(2020) 17:25
Aoki et al. Fluids Barriers CNS
https://doi.org/10.1186/s12987-020-00186-4

Fluids and Barriers of the CNS
Open Access

RESEARCH

Laminin 221 fragment is suitable
for the differentiation of human induced
pluripotent stem cells into brain microvascularendothelial‑like cells with robust barrier
integrity
Hiromasa Aoki†, Misaki Yamashita†, Tadahiro Hashita, Takahiro Iwao and Tamihide Matsunaga*

Abstract
Background: In vitro blood–brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived
brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drugcandidates. For the differentiation of iBMELCs, Matrigel, which is a gelatinous protein mixture, is often used as a coating substrate. However, the components of Matrigel can vary among lots, as it is obtained from mouse sarcoma cellswith the use of special technics and also contains various basement membranes. Therefore, fully defined substrates as
substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots.
Methods: iBMELCs were differentiated from human iPS cells on several matrices. The barrier integrity of iBMELCs
was evaluated based on transendothelial electrical resistance (TEER) values and permeability of fluorescein isothiocyanate-dextran 4 kDa (FD4) and Lucifer yellow (LY). Characterization of iBMELCs was conducted by RT-qPCR and immunofluorescence analysis. Functions of efflux transporters were defined by intracellular accumulation of the substrates
in the wells of multiwell plates.
Results: iBMELCs differentiated on laminin 221 fragment (LN221F-iBMELCs) had higher TEER values and lower permeability of LY and FD4 as compared with iBMELCs differentiated on Matrigel (Matrigel-iBMELCs). Besides, the geneand protein expression levels of brain microvascular endothelial cells (BMEC)-related markers were similar between
LN221F-iBMELCs and Matrigel-iBMELCs. Moreover, both Matrigel- and LN221F-iBMELCs had functions of P-glycoprotein and breast cancer resistance protein, which are essential efflux transporters for barrier functions of the BBB.
Conclusion: The fully defined substrate LN221F presents as an optimal coating matrix for differentiation of iBMELCs.
The LN221F-iBMELCs had more robust barrier function for a longer period than Matrigel-iBMELCs with characteristics
of BMECs. This finding will contribute the establishment of an iBMELC supply system for pharmacokinetic and pathological models of the BBB.

*Correspondence: tmatsu@phar.nagoya‑cu.ac.jp†
Hiromasa Aoki and Misaki Yamashita contributed equally to this work
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.

Aoki et al. Fluids Barriers CNS(2020) 17:25

Page 2 of 11

Keywords: Human induced pluripotent stem cell, Brain microvascular endothelial cell, Laminin 221, Differentiation,
Blood–brain barrier

Background
Brain microvascular endothelial cells (BMECs), which
are one of the constituents of the blood–brain barrier(BBB), regulate the transition of substances between thevasculature and brain parenchyma via strong intercellularadhesions and expression of multidrug efflux transporters [1]. In drug discovery, the robustness of the BBB often
abrogates drug development for the treatment of central
nervous system disorders because many candidates cannot reach the brain parenchyma. To predict drug permeability of the BBB, in vivo murine and monkey models areoften used [2]. However, drug permeability of the BBB ofthese models differs from that of humans [3] and the useof in vivo models is both expensive and time-consuming.
Therefore, it is difficult to predict the permeability of all
drug candidates using in vivo models. Hence, it is necessary to establish models that can easily and precisely
predict drug permeability of the BBB for pre-screening,
especially for drug discovery.
Various in vitro BBB models, including immortalized human BMECs and rodent-derived BMECs, havebeen reported [4]. However, immortalized BMECs haveweak barrier functions, and thus, are not suitable topredict drug permeability [5]. Besides, in case of usingrodent-derived BMECs, interspecies differences between
rodent and human BMECs are problematic. To addressthese problems, human induced pluripotent stem (iPS)
cell-derived brain microvascular endothelial-like cells(iBMELCs) were recently developed [6, 7]. Transendothelial electrical resistance (TEER), which is an index of therobustness of tight junctions, of iBMELCs is comparable
to that of the BBB in vivo [7]. Therefore, it is expectedthat drug permeability of the human BBB can be predicted with the use of iBMELCs.
It is necessary to establish a stable supply system oflarge numbers of iBMELCs as drug screening models.
Matrigel, which contains various basement membranecomponents such as laminin, collagen, and entactin, isoften used for the differentiation of iBMELCs [6–10].
However, the components of Matrigel can vary among
various batches because it is extracted from mouse sarcoma cells using special techniques. In addition, thequality of the Matrigel influences the barrier integrityof iBMELCs [11]. Moreover, Matrigel coating requires
complex manipulations because Matrigel solidifies withan increase in temperature. Therefore, it is difficult toensure the reproducibility of differentiation with theuse of Matrigel. Therefore, Matrigel is not suitable forreproducible production of large numbers of iBMELCs
as a fully defined substrate should be used for differentiation. Here, we show that coating of defined substrate
laminin 221 fragment (LN221F), which is a recombinantfragment with full integrin-binding activity, enhanced
the barrier integrity of iBMELCs. Moreover, gene andprotein expression levels of endothelial cell markers,
efflux transporters, tight junction-related proteins, andother BMEC-related markers in iBMELCs differentiatedon LN221F (LN221F-iBMELCs) were comparable tothose of iBMELCs differentiated on Matrigel (MatrigeliBMELCs). In addition, LN221F-iBMELCs had functionsof efflux transporters, which are essential for the barrier
functions of the BBB. Considering that coating a culture
plate with LN221F requires only simple manipulation,
the findings of this study are expected to contribute to
the establishment of a stable supply system of BBB models with robust barrier functions for a prolonged period.

