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Expression and functional characterization of drug transporters in brain microvascular endothelial cells derived from human induced pluripotent stem cells Toshiki Kurosawa, Yuma Tega, Kei Higuchi, Tomoko Yamaguchi, Takashi Nakakura, Tatsuki Mochizuki, Hiroyuki Kusuhara, Kenji Kawabata, and Yoshiharu Deguchi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00697 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Molecular Pharmaceutics

Expression and functional characterization of drug transporters in brain microvascular endothelial cells derived from human induced pluripotent stem cells

Toshiki Kurosawa1, Yuma Tega1, Kei Higuchi1, Tomoko Yamaguchi2, Takashi Nakakura4, Tatsuki Mochizuki3, Hiroyuki Kusuhara3, Kenji Kawabata2, and Yoshiharu Deguchi1*

1Laboratory

of Drug Disposition & Pharmacokinetics, Faculty of Pharma-Sciences,

Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan. 2Laboratory

of Stem Cell Regulation, National Institutes of Biomedical Innovation,

Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan 3Laboratory

of Molecular Pharmacokinetics, Graduate School of Pharmaceutical

Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 4Department

of Anatomy and Cell Biology, School of Medicine, Teikyo University, 2-

11-1 Kaga, Itabashi, Tokyo 173-8605, Japan

*Corresponding

author: Professor Yoshiharu Deguchi, Ph.D.

Laboratory of Drug Disposition and Pharmacokinetics, Faculty of Pharma-Sciences,

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 1 6 7 8 2 9 10 11 3 12 13 14 4 15 16 17 5 18 19 20 6 21 22 23 7 24 25 26 8 27 28 29 9 30 31 3210 33 34 3511 36 37 3812 39 40 4113 42 43 4414 45 46 4715 48 49 5016 51 52 5317 54 55 5618 57 58 59 60

Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan. Telephone: +81-03-3964-8246 FAX: +81-03-3964-8252 E-mail: [email protected]


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Abstract Brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs) have been proposed as a new blood-brain barrier model, but their transport function has not been fully clarified. Therefore, in this study, we investigated the gene expression and function of transporters in hiPS-BMECs by means of quantitative reverse transcription-PCR, in vitro transcellular transport studies, and uptake experiments. mRNAs encoding ABC and SLC transporters, such as BCRP, MCT1, CAT1, and GLAST, were highly expressed in hiPS-BMECs. Transcellular transport studies showed that prazosin, [14C]L-lactate, [3H]L-arginine, and [3H]L-glutamate (substrates of BCRP, MCT1, CAT1, and GLAST, respectively) were transported asymmetrically across the hiPS-BMECs monolayer.

Substrates of LAT1, OCTN2, CAT1, GLAST, MCT1, and

proton coupled organic cation (H+/OC) antiporter were taken up by hiPS-BMECs time-, temperature- and concentration-dependently, and the uptakes were markedly decreased by inhibitors of the corresponding transporter. These results indicate that hiPS-BMECs express multiple nutrient and drug transporters.


Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Keywords: human iPS cell, blood-brain barrier, gene expression, tight junction,

2

transporter

3 4

Abbreviations: BBB, blood-brain barrier; BMEC, brain microvascular endothelial cell;

5

CNS, central nervous system; iPS, induced pluripotent stem cells; TEER, transendothelial

6

electrical resistance


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Introduction Central nervous system (CNS) drug candidates have considerably lower success rates than other candidate drugs during development, and one of the reasons for this is the difficulty of predicting drug delivery efficiency to the brain in humans from data obtained in experimental models. This is mainly because the transport function across the bloodbrain barrier (BBB) is strongly species-dependent. Therefore, better experimental models are needed to enable prediction of drug transport characteristics at the human BBB. The BBB consists of brain microvascular endothelial cells (BMECs) linked by tight junctions, which provide a barrier function that contributes to homeostasis in the brain. This function is reflected in the high transendothelial electrical resistance (TEER) of the BBB in vivo (1). Moreover, the BBB contains many functional proteins, such as ATPbinding cassette (ABC) transporters, solute carrier (SLC) transporters, and receptors, which tightly regulate transport of their substrates into and out of the brain.

But, there

are substantial species differences between humans and other mammals in the expression of these proteins at the BBB (2, 3). It has been suggested that human primary-cultured BMECs could be useful to investigate the physiological functions of the human BBB, but they have significant disadvantages, such as variability from batch to batch, difficulty in obtaining them in quantity, and a short proliferative life span (4).


