cells were incubated with a 1:1000 dilution of
DAPI for 5 min. The cells were subsequently treated with
4% paraformaldehyde for 5 min and washed three times
with PBS.
Immunofluorescence images were observed using theOperetta High-Content Imaging System (PerkinElmer).
Positive cells were counted using the Harmony software, and the average number of cells from three wells (6fields/well) was considered. Positive cell ratio was calculated by dividing the number of positive cells by the total
cell number. Fluorescence intensity was also calculatedusing the Harmony software as the average fluorescenceintensity from three wells (6 fields/well). The mean fluorescence intensity was calculated by dividing the fluorescence intensity by the total cell number. The settings used
for the Opperetta High-Content Imaging System areshown in Additional file 1: Table S3.

Page 4 of 12

Vascular tube formation assay

Differentiated cells at day 8 were seeded onto 6-wellplates pre-coated with a mixture of 100 μg/mL fibronectin and 400 μg/mL collagen IV. At day 10, the cells
were plated onto 24-well plates pre-coated with 300 μL
Matrigel and incubated in Human Endothelial-SFM
supplemented with 1% PDS and 40 ng/mL VEGF, for
20–24 h. The cells were then treated with Calcein-AM
for 30 min, and tube formation was observed using an
ECLIPSE Ni microscope (NIKON, Tokyo, Japan).
Acetylated LDL uptake assay

iBMELCs were incubated with 10 μg/mL Ac-LDL (Alfa
Aesar) for 5 h and then incubated for 30 min with 10 μg/
mL Hoechst 33342. After washing four times with themedium, cells were analyzed using the Operetta HighContent Imaging System (PerkinElmer). Ac-LDL-positive
cells were counted using the Operetta High-Content
Imaging System as the average number in 6 fields/well inthree wells. Positive cell ratio was calculated by dividing
the number of positive cells by the total cell number. The
settings used for the Opperetta High-Content Imaging
System are shown in Additional file 1: Table S3.
Western blot analysis

The differentiated cells were lysed in 1 × sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
sample buffer. The protein samples were separated by
SDS-PAGE, transferred to polyvinylidene fluoride membranes, which were blocked with 4% Block-Ace solution,
washed with tris-buffered saline containing 0.1% Tween
20 (TBS-T), incubated with primary antibodies (Additional file 1: Table S3) at room temperature for 1 h followed by incubation with secondary antibodies at room
temperature for 30 min (Additional file 1: Table S4). Afterwashing with TBS-T, protein bands were detected using
an Amersham Imager 600 system (GE Healthcare Life
Sciences, Chicago, IL, USA).
Paracellular permeability assay

iBMELCs were cultured on a Transwell culture insert.
On day 10, the medium was replaced with transportbuffer (Hanks’ balanced salt solution containing 10 mM
HEPES), and the cells were cultured for 20 min at 37 °C.
Transport buffer containing 300 μM Lucifer Yellow wasadded to the apical side, and transport buffer alone wasadded to the basolateral side. After 60 min of incubation at 37 °C, 100 μL of the solution was collected fromthe basolateral side. The volumes of transport buffer in
the apical and basal sides were 300 and 800 μL, respectively. Fluorescent signals of LY were measured using theYamashita et al. Fluids Barriers CNS(2020) 17:36

Synergy HTX multimode plate reader and analyzed using
the Gen 5 data analysis software (BioTek Instruments,
Inc., Winooski, VA, USA).
Transport activity study

Differentiated cells at day 8 were seeded onto 24-wellplates pre-coated with a mixture of 100 μg/mL
fibronectin and 400 μg/mL collagen IV. At day 10, theculture medium was removed, and the cells were preincubated with the transport buffer at 37 °C for 15 min.
Uptake was initiated by replacement with transportbuffer containing 10 μM rhodamine 123 or 20 μM Hoechst 33342 at 37 °C in the presence or absence of 10 μM
Ciclosporin A or 20 μM Ko 143, which are the inhibitors of P-gp or BCRP, respectively. After 60 min, thecells were washed three times with PBS and lysed using
PBS with 5% Triton X-100 solution. Fluorescence intensity in the cells was measured using the Synergy HTX
multimode plate reader and analyzed using the Gen 5
data analysis software (Bio-Tek, Winooski, VA, USA).
The total protein of the differentiated cells was measured using Pierce™ BCA Protein Assay Kit (Thermo
Fisher Scientific).
TEER measurement