Materials and methods
Materials

The human iPS cell lines 610B1 and 648A1 derived
from human umbilical cord blood and human peripheral blood, respectively, were purchased from RIKEN
BioResource Center (Tsukuba, Japan). The immortalized human BMEC cell line hCMEC/D3 was purchasedfrom Merck Millipore (Burlington, MA, USA). The primary human BMECs and Complete Classic Medium
with Serum and CultureBoost™ were purchased from
Cell Systems Corporation (Kirkland, WA, USA). The primary BMECs derived from a monkey (Macaca irus) andthe culture medium for BMECs were purchased fromPharmaCo-Cell Co., Ltd. (Nagasaki, Japan). Fibronectin (FBN), ­StemSure® hPSC medium, l-glutamine, 1:1
mixture of Dulbecco’s modified Eagle’s medium andHam’s nutrient mixture F-12 (DMEM/F12), hydrocortisone, l-ascorbic acid phosphate magnesium saltn-hydrate, and MEM nonessential amino acids werepurchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). Endothelial Cell Basal Medium 2 waspurchased from Lonza (Basel, Switzerland). Porcine
skin gelatin, 2-mercaptoethanol, fetal bovine serum,and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) solution (1 M, pH 7.0–7.6) were purchasedfrom Sigma-Aldrich Corporation (St. Louis, MO, USA).
LN221F, laminin 411 fragment (LN411F), and laminin
511 fragment (LN511F) (imatrix 221, 411, and 511,
respectively) were purchased from Nippi Incorporated

Aoki et al. Fluids Barriers CNS(2020) 17:25(Tokyo, Japan). Gibco™ KnockOut™ Serum Replacement(KSR), Gibco™ Hank’s Balanced Salt Solution (HBSS)
with Calcium and Magnesium, No Phenol Red, Human
Endothelial-Serum-Free Medium (HE-SFM), Gibco™
Chemically Defined Lipid Concentrate, and VitronectinN (VTN-N) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fibroblast growth factor-2(FGF2) was purchased from PeproTech, Inc. (Rocky Hill,
NJ, USA). Penicillin–streptomycin solution was purchased from Biological Industries USA, Inc. (Cromwell,
CT, USA). An Agencourt RNAdvance Tissue Total RNA
Purification Kit was purchased from Beckman Coulter,
Inc. (Brea, CA, USA). ReverTra A­ ce® qPCR RT MasterMix was purchased from Toyobo Co., Ltd. (Osaka, Japan).
KAPA ­SYBR® FAST qPCR Master Mix (2×) was purchased from Nippon Genetics Co., Ltd. (Tokyo, Japan).
­Corning® ­Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, ­Falcon® Permeable Support fora 24-well plate with a 0.4-µm Transparent PET Membrane, and a F­ alcon® 24-well TC-treated Cell Polystyrene
Permeable Support Companion Plate and a lid were purchased from Corning Incorporated (Corning, NY, USA).
Platelet-poor plasma-derived bovine serum (PDS) and1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine
perchlorate acetylated low-density lipoprotein (Dil-AcLDL) were purchased from Alfa Aesar (Ward Hill, MA,
USA). All-trans retinoic acid (RA) was purchased from
Tocris Bioscience (Bristol, UK). Accutase™ was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Cell
Carrier-96 Black, Optically Clear Bottom microplates
were purchased from PerkinElmer, Inc. (Waltham, MA,
USA). Collagen type IV was purchased from Nitta Gelatin Inc. (Osaka, Japan). Total RNA from human primary BMECs (hBMECs) was purchased from ScienCell
Research Laboratories, Inc. (Carlsbad, CA, USA).
Coating before differentiation

Matrigel GFR was diluted (1:30) in iPS cell medium on
ice. The diluted solution was transferred into the wells of
a 6-well plate with a cold tip and incubated at 37 °C for
1 h. FBN, VTN-N, LN221F, LN411F, and LN511F, whichwere diluted (1 μg/cm2) with D-phosphate-bufferedsaline (D-PBS) (−), were transferred into the wells of sixwell plates and incubated at 37 °C for 1 h.
Cell culture

Human iPS cells were maintained in iPS cell medium(DMEM/F12 containing 20% KSR, 2 mM l-glutamine,
1 × MEM nonessential amino acids, and 0.1 mM 2-mercaptoethanol) supplemented with 5 ng/mL of FGF2. The
human iPS cells were cultured on mitomycin C-treatedmouse embryonic fibroblasts with daily mediumchanges. The primary human BMECs were cultured inPage 3 of 11

Complete Classic Medium with Serum supplemented
with CultureBoost. The cells were dissociated with TrypLE select and plated on dishes coated with Attachment
Factor at a 1:3 split ratio. Before conducting the TEER
measurement assay, the cells (passage 6) were seeded
onto transwell culture inserts coated with Matrigel,
FBN, VTN-N, LN221F, LN411F, or LN511F at a densityof 5.0 × 104 cells/well and cultured in HE-SFM-based
medium (HE-SFM containing 1% PDS and 1 × penicillin–streptomycin solution) supplemented with 20 ng/mLof FGF2. The medium was changed every alternate day.
For the TEER measurement assay of the primary monkey
BMECs, the cryopreserved cells (passage 1) were thawed
and seeded onto transwell culture inserts coated with
Matrigel, FBN, VTN-N, LN221F, LN411F, or LN511F at
a density of 2.0 × 104 cells/well and cultured in the culture medium for BMECs. The medium was changed
every alternate day. The hCMEC/D3 cells were cultured
in hCMEC/D3 medium: Endothelial Cell Basal Medium
2 supplemented with 5% fetal bovine serum, 5 μg/mL
l-ascorbic acid phosphate magnesium salt n-hydrate,
1% chemically defined lipid concentrate, 10 μM HEPES
solution, 1 × penicillin–streptomycin solution, 1.4 μM
hydrocortisone, and 1 ng/mL FGF2. The cells were dissociated with TrypLE select and plated at a 1:5 split
ratio. Before the TEER measurement assay, the cells
were seeded onto transwell culture inserts coated with
Matrigel, FBN, VTN-N, LN221F, LN411F, or LN511F at
a density of 5.0 × 104 cells/well and cultured in HE-SFMbased medium supplemented with 20 ng/mL of FGF2.
The medium was changed every alternate day.
Differentiation of iPS cells into iBMELCs

