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1521-009X/44/10/1662–1667$25.00
http://dx.doi.org/10.1124/dmd.116.069336DRUG METABOLISM AND
DISPOSITION Drug Metab Dispos 44:1662–1667, October 2016Copyright ª
2016 by The American Society for Pharmacology and Experimental
Therapeutics
Characteristic Analysis of Intestinal Transport in
Enterocyte-LikeCells Differentiated from Human Induced Pluripotent
Stem Cells
Nao Kodama, Takahiro Iwao, Takahiro Katano, Kinya Ohta, Hiroaki
Yuasa,and Tamihide Matsunaga
Department of Clinical Pharmacy, Graduate School of
Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
(N.K., T.I.,T.M.), Department of Biopharmaceutics, Graduate School
of Pharmaceutical Sciences, Nagoya City University, Nagoya,
Japan
(T.K., K.O., H.Y.)
Received January 6, 2016; accepted July 13, 2016
ABSTRACT
We previously demonstrated that differentiated enterocytes
fromhuman induced pluripotent stem (iPS) cells exhibited
drug-metabolizing activities and cytochrome P450 CYP3A4
inducibility.The aimof this studywas to apply human iPS
cell–derived enterocytesin pharmacokinetic studies by investigating
the characteristics ofdrug transport into enterocyte-like cells.
Human iPS cells cultured onfeeder cells were differentiated into
endodermal cells using activin A.These endodermal-like cells were
then differentiated into intestinalstem cells by fibroblast growth
factor 2. Finally, epidermal growthfactor and small-molecule
compounds induced the maturation of theintestinal stem cell-like
cells. After differentiation, we performedtransepithelial
electrical resistance (TEER) measurements, immuno-fluorescence
staining, and transport studies. TEER values increasedin a
time-dependentmanner and reachedapproximately 100V3cm2.
Efflux transport of Hoechst 33342, a substrate of breast
cancerresistance protein (BCRP), was observed and inhibited by the
BCRPinhibitor Ko143. The uptake of peptide transporter 1
substrateglycylsarcosine was also confirmed and suppressed when
thetemperature was lowered to 4�C. Using immunofluorescence
stain-ing, villin and Na+–K+ ATPase were expressed. These results
suggestthat human iPS cell–derived enterocytes had loose tight
junctions,polarity, as well as uptake and efflux transport
functions. In addition,the rank order of apparent membrane
permeability coefficient (Papp)values of these test compounds
across the enterocyte-like cellmembrane corresponded to the
fraction absorbance (Fa) values.Therefore, differentiated
enterocytes from human iPS cells mayprovide a useful comprehensive
evaluation model of drug transportand metabolism in the small
intestine.
Introduction
The small intestine is an important organ in the
pharmacokinetics oforally administered drugs owing to the presence
of drug transporters anddrug-metabolizing enzymes (Choi et al.,
2013; Li et al., 2013; Yoshidaet al., 2013; Kostewicz et al.,
2014). Thus, Caco-2 cells, a human coloncarcinoma cell line, are
widely used to evaluate the intestinal transport oforally
administered drugs. Although immortalized cells offer
manyadvantages, the extrapolation of data generated with these cell
lines toin vivo conditions is often difficult. This is because
these cells originatedfrom tumors and are therefore not
representative of the natural physi-ologic environment (Le Ferrec
et al., 2001). However, Caco-2 cells havedifferent characteristics
compared with human enterocytes owing todifferences in the
expression patterns of drug transporters (Sun et al.,2002; Harwood
et al., 2016). Additionally, the level of cytochrome P450
CYP3A4, a major drug-metabolizing enzyme in the small intestine,
isalso very low (Nakamura et al., 2002). Thus, it is difficult to
estimatedrug transport and metabolism in the small intestine
appropriately andcomprehensively using Caco-2 cells. To precisely
predict intestinalpharmacokinetics, it is desirable to use human
primary small intestinalepithelial cells. However, it is difficult
to obtain such cells, and there is noappropriate model that exists
for intestinal pharmacokinetic prediction.Human induced pluripotent
stem (iPS) cells (Takahashi et al., 2007)
have the potential to form almost any type of cell and are
expected to be auseful tool in regenerative medicine and drug
discovery research. Thehepatic differentiation of human iPS cells
has been frequently reported(Kondo et al., 2014; Takayama et al.,
2014; Faulkner-Jones et al., 2015;Ishikawa et al., 2015): however,
intestinal differentiation remainsrelatively unexplored in the
literature. Spence et al. (2011) reportedthe generation of
three-dimensional gut-like organoids from humaniPS cells and
demonstrated that the organoids had the morphologiccharacteristics
of intestinal-tract tissues. Ogaki et al. (2013, 2015)showed that
all four intestinal differentiated cell types
(absorptiveenterocytes, goblet cells, enteroendocrine cells, and
Paneth cells) couldbe efficiently differentiated from human
pluripotent stem cells. How-ever, the pharmacokinetic functions of
organoids and intestinal cells
This work was supported, in part, by Grants-in-Aid from the
Japan Society forthe Promotion of Science [Grant 23390036, Grant
25860120, Grant 26293036];Adaptable & Seamless Technology
Transfer Program through Target-Driven R&D(A-STEP) of Japan
Science and Technology Agency [AS262Z01122Q]; a grant ofthe
Nakatomi Foundation [NF2014-32].