TEER values were measured using Millicell-ERS(Merck Millipore, Burlington, MA, USA) or cellZscope(CellSeed, Tokyo, Japan) according to the corresponding manufacturer’s instructions. TEER values shown in
Fig. 2e were measured using cellZscope, whereas theothers were measured using Millicell-ERS. During themeasurement of the time-course of TEER values, themedium was switched to EC medium lacking FGF2,
RA, and TGF-β inhibitor at day 10, and the medium
was not changed thereafter. In this study, we definedvalues ≥ 1000 Ω × cm2 as high-TEER values.
Freezing–thawing of iBMELCs

At day 8, the cells were incubated with Accutase for
20 min at 37 °C after washing with PBS. The cells were
harvested with Human Endothelial-SFM and centrifuged at 100×g for 5 min. The cell pellets were resuspended with TC-protector (KAC, Kyoto, Japan) andfrozen at − 80 °C. After 60–90 min (Fig. 4) or 1 month(Additional file 1: Fig. S9), frozen cells were quickly
thawed with warm Human Endothelial-SFM. To
remove the cell preservation solution, cells were transferred into 50 mL tubes and centrifuged at 100×g for
5 min. The cells were then resuspended with 1 mL ofwarm EC medium and seeded onto tissue culture polystyrene plates or 0.3-cm2 Transwell-Clear permeable
inserts coated with a mixture of fibronectin and collagen IV. For subsequent operations, the procedure wasPage 5 of 12

the same as the above standard protocol on day 9 andlater.
Statistical analysis

The statistical significance of the differences between
the two groups was assessed using the unpaired Student’s t-test. The statistical significance in Fig. 2b wasassessed using one-way factorial analysis of variance(ANOVA) and Tukey’s HSD post hoc test for multiple
comparisons. The statistical significance in Figs. 2e, 4b,
Additional file 1: Figs. S4, S5 was assessed using twoway repeated measures ANOVA for multiple comparisons. SPSS statistics version 25.0 (IBM Japan, Tokyo,
Japan) was used for statistical analyses. A p-value< 0.05
(two-tailed) indicates a statistically significant
difference.

Results
The characteristics of TGF‑β inhibitor‑differentiated
iBMELCs

We differentiated iBMELCs using the protocol describedin Fig. 1a. To investigate the characteristics of iBMELCs
as vascular endothelial cells, we performed immunofluorescence with endothelial markers, the acetylated-low
density lipoprotein (Ac-LDL) uptake assay [30], and thetube formation assay. Expression of the vascular endothelial cell marker, VE-cadherin, was significantly increasedin the A-83-01-treated group compared to controls(Fig. 1b, Additional file 1: Fig. S1a). Expression of thetypical endothelial cell marker vWF was significantly
increased in the A-83-01-treated group compared withthat in controls, whereas the expression of the platelet
endothelial cell adhesion molecule-1 (PECAM1) in the
A-83-01-treated group was equivalent to that in controls(Additional file 1: Fig. S1b). Positive cell ratio of Ac-LDL
uptake was also significantly increased in the A-83-01treated group compared to controls (Fig. 1c, Additional
file 1: Fig. S1c). Although approximately 10 × 105 cells/
well were needed to form a blood vessel-like structurein the control group, less than 10 × 105 cells/well were
sufficient for this formation in the A-83-01-treatedgroup (Fig. 1d). TGF-β is known to mediate endothelial to mesenchymal transition (EndoMT) [31]. Thus,
we hypothesized that EndoMT related to the enhancement of endothelial characteristics, and confirmed theexpression level of the mesenchymal cell marker, neuralcadherin (N-cadherin). As a result, the expression levelof N-cadherin was significantly decreased when A-8301 was added, in agreement with our hypothesis (Additional file 1: Fig. S2). To assess the activation of TGF-βsignaling pathway using a standard protocol (control
group) during differentiation, we attempted to detect thePage 6 of 12

TEER (

cm 2 )