iBMELCs were differentiated from human iPS cells usinga modified protocol of a previous study [7]. Briefly, iPS
cells were seeded into the wells of a 6-well plate coatedwith Matrigel GFR, FBN, VTN-N, LN221F, LN411F, or
LN511F and cultured with StemSure hPSC medium supplemented with 35 ng/mL of FGF2 for 3 or 4 days. Whencell counts were needed, we prepared human iPS cells in
two culture dishes, one each for differentiation and cellcount. On day 0, after reaching 60–70% confluence, theculture medium was replaced with DMEM/F12-based
medium (iPS cell medium) and the cells were cultured foran additional 6 days. The medium was changed every day.
On day 6, the medium was switched to HE-SFM-based
medium supplemented with 10 μM RA and 20 ng/mL of
FGF2. On day 8, the differentiated cells were washed with
D-PBS (–) and dissociated with the use of Accutase celldetachment solution for 20 min at 37 °C. The cells were
centrifuged at 100×g, resuspended, seeded on a transwell
culture insert or into the wells of a multiwell plate coatedwith a mixture of FBN (100 μg/mL) and collagen type IV

Aoki et al. Fluids Barriers CNS(2020) 17:25(400 μg/mL), LN221F (100 μg/mL), or a mixture of FBN,
collagen type IV, and LN221F at a density of 3.0 × 105
cells/well, and cultured in HE-SFM-based medium supplemented with 10 μM RA and 20 ng/mL of FGF2. On
day 9, the medium was switched to HE-SFM-based
medium without 10 μM RA and 20 ng/mL of FGF2.
Cell viability assay

CCK-8 assay was conducted in accordance with the manufacturer’s instructions. The cells were incubated with
CCK-8 solution for 60 min.
TEER values were measured with the use of a M­ illicell®
ERS-2 V/Ωm (Merck Millipore) in accordance with the
manufacturer’s instructions.
TEER value measurement

Paracellular permeability assay

The iBMELCs were cultured on a transwell culture insert.
On day 10, the medium was replaced with transportbuffer (HBSS containing 10 mM HEPES solution) and thecells were cultured for 20 min at 37 °C. Transport buffer
containing 1 mg/mL of FD4 or 300 μM LY was added tothe apical side and transport buffer alone was added tothe basolateral side. After 60 min of incubation at 37 °C,
100 μL of the solution was collected from the basolateralside. The volumes of transport buffer in the apical andbasal sides were 300 and 800 μL, respectively. Fluorescentsignals of FD4 or LY were measured with a Synergy HTX
multimode plate reader and analyzed using Gen 5 data
analysis software (BioTek Instruments, Inc., Winooski,
VA, USA).

Page 4 of 11

RNA extraction and RT‑qPCR

Total RNA was purified using an Agencourt RNAdvance
Tissue Total RNA Purification Kit and reverse transcribed using ReverTra Ace qPCR RT Master Mix. Relative mRNA expression levels were measured using the
KAPA ­SYBR® FAST qPCR Kit with a ­LightCycler® 96
System (F. Hoffmann-La Roche, Ltd., Basel, Switzerland)and normalized to those of hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1). The RT-qPCR primers
are listed in Table 1.
Immunofluorescence analysis

For staining of cadherin 5 (CDH5, also known as VE-cadherin), claudin 5, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP), cells in the wells of Cellcarrier-96 microplates were washed thrice with D-PBS(−) containing 0.1% bovine serum albumin (BSA), fixedwith 4% paraformaldehyde for 15 min, washed thricewith D-PBS (–) containing 0.1% BSA, and permeabilizedwith D-PBS (−) containing 0.1% Triton X100 for 5 min.
After washing thrice with D-PBS (−) containing 0.1%
BSA, the cells were incubated overnight at 4 °C with a
primary antibody diluted in D-PBS (−) containing 0.1%
BSA. Afterward, the cells were washed thrice with D-PBS(−) containing 0.1% BSA and incubated with a secondary
antibody for 60 min at room temperature. After washing thrice with D-PBS (−) containing 0.1% BSA, the cells
were incubated with 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The cells were then treatedwith 4% paraformaldehyde for 5 min and washed thrice
times with D-PBS (−).
For staining of zonula occludens-1 (ZO-1) and occludin, cells in the wells of Cell carrier-96 microplates wereTable 1 RT-qPCR primer sequences
Genes

Forward primer sequence (5′ → 3′)

Reverse primer sequence (5′ → 3′)