dx.doi.org/10.1124/dmd.116.069336.
ABBREVIATIONS: A-83-01,
3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide;
BCRP, breast cancer resistanceprotein; ER, efflux ratio; Fa,
fraction absorbance; FGF, fibroblast growth factor; GFR-Matrigel,
Matrigel Matrix Growth Factor Reduced; HIEC, humansmall intestinal
epithelial cell; iPS, induced pluripotent stem cells; Ko143,
[(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[19,29:1,6]pyrido[3,4-b]indole-3-propanoic
acid 1,1-dimethylethylester; P-gp, P-glycoprotein; Papp,
apparentmembrane permeability coefficient; PBS, phosphate-buffered
saline; PD98059, 2-(2-amino-3-methoxyphenyl)4H-1-benzopyran-4-one;
PEPT1,peptide transporter 1; TEER, transepithelial electrical
resistance.
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were only briefly explored in these studies. Kauffman et al.
(2013) andOzawa et al. (2015) have reported the pharmacokinetic
characteristics ofhuman iPS–derived enterocyte-like cells, but the
characteristics ofintestinal transport have been insufficiently
investigated.We have previously established a method for
differentiating human
iPS cells into enterocytes (Iwao et al., 2014, 2015) and found
severalsmall-molecule compounds that were effective in promoting
thedifferentiation of human iPS cells. In addition, these
enterocytes wereassociated with a gain of pharmacokinetic function.
Moreover, we alsodemonstrated that the enterocyte-like cells had
various pharmacokineticfunctions such as drug-metabolizing
activities by cytochromes P450,UDP-glucuronosyltransferase, and
sulfotransferase, as well as CYP3A4induction by
1a,25-dihydroxyvitamin D3 and peptide uptake throughpeptide
transporters.In the present study, we investigated the
characteristics of drug
transport in enterocyte-like cells. The enterocyte-like cells
had tightjunctions and exhibited activities of efflux transporter
breast cancerresistance protein (BCRP). Moreover, our findings
indicated that theenterocyte-like cell membrane may be able to
predict fraction absor-bance (Fa) in humans. These results suggest
that the human iPS cell–derived enterocyte-like cells would be
useful for a human intestinalpharmacokinetic prediction that
included transport and metabolism.
Materials and Methods
Materials. Fibroblast growth factor (FGF) 2, activin A, and
epidermal growthfactor were purchased from PeproTech Inc. (Rocky
Hill, NJ). BDMatrigelMatrixGrowth Factor Reduced (GFR-Matrigel) was
purchased from BD Biosciences(Bedford, MA). KnockOut Serum
Replacement was purchased from InvitrogenLife Technologies/Thermo
Fisher Scientific (Carlsbad, CA).
(+)-(R)-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide
dihydrochloride
(Y-27632),2-(2-amino-3-methoxyphenyl)4H-1-benzopyran-4-one
(PD98059), 5-aza-29-deoxycytidine,
3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide
(A-83-01), ibuprofen, paraformaldehyde, and Hoechst33342were
purchased from Wako Pure Chemical Industries (Osaka, Japan).
Ko143was purchased from Sigma-Aldrich (St. Louis, MO). Anti-villin
and anti-BCRP/ABCG2 antibodies were purchased from Abcam
(Cambridge, UK).Anti-Na+–K+ ATPase antibody purchased from GeneTex,
Inc. (Irvine, CA).Anti–peptide transporter 1 (PEPT1) antibody was
purchased from Santa CruzBiotechnology, Inc. (Dallas, TX).