610B1
3500
3000
2500
2000
1500
1000

Control
A-83-01

648A1
3500
3000
2500
2000
1500
1000

***

500
0

c

3500
3000
2500
2000
1500
1000

***

500
0

Control
A-83-01

b

***

500
0

d

Lucifer Yellow

Pe ( 10 cm/sec)

Control
A-83-01

606A1

cm 2 )a(2020) 17:36

TEER (

Yamashita et al. Fluids Barriers CNS

500
0

ol

ntr

Co

ZO-1

6

***

3500
3000
2500
2000
1500
1000

Occludin

1

3-0

A-8

42

15

-43

SB

ox

pS

Re

Claudin-5

5

-6

3

Control

*

4
2
1
0

A-83-01

A-83-01

Control

e
610B1

3000

Control
A-83-01

cm2 )

2500
2000

1500

1000

TEER (

Control
A-83-01

2000

1500

1000

500
0

606A1

2500

***
8

9

10

11

12

13

14

15

16

17

18

19

Time (Day)

648A1

cm2 )

5000
4000

500
0

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25***

Time (Day)Control
A-83-01

TEER (

3000
2000
1000
0

8

9

10

11 12 13 14 15 16 17 18 19 20***

Time (Day)

Fig. 2 Tight junction analysis. a Effects of A-83-01 on the TEER values of BMECs derived from multiple iPSC lines. Statistical significance wascalculated using the unpaired Student’s t-test (***p < 0.001). Data are represented as the mean ± SD (610B1, n = 3; 648A1, 606A1 n = 4). b Effects
of TGF-β inhibitors (A-83-01, SB-431542, and RepSox) on the TEER values of BMECs. Statistical significance was calculated using one-way ANOVA
and Tukey test (***p < 0.001), vs. control. Data are represented as the mean ± SD (610B1, n = 3). c Lucifer Yellow permeability coefficient (Pe) in
iBMELCs. Statistical significance was calculated using the unpaired Student’s t-test (*p < 0.05). Data are represented as the mean ± SD (n = 3). d
Immunofluorescence for the tight junction markers (ZO-1, occludin, and claudin-5). Blue: DAPI. White arrows: fragile and discontinuous tightjunctions. Scale bar, 50 μm. e Time-course of the TEER values of BMECs derived from multiple iPSC lines. Statistical significance was calculated using
two-way repeated measures ANOVA (***p < 0.001). Data are represented as the mean ± SD (648A1, n = 6; 610B1, 606A1, n = 4)

phosphorylation of SMAD2 by Western blotting analysis. We confirmed that the phosphorylated SMAD2 wasdetected in iBMELCs on days 8 and 10 (control group),
whereas it was almost undetectable in A-83-01-treated
iBMELCs on day 10 (Additional file 1: Fig. S3).

The effects of TGF‑β inhibitors on tight junction formation

TEER values, an index of the intensity of tight junctions, were measured in three different iPSC line-derived
iBMELCs at day 10. In all cell lines, A-83-01 significantly increased the TEER value of iBMELCs (Fig. 2a).