CDH5

GAT​T TG​GAA​CCA​GAT​GCA​CA

ACT​TGG​CAT​TCT​TGC​GAC​TC

MDR1

CCC​ATC​ATT​GCA​ATA​GCA​GG

TGT​TCA​AAC​T TC​TGC​TCC​TGA​

BCRP

AGA​TGG​GTT​TCC​AAG​CGT​TCAT​CCA​GTC​CCA​GTA​CGA​C TG​TGACA​GLUT1GAA​GAG​AGT​CGG​CAG​ATG​ATG​

GGA​GTA​ATA​GAA​GAC​AGC​GTT​GAT​G

Occludin

TCC​AAT​GGC​AAA​GTG​AAT​GAGCA​GGT​GCT​C TT​T TT​GAA​GG

ZO-1

CGA​GGG​ATA​GAA​GTG​CAA​GTAGA​TAT​TCT​TCA​T TT​T TC​CGG​GATTT​
CGT​AGA​GCA​GCG​TCA​TCA​CALAT1AAT​GGG​TCC​C TG​T TC​ACA​TC

HPRT1

CTT​TGC​T TT​CCT​TGG​TCA​GG

TCA​AGG​GCA​TAT​CCT​ACA​ACA​

PECAM1AGT​CGG​ACA​GTG​GGA​CGT​AT

ATG​ACC​TCA​AAC​TGG​GCA​TC

vWF

CGG​C TT​GCA​CCA​T TC​AGC​TA

TGC​AGA​AGT​GAG​TAT​CAC​AGC​CAT​CKDR

CTG​CAA​ATT​TGG​AAA​CCT​GTC​GAG​C TC​TGG​C TA​C TG​GTG​ATG​

TBXT

ACC​CAG​T TC​ATA​GCG​GTG​ACCAA​T TG​TCA​TGG​GAT​TGC​AG

KDR

CTG​CAA​ATT​TGG​AAA​CCT​GTC​GAG​C TC​TGG​C TA​C TG​GTG​ATG​

OCT-4

AGC​GAA​CCA​GTA​TCG​AGA​ACTTA​CAG​AAC​CAC​ACT​CGG​AC

NANOG

CTG​CAG​AGA​AGA​GTG​TCG​CAACC​AGG​TCT​TCA​CCT​GTT​TG

Aoki et al. Fluids Barriers CNS(2020) 17:25

Page 5 of 11

fixed with 4% paraformaldehyde for 15 min, washedthrice with D-PBS (−) containing 10 mM glycine, permeabilized in D-PBS (−) containing 0.1% Triton X100 for
25 min, and stored in D-PBS (−) containing 0.1% ­NaN3
at 4 °C. The stored cells were blocked with 5% donkey
serum for 20 min, incubated with a primary antibody for
120 min, and then incubated with a secondary antibody
and 1 µg/mL DAPI for 60 min.
The stained samples were visualized using an Operetta High-Content Imaging System (PerkinElmer, Inc.,
Waltham, MA, USA). The antibodies used in this study
are shown in Table 2. The fluorescence intensities werealso calculated using ­Harmony® high-content analysis
software (PerkinElmer, Inc., Waltham, MA, USA). The
mean fluorescence intensities were calculated by dividing
the fluorescence intensity by the total cell number.
Dil‑Ac‑LDL uptake assay

Differentiated cells were first incubated with 10 μg/mL
Dil-Ac-LDL for 5 h, then incubated with 10 µg/mL Hoechst 33342 at room temperature for 10 min, and washedfour times with medium. The samples were visualizedusing an Operetta High-Content Imaging System. The
fluorescence intensities of Dil were also calculated using­Harmony® high-content analysis software. The mean
fluorescence intensities were calculated by dividing theintensity of Dil by the total cell number.
Functional analysis of efflux transporters

The culture medium was removed and the cells in thewells of a 96-well plate were pre-incubated with transport buffer (HBSS containing 10 mM HEPES buffer) at
37 °C for 15 min. Intracellular accumulation was initiatedby the replacement of transport buffer containing 10 μM
rhodamine 123 or 10 μM Hoechst 33342 at 37 °C in thepresence or absence of 10 μM cyclosporine A (CsA) or
20 μM Ko 143, which are inhibitors of P-gp and BCRP,
respectively. After 60 min, the cells were washed thricewith D-PBS (−) and lysed using D-PBS (−) containing

5% Triton X-100. The fluorescence intensities were measured using a Synergy HTX multimode plate reader andanalyzed with Gen 5 data analysis software.
Western blot analysis

The differentiated cells on day 10 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 (Table 3) at room temperature for 2 h,and then incubated with secondary antibodies at room
temperature for 30 min (Table 3). After washing with
TBS-T, Protein bands were detected using an Amersham
Imager 600 system (GE Healthcare Life Sciences, Chicago, IL, USA). The protein bands were quantified using
Amersham Imager 600 analysis software. Backgroundsignals were removed with the Rolling Ball algorithm(Amersham Imager 600 analysis software).
Statistical analysis

For all experiments, ‘‘n’’ represents the number of biological replicates (three or six wells were analyzed).
Each experiment was repeated at least twice. Data are

Table 3 Antibodies for Western blotting analysis
Targets

Manufacturer

CatalognumberSpecies Dilution

OccludinFisher Scientific 71-1500

Rabbit

1:500

ZO-1

Fisher Scientific 33-9100

Mouse

1:500

Claudin-5

Fisher Scientific 35-2500

Mouse

1:500

GAPDH (HRP)

Wako

35-2500

Mouse

1:1000Anti-Mouse
(HRP)

Abcam

ab6789Goat

1:5000Anti-Rabbit
(HRP)