[N-methyl-14C]Antipyrine was purchased fromAmerican Radiolabeled
Chemicals, Inc. (St. Louis, MO). [Ring-3H]Atenolol,[3H]metoprolol,
and [3H]glycylsarcosine were purchased from Moravek Bio-chemicals,
Inc. (Brea, CA). (2)-[Methoxy-3H]sulpiride and
[14C]polyethyleneglycol 4000 were purchased from PerkinElmer, Inc.
(Boston, MA). All otherreagents were of the highest quality
available. Triton X-100 was purchased fromAMRESCO (Cleveland,
OH).
Human iPS Cell Culture. The human iPS cell lineWindy, whichwas
derivedfrom the human embryonic lung fibroblast cell line MRC-5,
was provided by Dr.Akihiro Umezawa of the National Center for Child
Health and Development(Tokyo, Japan). Human iPS cells were
maintained in a 1:1 mixture of Dulbecco’smodified Eagle’s medium
and Ham’s nutrient mixture F-12 (DMEM/F12)containing 20% KnockOut
Serum Replacement, 2 mM L-glutamine, 1% minimal
essential medium nonessential amino acid solution (NEAA), 0.1
mM2-mercaptoethanol, and 5 ng/ml of FGF2 at 37�C in humidified air
with 5% CO2.The human iPS cells were cultured on a feeder layer of
mitomycin C-treatedmouse embryonic fibroblasts, and the medium was
changed daily.
Differentiation into Enterocyte-Like Cells. The human iPS cells
weredifferentiated into enterocytes on the basis of our previous
report (Iwao et al.,2015). Briefly, human iPS cells were
differentiated into endodermal cells byincubating the cells in the
presence of 100 ng/ml activin A for 72 hours, and
theseendodermal-like cells were then differentiated into intestinal
stem cells via250 ng/ml FGF2 for 96 hours. Finally, the cells were
passaged on GFR-Matrigel–coated 24-well plates or cell culture
inserts and cultured in medium containing20 ng/ml epidermal growth
factor. Subsequently, 20 mM PD98059, 5 mM 5-aza-29-deoxycytidine,
and 0.5 mM A-83-01 were also added to the medium on day14 after
differentiation. The medium was subsequently changed every 3
days.Transepithelial electrical resistance (TEER) values were
obtained to check theintegrity of the membrane before the transport
assay.
Uptake Assay. The culture medium was removed, and the
differentiated cellswere preincubatedwith the transport buffer
(Hank’s balanced salt solution containing10 mMMES, pH 6.0) at 37�C
for 15 minutes. Uptake assays were initiated by thereplacement of a
transport buffer containing 135 nM [3H]glycylsarcosine at 37�C
inthe presence or absence of 3 mM ibuprofen, a known PEPT1
inhibitor (Omkvistet al., 2010) or at 4�C. Assays were stopped by
the addition of ice-cold transportbuffer, and the cells were washed
twice with the same buffer. The cells weresolubilized with 0.2 M
NaOH solution (0.5 ml) containing 0.5% sodium dodecylsulfate.
Radioactivity was measured by liquid scintillation counting using 3
ml ofClear-sol I (Nakarai Tesque, Kyoto, Japan) as a scintillation
fluid.
To correct for the uptake of glycylsarcosine, the total protein
of the differen-tiated cells was measured using a Pierce BCA
Protein Assay Kit (Thermo FisherScientific Inc., Waltham, MA),
according to the manufacturer’s instructions.
Immunofluorescence Staining. Differentiated cells were washed
three timeswith phosphate-buffered saline (PBS) with 1 mM CaCl2 and
1 mM MgCl2,following which they were fixed and permeabilized in
methanol (220�C) for5 minutes at 4�C for staining villin, Na+–K+
ATPase, and PEPT1. Differentiatedcells were washed three times with
PBS, following which they were fixed in a 4%(w/v) paraformaldehyde
solution for 30 minutes at room temperature, andpermeabilized in a
Triton X-100 solution for 5 minutes at room temperature forstaining
BCRP. After washing three times with PBS, the cells were blocked
inPBS containing 2% skim milk for 20 minutes at room temperature.