Yamashita et al. Fluids Barriers CNS(2020) 17:36

Similar effects were observed when using two other
TGF-β inhibitors, SB-431542 and RepSox (Fig. 2b).
iBMELC paracellular permeability (Pe) for Lucifer Yellow
was also significantly lower in A-83-01-treated iBMELCs
than in control (Fig. 2c). To examine the effect of TGF-βinhibitor on relatively mature endothelial cells, immortalized human BMEC, hCMEC/D3, and human umbilicalvein endothelial cells (HUVEC) were treated with A-8301. However, A-83-01 had little effect on the TEER valuesof hCMEC/D3 and HUVEC (Additional file 1: Fig. S4).
The effects of TGF-β inhibitor addition on TEER values
before and after the end of differentiation were investigated. Although the TEER values in days 10–12 A-83-01treated group were significantly higher than those in the
non-treated group, TEER values in days 8–10 A-83-01treated and days 8–12 A-83-01-treated groups were notsignificantly different. The two groups treated with A-8301 from days 8 to 10 (days 8–10-treated group and days
8–12-treated group) showed higher TEER values thanthe other groups (Additional file 1: Fig. S5). Expression ofseveral tight junction markers, such as zonula occludens
protein 1 (ZO-1), occludin, and claudin-5, revealed fragile and discontinuous tight junctions in controls (whitearrows in Fig. 2d) and continuous tight junctions in the
A-83-01-treated group (Fig. 2d). In addition, the mRNA
expression level of matrix metallopeptidase 9 (MMP9),which increases BBB permeability, was significantly
decreased in the A-83-01-treated group compared tocontrols (Additional file 1: Fig. S6).
To use iBMELCs in pharmacokinetic studies, stable maintenance of high-TEER values is essential. Thus,
TEER values were measured every 6 h for approximately
2 weeks after seeding the cells on Transwell inserts.
TEER values in the human brain have not been reported,unlike those reported in the rat brain [11]. Based on thesevalues, we defined high-TEER as values ≥ 1000 Ω × cm2.
In the A-83-01-treated group, high-TEER values weremaintained for a longer period than in the control groupin all cell lines (Fig. 2e). In addition, the positive cell ratioof caspase-3 to total number of cells was similar with orwithout A-83-01 at day 10, but significantly decreased in
the A-83-01-treated group at day 19 (Additional file 1:
Fig. S7).
The effects of TGF‑β inhibitor on the expression
and activities of drug transporters

Drug transporters play an important role in the selective
transfer of substances to the brain. In the present study,
we focused on the expression of the drug transporters GLUT1, BCRP, and P-gp because it was previously
shown that these proteins are highly expressed in human
BMECs in descending order of protein expression levels[32]. Addition of A-83-01 did not change the expression

Page 7 of 12

of P-gp, but significantly increased that of GLUT1 and
BCRP compared to that in controls (Fig. 3a). To evaluate
the activities, each substrate accumulation into iBMELCs
was measured. The intercellular accumulation of BCRP
or P-gp substrates was observed in both groups and theamounts were significantly increased by each inhibitor(Fig. 3b).
Effects of TGF‑β inhibitor on cryopreservation of iBMELCs

Cell cryopreservation is required for establishing any
process as a practical in vitro model. Therefore, theeffect of freezing–thawing on iBMELC was investigated. iBMELCs were frozen on day 8, and the cells were
thawed and seeded on Transwell inserts after 60–90 min
or 1 month. TEER values on the second day after seeding were significantly decreased in frozen cells comparedwith non-frozen cells in the control group; however, thedecrease was suppressed in the A-83-01-treated group(Fig. 4a, Additional file 1: Fig. S8). Furthermore, TEER
values in the A-83-01-treated group were similar among
two groups, frozen and non-frozen cells, between thesecond and eleventh day after seeding on the inserts(Fig. 4b). Immunofluorescence with tight junction markers showed that continuous tight junctions were observedin the A-83-01-treated group irrespective of freezing–
thawing (Fig. 4c). The fluorescence intensity of P-gp and
BCRP staining hardly changed between the non-frozen
and frozen group in both the control and A-83-01-treatedgroups (Fig. 4d). The mRNA expression levels of VE-cadherin, multiple drug resistance 1 (MDR1), BCRP, GLUT1,
occludin, and ZO-1 were also similar between the frozen
and non-frozen group (Fig. 4e). Furthermore, TEER value
was also increased by A-83-01 even in iBMELCs cryopreserved for 1 month. Furthermore, the TEER value in
the A-83-01-treated group (approximately 2500 Ω × cm2)
was equivalent to that of non-frozen cells (Additional
file 1: Fig. S9).

Discussion
Previous reports have shown that a co-culture systemwith BBB-related cells, such as pericytes, astrocytes,
or neurons, enhances the barrier property of iBMELCs[15–21]. However, these systems require complex
manipulation and high costs and are easily affected by
lot-to-lot differences in co-cultured cells. Thus, thesesystems are useful models for the study of neuron toxicity, development of BBB, or diseases related to central nervous system; however, they are not suitable forpredicting drug permeability using high throughput
screening. Therefore, in this study, we focused on a differentiation method using small molecule compounds,
which exhibit less lot-to-lot variability, high stability, and low-costs compared with co-culture systems