Abcam

ab6721Goat

1:5000

Table 2 Antibodies for immunofluorescence analysis
Targets

Manufacturer

Catalog number

Species

Dilution

CDH5

Santa Cruz

sc-9989

Mouse

1:25

P-gp

Abcam

ab10333

Mouse

1:25

BCRP

Abcam

ab3380

Mouse

1:50

OccludinFisher Scientific71-1500

Rabbit1:50

ZO-1

Fisher Scientific33-9100

Mouse

1:100

Claudin-5

Fisher Scientific35-2500

Mouse

1:50

Anti-Rabbit (Alexa Flour 488)

Fisher Scientific

A-21206

Donkey1:200

Anti-Mouse (Alexa Fluor 568)

Fisher Scientific

A-11004

Goat

1:200

Aoki et al. Fluids Barriers CNS(2020) 17:25

Page 6 of 11

presented as the mean ± standard deviation (SD). The
two-tailed Student’s t-test was used for comparisons
between two groups. Two-way repeated measures
analysis of variance was used for comparisons of TEER
values measured after a prolonged period. One-way
analysis of variance followed by Tukey’s honestly significant difference test or the Games–Howell test wasused for comparisons of all six groups. Levene’s test
was used to assess the equality of variances. All statistical analyses were performed using IBM SPSS Statistics
for Windows, version 25.0 (IBM Corporation, Armonk,
NY, USA). A probability (p) value of < 0.05 was considered statistically significant.

a

Day 0

iPS cells

Results
LN221F coating enhanced the barrier integrity of iBMELCs

We attempted to identify basement membranes thatwere more suitable to promote the differentiation of
iBMELCs than Matrigel using previously reported protocols (Fig. 1a). As shown in Fig. 1b, LN221F-iBMELCs had
higher TEER values than the Matrigel-iBMELCs. In contrast, the TEER values of differentiated iBMELCs, whichwere cultured on Matrigel until day 8, were not influenced by coating LN221F from days 8 to 10 (Additional
file 1: Figure S1A). To examine the effect of LN221F onhuman mature BMECs, we measured the TEER valuesof primary BMECs derived from human and monkey,
and hCMEC/D3 (immortalized cell line) cells on several

Day 6

Day 9

Day 8

HE-SFM based medium+Retinoic acid
Replating+FGF2

DMEM/F12 based medium

HE-SFM based medium

iPS cell-derived
BMELCson Fibronectin/
Collagen typeon Matrigel
modification

c
2500

1500

2

*

1.5

cm )

**

2.0

1.0

*

0.5

1000

**

41
1F
LN
51
1F

1

2

3

4

5

6

7

8

9

10

Day after seeding

LN

22
1F

-N

LN

N

N

VT

FB

ig

: LN221F

2000

0

at
r

: Matrigel

500

0

M

610B1 iBMELCs

3000

TEER (

2.5

elRelative TEER valueb648A1 iBMELCs

TEER (

2

cm )

3500

: Matrigel

3000: LN221F

2500
2000
1500
1000

**

500
0

1

2

3

4

5

6

7

8

9

10

Day after seeding

Fig. 1 Evaluation of TEER values in LN221F- and Matrigel-iBMELCs. a Scheme for the differentiation of iBMELCs. b Relative TEER values of
610B1-derived iBMELCs on day 10 on Matrigel, FBN, VTN-N, LN221F, LN411F, or LN511F. The relative TEER value of iBMELCs on Matrigel was definedas 1. Data are presented as the mean ± SD (n = 3; *p < 0.05, **p < 0.01; Tukey’s honestly significant difference test, Matrigel group vs. others). c
Measurement of TEER values of LN221F- and Matrigel-iBMELCs derived from 610B1 and 648A1 over a long period (from day 9 to 18). Data arepresented as the mean ± SD (n = 6; **p < 0.01; two-way repeated measures analysis of variance)

(2020) 17:25

Page 7 of 11

648A1

0.2

0

0
22
1F

0.4

0.2

LN

M

at
r

ig

1F
22
LN

ig
at
r
M

0.6

0.4

el

0

0.8

0.6

ig

0

22

0.1

el

0.1

LN

0.2

1F

0.2

1.0

0.8

at
r

0.3

0.3

**

1.4

M

0.4

648A1

1.2

1.0

el

0.4

**

1.2

Papp (10-6cm/sec)

0.5

610B1

**

0.6
0.5

el

Papp (10-6cm/sec)

0.6

LY permeability assay

b

rig

**

0.7

To assess whether LN221F-iBMELCs retain the characteristics of BMECs, gene expression levels in LN221FiBMELCs, Matrigel-BMECs, and hBMECs were analyzed
using RT-qPCR. As shown in Fig. 3a, the expression levels of CDH5 (a typical endothelial cell marker), MDR-1(encoding P-gp) and BCRP (efflux transporters), ZO-1
and occludin (tight junction-related proteins), and glucose transporter 1 (GLUT1) and l-type amino acidtransporter 1 (LAT1) (BMEC-related transporters) weresimilar in LN221F-iBMELCs and Matrigel-iBMELCs.
However, CDH5 expression levels in both LN221F- andMatrigel-iBMELCs were lower than that in hBMECs.

at

610B1

LN221F‑iBMELCs expressed BMEC‑related markers

22
1F

FD4 permeability assaya

iPS cells compared with Matrigel (Additional file 1: Figure S2A, B). The LN221F-iBMELCs derived from two
iPS cell lines, 610B1 and 648A1, maintained high TEER
values for a longer period than the Matrigel-iBMELCs(Fig. 1c). An important characteristic of BMECs is strict
control of the permeability of substances via the paracellular route. Therefore, paracellular permeability of both
LN221F- and Matrigel-iBMELCs was investigated. The
results showed that the permeabilities of FD4 and LY
were significantly lower in LN221F-iBMELCs derived
from the two iPS cell lines as compared with that of the
Matrigel-iBMELCs (Fig. 2a, b).