Following theblocking step, the cells were incubated for 60 minutes
at room temperature, withanti-villin 1 and Na+–K+ ATPase antibody
diluted at 1:100. The cells wereincubated overnight at 4�C,with the
Na+–K+ATPase antibody diluted at 1:100, orBCRP and PEPT1 antibodies
diluted at 1:50. The cells were washed three timeswith PBS and
incubated with a 1:500 dilution ofAlexa Fluor 488- and
568-labeledsecondary antibody for 60 minutes at room temperature.
After washing threetimes with PBS, the cells were incubated with 1
mg/ml of 49,6-diamidino-2-phenylindole (DAPI) for 5 minutes at room
temperature and washed with PBS.The cells were mounted on a glass
slide using a 9:1 mixture of glycerol and PBS(without magnesium and
calcium) and were viewed using an LSM 510 Metaconfocal microscope
(Carl Zeiss Inc., Oberkochen, Germany).
TEER Value Measurements. The TEER values of the human iPS
cell–derived enterocyte-like cell membrane on cell culture inserts
were measured byMillicell ERS-2 (Millipore, Bedford, MA).
Membrane Transport Assay. The culture medium was removed, and
thedifferentiated cells were preincubated with transport buffer
(Hank’s balanced saltsolution containing 10 mM HEPES, pH 7.4) at
37�C for 15 minutes. Transport
Fig. 1. Immunofluorescence staining analysis of PEPT1in the
differentiated enterocyte-like cells. After differen-tiation, the
cells were stained with PEPT1 (red) andDAPI (blue). Scale bar, 50
mm.
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assays were performed by replacing with transport buffer
containing substratessuch as 1.15mM [14C]antipyrine, 87.1 nM
[3H]atenolol, 9.39 nM [3H]metoprolol,7.70 nM [3H]sulpiride, or 1.31
nM [14C]PEG4000 on the apical chambers. Thesolution was collected
from the basal chambers at 30, 60, and 120 minutes.Radioactivity
was measured by liquid scintillation counting using 3 ml Clear-sol
I(Nakarai Tesque, Kyoto, Japan) as a scintillation fluid. In a
bidirectional transportassay using Hoechst33342, after
preincubation, the transport buffer containing20 mM Hoechst33342
was added to the apical or basal chambers, and the cellswere
incubated at 37�C for 120 minutes in the presence or absence of 10
mMKo143. Samples were collected from the receiver chambers. The
intensity of theHoeschst33342 fluorescence was measured using a
fluorescence plate reader(ARVO MX 1420 Multilabel Counter; Perkin
Elmer Inc., Waltham, MA) usingthe wavelengths 355 nm for excitation
and 460 nm for emission.
Analysis of Apparent Membrane Permeability. The apparent
membranepermeability coefficient (Papp) in transport assay was
calculated as follows:
Papp ¼ dQdt ×1
A� C0 ð1Þ
where dQ/dt is the amount of the compound permeated per unit of
time, A is thesurface area of Transwell membrane (0.3 cm2), and C0
is the initial compoundconcentration in the donor chamber. Efflux
ratio (ER) of Hoechst33342 was
calculated by dividing Papp of the basal-to-apical transport by
that of the apical-to-basal transport.
Statistical Analysis. The level of statistical significance was
assessed usingStudent’s t test. The correlation between the Papp
value and Fa of the fivecompounds was estimated by a P and R
values. The best-fitting curves werecalculated by nonlinear
regression using PASW Statistics 18 system software(IBM, Armonk,
NY).
Results
Uptake of Glycylsarcosine in the Differentiated
Enterocyte-LikeCells. The oligopeptide transporter SLC15A1/PEPT1 is
expressed in thesmall intestine and plays an important role in
peptide transport from thelumen (Liang et al., 1995; Giacomini et
al., 2010). In previous studies,we confirmed mRNA expression, but
not protein expression, of PEPT1in enterocyte-like cells.
Therefore, we conducted immunofluorescencestaining of PEPT1,
indicating that the protein was also expressed (Fig.1). Moreover,
we performed uptake analyses of glycylsarcosine,which is a
substrate of PEPT1 (Nakanishi et al., 1997). The uptake
ofglycylsarcosine in the enterocyte-like cells was increased in a
time-dependent manner at 37�C (Fig. 2). When the uptake temperature
waslowered to 4�C, the uptake was significantly suppressed and
reacheda plateau after 30 minutes. Moreover, at 37�C, in the
presence ofibuprofen, the uptake was significantly suppressed to a
similar extent asthat at 4�C. Therefore, these findings indicate
that PEPT1-mediatedactive transport was quantitatively evaluated in
the enterocyte-like cells.Characteristics of Enterocyte-Like Cells.