Yamashita et al. Fluids Barriers CNS

a(2020) 17:36

Page 8 of 12

P-gp

BCRP

A-83-01

Control

GLUT1

Mean fluorescenceintensity

GLUT1

*

1.5

1.5

P-gp

**

1.5

1.0

1.0

1.0

0.5

0.5

0.5

0

0

0

b
Normalized accumulation

BCRP

Hoechst 33342
2.0
1.5

Rhodamine 123
2.0

***

***

1.5

1.0

1.0

0.5

0.5

0

Control

A-83-01

Control
A-83-01

0

***

***
VehicleInhibitor

Control

A-83-01

Fig. 3 Protein expression and activities of efflux transporters in iBMELCs. a Immunofluorescence and mean fluorescence intensity of transporters(GLUT1, BCRP, and P-gp). Blue: DAPI. Scale bar, 100 μm. Statistical significance was calculated using the unpaired Student’s t-test (*p < 0.05,**p < 0.01), control = 1. Data are represented as the mean ± SD (n = 3). b Intercellular accumulation of each substrate of efflux transporters. iBMELCs
were incubated with Hoechst 33342 (20 μM) or rhodamine 123 (10 μM) in the absence or presence of Ko143 (20 μM) or cyclosporin A (10 μM),
respectively for 60 min at 37 °C. Relative fluorescence intensity values were normalized to protein content and self-normalized to the conditions
without inhibitor. Data are represented as the mean ± SD (n = 3). Statistical significance was calculated using the unpaired Student’s t-test(***p < 0.001), vehicle of control = 1

or culture systems that use recombinant proteins.
Here, we demonstrated that TGF-β signaling pathway
inhibitor dramatically enhanced barrier integrity andendothelial characteristic of iBMELCs.

The TGF-β inhibitor we used remarkably increased
the protein expression level of vascular endothelial
cell marker VE-cadherin and the positive cell ratio of
Ac-LDL uptake cells. These results suggest that TGF-βYamashita et al. Fluids Barriers CNS

a

b

610B1
Non-frozen
Frozen**500
0Control

cm 2 )

1000

1500

Page 9 of 12

3500
3000
2500

TEER (

cm 2 )

2000

TEER (

2500(2020) 17:36

2000
1500
1000
500
0

A-83-01

A-83-01
Non-frozen
Frozen

10

13

10

19

Time (Day)c

A-83-01

A-83-01
1.5

P-gp

1.0
0.5
0

1.5

Control A-83-01

BCRP

1.0
0.5
0

Control A-83-01
Non-frozen
Frozene
Relative mRNA expressions

Mean fluorescence intensitydOccludinNon-frozen
FrozenFrozenControl

ZO-1

Control

Non-frozen

1.5

MDR1

2.0
1.5

1.0

BCRP

*

1.5

GLUT1

1.0

1.0
0.5
0

1.5

0.5

0.5
0

VE-cadherin

1.50Occludin

1.5

1.0

1.0

1.0

0.5

0.5

0.5

0

0

0

ZO-1

Non-frozen
FrozenFig. 4 The effect of TGF-β inhibitor on freezing–thawing of BMECs. a Freezing–thawing was performed at day 8 and the TEER values were measured
at day 10. Statistical significance was calculated using the unpaired Student’s t-test (**p < 0.01). Data are represented as the mean ± SD (n = 3). b
Temporal changes in TEER values were observed in the BMECs with TGF-β inhibitor. Statistical significance was calculated using two-way repeatedmeasures ANOVA. Data are represented as the mean ± SD (n = 3). c Immunofluorescence for the tight junction markers (ZO-1 and occludin). Blue:
DAPI. White arrows: discontinuous tight junctions. Scale bar, 50 μm. d Mean fluorescence intensities of P-gp and BCRP staining were calculated.
Statistical significance was calculated using the unpaired Student’s t-test, non-frozen of control = 1. Data are represented as the mean ± SD (n = 3).
e Relative gene expression levels of MDR1, BCRP, GLUT1, VE-cadherin, occludin, and ZO-1. Statistical significance was calculated using the unpaired
Student’s t-test (*p < 0.05), non-frozen = 1. Data are represented as the mean ± SD (n = 3)