LN

coating substrates including LN221F. The TEER valueof human BMECs on LN221F was lower than those ofhuman BMECs on LN511F (Additional file 1: Figure S1B).
The TEER value of monkey BMECs on LN221F was lower
than those of monkey BMECs on Matrigel, FBN, VTNN, and LN511F (Additional file 1: Figure S1C). The TEER
values of both human and monkey BMECs on LN511F
were the highest among the six coating substrates. The
TEER value of hCMEC/D3 cells on LN221F was almost
equivalent to those of hCMEC/D3 cells on other matrices except Matrigel (Additional file 1: Figure S1D). We
examined the TEER values of iBMELCs differentiatedfrom human iPS cells at different seeding densities before
start of differentiation because previous studies haveshown that seeding density may affect barrier functions
in iBMELCs [12]. The barrier function of LN221F wasincreased by a seeding cell density of < 60–70% confluency (the standard protocol) (Additional file 1: Figure
S1E). Considering the possibility that coating substrates
affected the state of undifferentiated iPS cells (days − 3 to0), the gene expression levels of undifferentiation markers [octamer-binding transcription factor 4 (OCT-4)
and NANOG] and cell number of iPS cells on day 0 wereanalyzed. These coating substrates hardly affected thestate of human iPS cells, although LN221F and LN511F
slightly increased NANOG mRNA expression in human

M

Aoki et al. Fluids Barriers CNS

Fig. 2 Analyses of paracellular permeability of LN221F- and Matrigel-iBMELCs. a FD4 permeability assay of LN221F- and Matrigel-iBMELCs on day
10. ­Papp, apparent permeability coefficient. Data are presented as the mean ± SD (n = 6; **p < 0.01; Student’s t-test). b LY permeability assay. Data arepresented as the mean ± SD (n = 6; **p < 0.01; Student’s t-test)(See figure on next page.)
Fig. 3 Characterization of LN221F- and Matrigel-iBMELCs. a Relative mRNA expression levels of CDH5, MDR1, BCRP, ZO-1, occludin, GLUT1, and
LAT1 in LN221F- and Matrigel-iBMELCs derived from 610B1 on day 10. The values are normalized to those of HPRT1. The relative mRNA expression
levels of Matrigel-iBMELCs were defined as 1. Data are presented as the mean ± SD (n = 3; N.S. = not significant; Student’s t-test). hBMECs: n = 1. b
Immunofluorescence analysis of CDH5 (orange), P-gp (orange), BCRP (orange), ZO-1 (orange), occludin (green), and claudin 5 (orange) expression in
LN221F- and Matrigel-iBMELCs derived from 610B1 on day 10. DAPI = blue. Scale bars = 50 μm

LN221F-iBMELCs

Matrigel-iBMELCs

b
s

EC

M

1F

22

el

0

LAT1

2.0

1.5
N.S.

1.0

0.5

0

CDH5
P-gp
BCRP

ZO-1
OccludinClaudin 5

1
hB F
M
EC
s

0.5

el

1.0

LN
22

1.5

rig

2.0

at

BCRP

N.S.

M

1
hB F
M
EC
s

LN
22

2.5

hB

1.0

LN

GLUT1
N.S.

rig

0

at

0.5

el

N.S.

M

1.0

rig

20

at

1
hB F
M
EC
s

MDR1

N.S.

M

0

s

0

EC

1.5

1F

el

1.5

M

0.5

LN
22

rig

at

CDH5

hB

0.5

22

1.0

LN

Occludin

N.S.

M

0

el

10

rig

15

at

1
hB F
M
EC
s

el

mRNA relative expression
25

M

s

EC

M

1.5

1F

22

rig

at

LN
22

M
5

hB

LN

mRNA relative expression
el

rig

at

M

a

LN221F-iBMELCs Matrigel-iBMELCs

Aoki et al. Fluids Barriers CNS(2020) 17:25
Page 8 of 11

2.0
ZO-1

1.5
N.S.

1.0

0.5
0

(2020) 17:25

Page 9 of 11

Other gene expression levels in LN221F- and MatrigeliBMELCs were similar or greater as compared tohBMECs. Considering the low expression of CDH5, we
additionally analyzed the expression levels of endothelial linage cell markers [platelet endothelial cell adhesion
molecule (PECAM1), von Willebrand factor (vWF), andkinase insert domain receptor (KDR)] in both LN221Fand Matrigel-iBMELCs. The expression levels of thesegenes in both LN221F-iBMELCs and Matrigel-iBMELCs
were lower than those in hBMECs (Additional file 1:
Figure S3A). Besides, to assess whether both LN221FiBMELCs and Matrigel-iBMELCs have the functionof endothelial cells, we conducted Dil-Ac-LDL uptake
assay. As a result, both LN221F-iBMELCs and MatrigeliBMELCs incorporated Dil-Ac-LDL (Additional file 1:
Figure S4A). To analyze the protein expression, we conducted immunofluorescence analysis. In visual observation, the protein localization and expression levels of
CDH5, P-gp, BCRP, ZO-1, occludin, and claudin 5 in
LN221F-iBMELCs were almost equivalent to those in
Matrigel-iBMELCs (Fig. 3b). Furthermore, the cytosol
fluorescence intensities of CDH5, P-gp, and BCRP immunostaining were almost equivalent to those in MatrigeliBMELCs (Additional file 1: Figure S4B). Besides,
Western blotting analysis confirmed that the protein
expression levels of tight junction-related proteins (ZO1, occludin, and claudin 5) in LN221F-iBMELCs were
almost equivalent to those in Matrigel-iBMELCs (Additional file 1: Figure S4C). These results indicate that
LN221F-iBMELCs exhibit characteristics of BMECs as