To investigate whether
the enterocyte-like cell membrane was available for drug
permeabilitystudies, we characterized the enterocyte-like cells.
After seeding on cellculture inserts, the TEER values were
increased in a time-dependentmanner and finally reached a plateau
at approximately 100 V � cm2(Fig. 3). Therefore, these results
suggest that the enterocyte-like cellsformed a membrane with a
loose tight junction. Using immunofluores-cence staining, we found
that almost all the cells expressed the intestinalepithelial marker
villin, whereas Na+–K+ ATPase was located only onthe basal side
(Fig. 4).Bidirectional Transport across the Enterocyte-Like
Cell
Membrane. It has been found that BCRP is highly expressed in
theapical membrane of the small intestinal epithelium and plays an
importantrole in intestinal absorption of drug substrates
(Giacomini et al., 2010).Thus, we used a bidirectional transport
assay to examine the enterocyte-like cell membrane on the cell
culture inserts. Apical-to-basal Papp valueswere 3.32 6 1.09 and
11.53 6 0.82 (� 1026 cm/s) in the absence orpresence of Ko143,
respectively (n = 4). The basal-to-apical values were49.966 7.98
and 33.726 9.55 (� 1026 cm/s) in the absence or presence
Fig. 2. Uptake of glycylsarcosine in the differentiated
enterocyte-like cells.Following differentiation, the
enterocyte-like cells were incubated with a transportbuffer (pH
6.0) containing glycylsarcosine at 37�C, with or without 3 mM
ibuprofen,or at 4�C. Data are represented as the mean 6 S.D. (A, n
= 3; B, n = 4). Open andclosed symbols bars show the uptake at 37�C
and 4�C, respectively, and the gray barshows the uptake with 3 mM
ibuprofen at 37�C. Levels of statistical significancecompared with
the uptake at 37�C: A, **P , 0.01; B, *P , 0.05.
Fig. 3. Time-dependent changes of TEER values in the
enterocyte-like cell mem-brane. The enterocyte-like cells were
seeded on GFR-Matrigel-coated cell cultureinserts. TEER values were
measured every 3 days from day 4 after seeding. Datawere
represented as the mean 6 S.D. (n = 22).
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of Ko143, respectively (n = 4). As shown in Fig. 5, Papp values
ofHoechst 33342, a substrate of BCRP (Doyle and Ross, 2003), in
thebasal-to-apical direction was significantly higher than for the
apical-to-basal direction. The ER, calculated from the ratio of
Papp in the basal-to-apical direction to that in the
apical-to-basal direction, was 15. From theaddition of Ko143, a
BCRP inhibitor (Allen et al., 2002), the basal-to-apical Papp
values were decreased and the apical-to-basal Papp valueswere
increased. Additionally, the ER value was reduced to 3. Moreover,in
immunofluorescence staining, it was indicated that BCRPwas
locatedon the apical side (Fig. 6). Therefore, these results
indicate that theenterocyte-like cell membrane had also efflux
transporter BCRPactivity.Permeability of Test Compounds across an
Enterocyte-Like Cell
Membrane. We performed a membrane transport study using five
testcompounds with various Fa (1–97%) values in humans. The Papp
valuesof the five test compounds ranged from 2.04 to 9.99 (� 1026
cm/s;Table 1). The rank order of Papp values of these test
compoundscorresponded to those of the Fa values. Moreover, the
sigmoidalrelationship between the Papp values and the Fa values of
the fivecompounds were observed in differentiated cells (Fig. 7).
These resultssuggest that drug permeability in the enterocyte-like
cell membrane maybe able to predict Fa in humans.