Yamashita et al. Fluids Barriers CNS(2020) 17:36

inhibition promotes differentiation of human iPSCs
into BMELCs and increases proportion of endothelial
cells among differentiated cells. These positive effects
are reflected in our results by an enhancement in theability of these cells to form blood vessel-like structures. In addition, iBMELCs showed significantly
high-TEER values, low-permeability coefficient of Lucifer Yellow, and formed tighter continuous junctions
when adding the TGF-β inhibitor compared to controls. TGF-β is known to mediate EndoMT [31], which
increases BBB permeability [33]. Therefore, we hypothesized the enhancement of TEER occurred because of
EndoMT suppression. However, TGF-β inhibitor hardly
affected relatively mature endothelial cells, such as
HUVECs and hCMEC/D3, in which EndoMT was previously observed [31, 34]. On the other hand, TGF-βinhibitor decreased the protein expression level of themesenchymal cell marker, N-cadherin. Additionally, the
TEER value was slightly increased by TGF-β inhibitortreatment after the end of differentiation. Furthermore,
iBMELCs treated with TGF-β inhibitor before the endof differentiation showed high-TEER values notably.
Taken together, the increase of TEER might be caused
not only by EndoMT suppression, but specifically by
the promotive effect of TGF-β signaling pathway inhibition on iBMELCs during endothelial differentiation.
Further, according to a previous study, retinoic acidsupplementation also enhances the protein expression
level of VE-cadherin, which is a typical adherence junction maker and has barrier properties [16]. Considering that VE-cadherin is a positive regulator of tightjunctions [35], it was suggested that an increase in
the expression of VE-cadherin by supplementation of
TGF-β inhibitor correlated with a significant increase
in TEER value. Alternatively, it was also reported that
TGF-β upregulates MMP9, which is a matrix metalloprotease that disrupts tight junctions, and increases
the permeability of drugs in BBB [27]. In our study, therelative gene expression level of MMP9 was markedly
decreased by the addition of TGF-β inhibitor. These
results imply that suppression of MMP9 by the TGF-βinhibitor might contribute to robust tight junction formation. The fact that iBMELCs, treated with TGF-βinhibitor, maintained high-TEER value longer than thecontrol group may also be involved in the suppressionof MMP9. The effect of TGF-β inhibitor to maintainhigh-TEER value might be related to a TGF-β inhibitor
apoptosis suppression, in accordance with the results of
caspase-3 expression levels. From the results of efflux
transport analysis, the activities could be evaluated inboth control and A-83-01-treated groups. Although
TGF-β inhibitor hardly affected activities of several
drug transporters, it slightly increased the protein

Page 10 of 12

expression levels of BCRP and GLUT1. These results
indicate that the characteristics of cells differentiatedusing TGF-β inhibitor became close to those of BMECs.
Our results suggested that the TGF-β inhibitor is a critical modulator of BMEC properties during differentiation
to direct human iPSCs into BMELCs; it is also useful forthe establishment of in vitro BBB models.
Cell cryopreservation is required for the application of a process to an in vitro model. The TEER values
were significantly decreased in frozen cells in the control
group. Wilson et al. have reported that cryopreservation impaired the barrier function of iBMELC [24]. Our
results were consistent with this report. However, byadding the TGF-β inhibitor, we succeeded in maintaining
robust tight junctions. Even when iBMELCs were cryopreserved for 1 month, high-TEER values could be maintained by the TGF-β inhibitor. Wilson et al. also reported
that Y-27632 suppressed the decrease of TEER [24]. Thus,
apoptosis inhibition may be one of the mechanisms thatlead to strengthening of tight junctions. However, in thepresent study, the positive cell ratio of caspase-3 to total
number of cells was not changed under non-frozen conditions, independently of whether we used or not A-8301 at day 10. Thus, this phenomenon may be due to othereffects of TGF-β inhibitors.
We confirmed the effect of TGF-β inhibition on
iBMELCs differentiated by a specific protocol. However,there are several protocols, including Quian’s protocol[19] and Hollmann’s protocol [20], for the differentiation of iBMELCs. We speculate that TGF-β inhibitor also
promotes the differentiation of iBMELCs in Hollmann’s
protocol because the cells differentiated by Hollmann’s
protocol are relatively similar to those differentiated by
Lippmann’s protocol. In contrast, the characteristics
of iBMELCs differentiated by Quian’s protocol are dissimilar to those differentiated by Lippmann’s protocol,and the effect of TGF-β inhibition in their protocol is
not predictable. Therefore, future studies are warranted
to address whether TGF-β inhibition can be applied toother protocols.