Rhodamine 123(Substrate of P-gp)aNormalized accumulation

Matrigel
1.6
1.4

**

1.8
1.6

**

1.2

1.0

1.0

0.8

0.8
0.6

0.6
0.4

0.4
0.2
0

0.2
0

CsA

LN221F‑iBMELCs have functions of efflux transporters

To determine whether LN221F-iBMELCs have functions of efflux transporters, which are essential for BBB
functions, an intracellular accumulation assay of the substrates was conducted. The results showed that accumulation of rhodamine 123 (a P-gp substrate) and Hoechst
33342 (a BCRP substrate) in both Matrigel- and LN221FiBMELCs was increased by the inhibitors CsA and Ko
143, respectively (Fig. 4a, b), which confirmed that bothcell types had functions of P-gp and BCRP.

Discussion
The results of the present study showed that fully
defined coating matrix LN221F is more suitable for thedifferentiation of iBMELCs than Matrigel. The components of Matrigel can vary among lots and coating
requires complex manipulation, whereas there is limited

Hoechst 33342(Substrate of BCRP)b

Matrigel

LN221F

1.4

1.2

well as Matrigel-iBMELCs. In addition, we analyzed theexpression levels of mesodermal markers [T-box transcription factor T (TBXT) and KDR (both endothelial
linage cell and mesodermal marker)] in the intermediate state of LN221F-iBMELCs and Matrigel-iBMELCs.
The expression levels of these genes on day 3 in LN221FiBMELCs were higher than those of Matrigel-iBMELCs.
In contrast, the expression levels of these genes on day 6
in LN221F-iBMELCs were lower than those of MatrigeliBMELCs (Additional file 1: Figure S3B). These results
suggest that LN221F increases the rate of differentiationfrom iPS cells to iBMELCs.

CsA

Normalized accumulation

Aoki et al. Fluids Barriers CNS

1.6
1.4

**

LN221F
1.8
1.6

**

1.4

1.2

1.2

1.0

1.0

0.8

0.8
0.6

0.6
0.4

0.4
0.2
0

0.2
0

Ko 143

Ko 143Fig. 4 Analyses of P-gp and BCRP functions in LN221F- and Matrigel-iBMELCs. a Relative intracellular accumulation of rhodamine 123. The iBMELCs
were incubated with 10 μM rhodamine 123 in the absence or presence of 10 μM CsA for 60 min at 37 °C. Relative fluorescence intensity values
were normalized to the condition without inhibitor (set to 1). Data are presented as the mean ± SD (n = 6; **p < 0.01; Student’s t-test). b Relative
intracellular accumulation of Hoechst 33342. The iBMELCs were incubated with 10 μM Hoechst 33342 in the absence or presence of 20 μM Ko 143
for 60 min at 37 °C. Relative fluorescence intensity values were normalized to the condition without inhibitor (set to 1). Data are presented as themean ± SD (n = 6; **p < 0.01; Student’s t-test)

Aoki et al. Fluids Barriers CNS(2020) 17:25

variation among lots of LN221F (imatrix 221). Besides,
LN221F-iBMELCs maintained robust barrier function
for a longer period than Matrigel-iBMELCs, whereas
mRNA and protein expression levels of BMEC-related
markers were similar to those of Matrigel-iBMELCs.
Moreover, LN221F-iBMELCs as well as MatrigeliBMELCs had functions of P-gp and BCRP. To enhancebarrier integrity, iBMELCs are often co-cultured with
BBB components, such as brain pericytes, astrocytes,
and neurons [9, 13]. In this study, barrier integrity was
successfully enhanced just by differentiation with theuse of LN221F without a co-culture system. Based onthese findings, we concluded that coating with LN221F
is more suitable for iBMELC differentiation thanMatrigel.
Laminin 221 is abundant in skeletal and heart muscles, as well as neuromuscular junctions [14, 15]. It has
been reported that laminin 221 also induces differentiation of human embryonic stem cells to cardiovascularprogenitors in vitro [15]. However, to our knowledge,
few studies have investigated the relationship between
laminin 221 and BBB development and maintenance.
Therefore, before conducting the experiments, we
speculated that basement membranes componentsthat are abundant in BBB, such as FBN, LN411F, and
LN511F [16], promote the differentiation of iBMELCs.
However, unlike LN221F, these basement membranes
components did not enhance the barrier function of
iBMELCs as compared to Matrigel. The application of
LN221F to mature iBMELCs from days 8 to 10 did not
induce a tighter barrier. The TEER values of primary
BMECs derived from human and monkey on LN221F
were lower than those on some coating substrates. Of
note, the TEER values of the primary cells on LN511F
were the highest among the six coating substrates, suggesting that laminin 511 (or LN511F) was required formaintaining the integrity of the tight junction in mature
BMECs. Besides, the TEER value of hCMEC/D3 cells on
LN221F was higher than that on Matrigel but did not
differ from those on other basement membranes such as
FBN and LN511F. These findings suggest that LN221F
is not always necessary as a basement membrane toenhance the barrier integrity of the mature BMECs
and LN221F is particularly effective on the differentiation process of iBMELCs. There are two possible reasons why the contact between LN221F and human iPS
cells during differentiation enhanced the barrier integrity of iBMELCs. First, the difference in the differentiation processes in vitro versus in vivo and iBMELCs
require different factors for differentiation. Infact,
iBMELCs expressed low levels of endothelial markers and have characteristics of both endothelial andepithelial cells [17]. In this study, iBMELCs were able