Discussion
We previously reported that human iPS cell–derived
enterocyte-likecells had metabolic functions, such as
drug-metabolizing enzymeactivities and CYP3A4 inducibility (Iwao et
al., 2015). Moreover, inthis study, we demonstrated the drug
transport characteristics in theenterocyte-like cells.Human peptide
transporter PEPT1 is primarily responsible for the
transport of dietary di- and tripeptides from the lumen of the
small
intestine. PEPT1 has been exploited with prodrugs designed to
introducepeptide and peptide bond-like moieties onto the parent
molecule. Thismethod was demonstrated to significantly increase the
absorption ofdrugs with poor oral bioavailability (Gomez-Orellana,
2005; Hammanet al., 2005; Leonard et al., 2006; Majumdar and Mitra,
2006). In the
Fig. 4. Immunofluorescence staining analysis of villin and
Na+–K+ ATPase in the differentiated enterocyte-like cells. The
enterocyte-like cells were seeded on GFR-Matrigel–coated cell
culture inserts. After differentiation, the cells were stained with
villin (green), Na+–K+ ATPase (red), and DAPI (blue). Scale bar, 50
mm. I and II arecross-sectional views along the red and green
lines, respectively. A, apical side; B, basal side.
Fig. 5. Bidirectional permeability of Hoechst33342 across the
enterocyte-like cellmembrane. The enterocyte-like cells were seeded
on GFR-Matrigel–coated cell cultureinserts. After differentiation,
the cells were incubated with the transport buffer (pH
7.4)containing Hoechst33342 (20 mM) for 120 minutes at 37�C in the
presence or absenceof Ko143 (10 mM). Data are represented as the
mean 6 S.D. (n = 4). White or blackbars show apical-to-basal or
basal-to-apical Papp values, respectively. ER ofHoechst33342 was
calculated by dividing Papp of the basal-to-apical transport bythat
of the apical-to-basal transport. Levels of statistical
significance compared with theeach Papp value in the absence of
Ko143: *P , 0.05, **P , 0.01; and compared witheach Papp values for
apical-to-basal transport:
†P , 0.01.
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enterocyte-like cells, we confirmed the expression of the PEPT1
protein(Fig. 1) and also quantitatively evaluated the
PEPT1-mediated uptakeactivity of glycylsarcosine (Fig. 2). However,
PEPT1-mediated uptakeusing fluorescence-labeled di- or tripeptides
was also qualitativelyevaluated in previous reports (Iwao et al.,
2014; Ozawa et al., 2015).Therefore, it was considered that the
enterocyte-like cells may be used asa quantitative evaluation model
of uptake transporters such as PEPT1.To our knowledge, there are
few reports involving drug membrane
permeability using human iPS–derived enterocytes (Kauffman et
al.,2013; Ozawa et al., 2015). However, this evaluation used only
TEERmeasurements and the permeability of a nonabsorbable marker
(fluo-rescein isothiocyanate-dextran, molecular weight 4 or 150
kDa) in thesereports. Thus, we performed a bidirectional transport
assay and drugmembrane permeability assay to characterize the
enterocyte-like cells.TEER values of the enterocyte-like cell
membrane on the cell cultureinserts were gradually increased and
finally reached a plateau atapproximately 100 V � cm2 (Fig. 3). It
was reported that TEER valuesin the human small intestine were
approximately 40 V � cm2 (Sjoberget al., 2013). In addition, we
previously reported that the values in humansmall intestinal
epithelial cell (HIEC) monolayer were 98.9 V � cm2,even lower in
the Caco-2 cell monolayer (900V�cm2) (Takenaka et al.,2014). It was
indicated that the TEER value of the enterocyte-like cellmembrane
was comparable to that of the HIEC monolayer and thehuman small
intestine. We were also able to use immunofluorescencestaining to
visualize the presence of the intestinal epithelial marker
villinand Na+–K+ ATPase located in the cytoplasm and on the basal
side ofthe enterocyte-like cell membrane, respectively (Fig. 4).