Conclusions
In this study, we succeeded in significantly enhancing
the function and endothelial characteristics of iBMELCs
by adding a small molecular compound, a TGF-β inhibitor. Moreover, the iBMELCs could maintain high barrier
function even after freezing–thawing. Therefore, we suggest that using a TGF-β inhibitor can help in developing
high functional in vitro BBB models from iPSCs. Furthermore, iBMELCs differentiated using a TGF-β inhibitor can be potentially used as tools in pharmaceuticalresearch in the future.

Yamashita et al. Fluids Barriers CNS(2020) 17:36

Supplementary information
Supplementary information accompanies this paper at https​://doi.
org/10.1186/s1298​7-020-00197​-1.
Additional file 1: Fig. S1. Endothelial characteristics of BMECs derived
from multiple iPSC lines. Fig. S2. Protein expression levels of N-cadherin.
Fig. S3. Phosphorylation levels of SMAD2. Fig. S4. Effect of TGF-β inhibitor
on human immortalized BMEC (hCMEC/D3) and umbilical vein endothelial cells (HUVEC). Fig. S5. Effect of TGF-β inhibitor addition on TEER
values before and after the end of differentiation. Fig. S6. Relative gene
expression of MMP9 in A-83-01-treated cells. Fig. S7. Expression levels of
caspase-3. Fig. S8. The effect of TGF-β inhibitor on freezing–thawing of
BMECs derived from another iPSC line. Fig. S9. Effect of TGF-β inhibitoron the cryopreservation of iBMELCs. Table S1. PCR primer sequences.
Table S2. Antibodies for immunofluorescence analysis. Table S3. Setting
for the Opperetta High-Content Imaging System and minimum intensityof positive cells. Table S4. Antibodies for Western blotting analysis. Additional Experimental Procedures.
Abbreviations
Ac-LDL: Acetylated-low density lipoprotein; RA: All-trans retinoic acid; FGF2:
Basic fibroblast growth factor; BSA: Bovine serum albumin; BCRP: Breast cancer
resistant protein; DAPI: 4′,6-diamidino-2-phenylindole; EndoMT: Endothelial
to mesenchymal transition; GLUT1: Glucose transporter 1; Matrigel: Growth
factor reduced matrigel; HUVEC: Human umbilical vein endothelial cells; HPRT:
Hypoxanthine phosphoribosyltransferase; iPSC: Induced pluripotent stem
cells; iBMELCs: iPSC-derived BMECs; MMP9: Matrix metallopeptidase 9; MDR1:
Multiple drug resistance 1; N-cadherin: Neural cadherin; PECAM1: Platelet
endothelial cell adhesion molecule-1; P-gp: P-glycoprotein; PBS: Phosphatebuffered saline; PDS: Platelet-poor plasma derived bovine serum; SDS-PAGE:
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TEER: Transendothelial electrical resistance; TGF-β: Transforming growth factor beta; UM:
Unconditioned medium; VE-cadherin: Vascular endothelial cadherin; vWF: Von
Willebrand factor; ZO-1: Zonula occludens protein 1.
Acknowledgements
We acknowledge the assistance of the Research Equipment Sharing Center at
the Nagoya City University.
Authors’ contributions
MY, TH, and TM contributed to the conception and design of the work. MY
and HA collected and assembled data. MY and HA also performed data
analysis and interpretation. All authors contributed to the writing of the final
manuscript. All authors read and approved the final manuscript.
Funding
This work was supported, in part, by AMED (Grant Number JP19be0304322)
and JSPS KAKENHI (Grant Number JP17K08422).
Availability of data and materials
The datasets used and/or analyzed in the current study are available from thecorresponding author upon reasonable request.
Ethics approval and consent to participate
No applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Received: 7 February 2020 Accepted: 15 May 2020

Page 11 of 12

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