Page 10 of 11

to incorporate Dil-Ac-LDL, indicating that iBMELCs
have the characteristics of endothelial cells. In contrast, CDH5, PECAM1, vWF, and KDR expression levels were lower in iBMELCs than hBMECs. Besides, theresults of immunofluorescence staining showed that
CDH5 was localized to the cytosol in iBMELCs, but isreportedly localized on the cellular membrane in various endothelial cell lineages, such as human umbilicalvein endothelial cells [18], iPS cell-derived endothelial
progenitor cells [19], human immortalized BMECs [20],and mouse BMECs [21]. Considering these findings,
iBMELCs differentiated by a previously method or themodified method may not completely reflect the in vivo
characteristics of endothelial cell lineages. As a second
possibility, laminin 221 plays important roles in thedevelopment of the human BBB in vivo and influences
the differentiation of iBMELCs in vitro. Indeed, laminin
α2, which is a component of laminins 211 and 221, is
indispensable for the development of the BBB as well
as for maintaining barrier integrity [22]. Therefore, it isquite possible that laminin 221 has a positive effect on
the differentiation and barrier function of iBMELCs in alaminin α2-dependent manner.
The results of the present study also confirmed that
LN221F-iBMELCs have characteristics of BMECs, suchas expression of BMEC-related markers and the functionsof efflux transporters. However, the expression levels ofgenes and proteins related to tight junctions in LN221FBMECs were equivalent to those of Matrigel-iBMELCs,
even though LN221F-iBMELCs exhibited more robust
barrier integrity than Matrigel-iBMELCs. The reasons forthese findings will be elucidated in future studies.

Conclusion
In this study, we succeeded in identifying basement
membrane LN221F suitable for the differentiation of
iBMELCs. LN221F-iBMELCs had a more robust barrier function for a longer period than that of MatrigeliBMELCs. Furthermore, the gene and protein expression
levels of BMEC-related markers in LN221F-iBMELCs
were comparable to those of Matrigel-iBMELCs. Like
Matrigel-iBMELCs, LN221F-iBMELCs also had functions of P-gp and BCRP, which are important efflux
transporters of the BBB. In addition, the componentsof Matrigel can vary among various batches, whereas
limited variation occurs among the batches of LN221F.
Therefore, we consider that coating with LN221F,
which requires simple manipulation, as an alternative
to Matrigel is useful for the reproducible production oflarge numbers of iBMELCs for pharmacokinetic andpathological models of the BBB.

Aoki et al. Fluids Barriers CNS(2020) 17:25

Supplementary information

Page 11 of 11

3.

Supplementary information accompanies this paper at https​://doi.
org/10.1186/s1298​7-020-00186​-4.
4.
Additional file 1. Additional figures.
Abbreviations
BCRP: Breast cancer resistance protein; BSA: Bovine serum albumin; CDH5:
Cadherin 5; CsA: Cyclosporine A; Dil-Ac-LDL: 1,1′-Dioctadecyl-3,3,3′,3′tetramethyl-indocarbocyanine perchlorate acetylated low-density lipoprotein; DMEM/F12: 1:1 mixture of Dulbecco’s modified Eagle’s mediumand Ham’s nutrient mixture F-12; D-PBS: D-phosphate-buffered saline; FD4:
Fluorescein isothiocyanate-dextran 4 kDa; FGF2: Fibroblast growth factor 2;
GLUT1: Glucose transporter 1; HBSS: Hank’s Balanced Salt Solution; HEPES:
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid; HE-SFM: Human endothelial-SFM; HPRT1: Hypoxanthine guanine phosphoribosyltransferase 1; iPS
cells: Induced pluripotent stem cells; KDR: Kinase insert domain receptor; KSR:
KnockOut Serum Replacement; LAT1: l-type amino acid transporter 1; LN221FiBMELCs: Human induced pluripotent stem cell-derived brain microvascularendothelial-like cells differentiated on laminin 221 fragment; LA221F: Laminin
221 fragment; LN411F: Laminin 411 fragment; LN511F: Laminin 511 fragment;
LY: Lucifer yellow; Matrigel-iBMELCs: Human induced pluripotent stem cellderived brain microvascular endothelial-like cells differentiated on Matrigel;
MDR1: Multiple drug resistance 1; PECAM1: Platelet endothelial cell adhesion
molecule; P-gp: P-glycoprotein; RA: All-trans retinoic acid; SFM: Serum-free
medium; TBXT: T-box transcription factor T; TEER: Transendothelial electricalresistance; vWF: Von Willebrand factor; ZO-1: Zonula occludens-1.
Acknowledgements
We acknowledge the assistance of the Research Equipment Sharing Center at
the Nagoya City University.
Authors’ contributions
HA, TH, and TM conceived the project and designed the research. HA and MY
performed the experiments. HA and MY analyzed and interpreted the data.
HA, MY, TH, TI, and TM wrote the manuscript. All authors read and approved
the final manuscript.
Funding
This research was supported by grants from AMED (JP19be0304322) and JSPS
KAKENHI (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
Not applicable.

5.
6.
7.
8.
9.
10.
11.
12.

13.

14.
15.
16.
17.
18.

Consent for publication
Not applicable.

19.

Competing interests
The authors declare that they have no competing interests.

20.

Received: 7 November 2019 Accepted: 17 March 2020

21.
22.

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