Taken together,these results suggest that the enterocyte-like cell
membrane formed a
loose tight junction similar to the small intestine and also
exhibited apolarity.In the intestine, transporters are localized on
the brush border
membrane and the basal side of intestinal cells. Four major
ATP-binding cassette efflux transporters have been shown to
localize at theapical/luminal membrane of enterocytes. These are
thought to form abarrier to intestinal absorption of the substrate
drugs: P-glycoprotein(P-gp), BCRP, multidrug resistance–associated
protein (MRP)2, andMRP4 (Englund et al., 2006; Takano et al.,
2006;Maubon et al., 2007). Theexpression levels of these substrate
drugs differed between the segmentsof the intestine. In general,
BCRP,MRP2, and P-gp are expressed at highlevels in the small
intestine and are considered to be a limiting barrier tooral drug
absorption (Shirasaka et al., 2008; Giacomini et al., 2010).In the
Food and Drug Administration (FDA) and the Ministry of HealthLabor
andWelfare guidelines for drug interactions
(http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf),
it is indicated that drug candidates should be evaluatedto
determine whether they are substrates of efflux transporters such
asP-gp and BCRP. Thus, we examined the BCRP-mediated
transportactivity in the enterocyte-like cells. The basal-to-apical
Papp values of
Fig. 6. Immunofluorescence staining analysis of BCRP in the
differentiated enterocyte-like cells. The enterocyte-like cells
were seeded on GFR-Matrigel–coated cell cultureinserts. After
differentiation, the cells were stained with BCRP (green) and DAPI
(blue). Scale bar, 50 mm. I and II are cross-sectional views along
the red and green lines,respectively. A, apical side; B, basal
side.
TABLE 1
Papp values of test compounds in the enterocyte-like cell
membrane
Compounds Papp (Mean 6 S.D.) Faa
�1026 cm/sec %Antipyrine 9.99 6 2.59 97Metoprolol 7.54 6 1.51
85Atenolol 6.10 6 1.19 50Sulpiride 5.41 6 2.74 44PEG4000 2.04 6
0.43 .1
Papp values were represented as the mean 6 S.D. (n = 4).aFa
values were obtained from published data (Rozehnal et al.,
2012).
Fig. 7. Relationship between Fa values and Papp of test
compounds across theenterocyte-like cell membrane. The
enterocyte-like cells were seeded on GFR-Matrigel–coated cell
culture inserts. Following differentiation, the cells wereincubated
with the transport buffer (pH 7.4) containing antipyrine,
atenolol,metoprolol, sulpiride, or PEG4000 for 120 minutes at 37�C.
The correlation curvewas fitted by using the following formula: Fa
= 1 / (0.01 + 5.28 * 0.36
Papp). Datawere represented as the mean 6 S.D. (n = 4). P ,
0.01; R = 0.99.
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-
Hoechst33342, a substrate of BCRP, were higher than the
apical-to-basal Papp values (Fig. 5). By addition of Ko143, a BCRP
inhibitor,basal-to-apical Papp values were decreased and
apical-to-basal Pappvalues were increased. ER values were also
decreased by the presenceKo143. In addition, BCRP was located on
the apical side (Fig. 6). Theseresults demonstrated that the
enterocyte-like cells had BCRP-mediatedtransport activity and that
the cell membrane was available for theevaluation of efflux
transport.In our membrane permeability study, the rank order of
Papp values of
these test compounds corresponded to those of the Fa values
(Fig. 7).Takenaka et al. (2014) reported that the HIEC monolayer
with loosetight junctions was able to accurately predict the oral
absorption ofparacellularly absorbed compounds. The enterocyte-like
cell membranecould also be useful for the prediction of the Fa
values of drugs,including such compounds.In conclusion, we
demonstrated that the enterocyte-like cells
exhibited: 1) functions of uptake and efflux transporters; 2)
loose tightjunctions similar to the human small intestine; and 3)
apical/basalpolarity. Moreover, it was indicated that the Fa of
drugs in humans canbe estimated from permeability data of the
enterocyte-like cellmembrane. In our previous study, we found that
the differentiated cellsperformed drug-metabolizing enzyme
activities and CYP3A4 induc-ibility (Iwao et al., 2015). Taken
together, the enterocyte-like cells maybe useful as an appropriate
model to comprehensively predict drugtransport and metabolism in
the intestine.
Acknowledgments
The authors thank Drs. Hidenori Akutsu, Yoshitaka Miyagawa,
Hajime Okita,Nobutaka Kiyokawa, Masashi Toyoda, and Akihiro Umezawa
for providinghuman iPS cells. The authors also thank Enago
(www.enago.jp) for the Englishlanguage review.
Authorship ContributionsParticipated in research design: Kodama,
Iwao, Ohta, Yuasa, Matsunaga.Conducted experiments: Kodama, Iwao,
Katano, Ohta.Performed data analysis: Kodama, Iwao, Katano,
Ohta.Wrote or contributed to the writing of the manuscript: Kodama,
Iwao, Ohta,
Yuasa, Matsunaga.
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Address correspondence to: Dr. Takahiro Iwao, Department of
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