In contrast,


immortalized human BMECs can be passaged for many generations, and their usefulness for drug transport studies has been demonstrated in numerous laboratories worldwide (5). However, they frequently show attenuation of barrier and/or transport functions as a result of decreased expression of the relevant proteins (6, 7). Recently, some human in vitro BBB models have been constructed from stem cells (8-15). Lippmann et al. reported that brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs) by co-differentiation with neural cells showed strong tight-junction formation and expressed some transporter proteins (8, 9).

However, the function of transporters, especially SLC transporters, in hiPS-BMECs

has not yet been fully evaluated, and this information would be essential for construction of a high-quality BBB model that mimics the in vivo human BBB. Therefore, the purpose of the present study was to clarify the expression and function of nutrient and drug transporters in hiPS-BMECs derived from induced pluripotent stem (iPS) cells (IMR90-4) by means of quantitative PCR and cellular uptake/transcellular transport studies.


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Experimental Section Reagents Reagents (reagent grade) were purchased from Wako Pure Chemical Industries (Osaka Japan), or Sigma-Aldrich Company (St. Louis, MO). Arginine, L-[2,3,4-3H] ([3H]L-arginine, 40 Ci/mmol) and glutamic acid, L-[2,3,4-3H] ([3H]L-glutamate, 60 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). Lactic acid sodium salt, L-[14C(U)] ([14C]L-lactate, 150.6 mCi/mmol) was purchased from PerkinElmer (Waltham, MA). Details of buffers and media are given in the supporting information.

Cell culture The human iPS cell line IMR90-C4 was purchased from WiCell Research Institute (Madison, WI), and cultured in mTeSR1 medium on Matrigel-coated dishes. hCMEC/D3 cells were supplied by Dr. Pierre-Oliver Couraud (Institut Cochin, Paris, France) under license from INSERM. hCMEC/D3 cells were maintained on collagen I-coated dishes as reported previously (16). Details of the cell culture conditions are given in the supporting information.



Differentiation to hiPS-BMECs Human iPS cells were differentiated to BMECs as previously reported (8, 9). Briefly, iPS cells were treated with Accutase (Merck-Millipore, Billerica, MA) to dissociate them into single cells, which were seeded on Matrigel-coated 6-well dishes (Thermo Scientific, Waltham, MA) at a density of 1-1.5 × 105 cells/well, and cultured in mTeSR1 medium containing 10 µM Y27632 (a ROCK inhibitor, Wako) (day -3). After 24 h, the medium was replaced with fresh mTeSR1 medium without Y27632 (day -2). On day 0, the medium was replaced with unconditioned medium (UM) (See Supporting Information for details of the components). On day 6, the medium was changed to human endothelial serum-free medium (EM; Life Technologies, Carlsbad, CA) containing 1% human platelet-derived serum (hPDS; Sigma-Aldrich Company), 10 µM all-trans-retinoic acid (RA; Wako), and 20 ng/ml human fibroblast growth factor 2 (FGF2; Sigma-Aldrich Company). On day 8, the cells were dissociated by treatment with Accutase, and the resulting single cells were seeded onto fibronectin/collagen IV (PharmaCo-Cell, Nagasaki, Japan)-coated cell culture inserts having a PET membrane for 24-well plates (0.4 mm pore size; Corning, NY) at a density of 3.3 × 105 cells/cm2 or on collagen Icoated 24-well plates (BD Biosciences, Franklin Lakes, NJ) at a density of 1 × 105 cells/well, followed by culture in EM with hPDS, RA, and FGF2. On day 9, floating cells


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were removed by washing with EM, and the medium was replaced with fresh EM with hPDS.

Quantitative reverse transcription-PCR (qRT-PCR) Total RNA was isolated from the iPS-BMECs cultured on fibronectin/collagen IVcoated inserts or hCMEC/D3 cells cultured on collagen I-coated 24-well plates using an RNeasy Mini Kit (QIAGEN, Hilden, Germany), and cDNA was prepared from total RNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA) and random primers according to the manufacturer's protocol. PCR was performed with Power SYBR Green PCR master mix (Thermo Fisher Scientific) using 5.25 µL of cDNA, which is equivalent to 2 ng total RNA, and 5 pmol sense/antisense primers in 12.5 µL final volume. Primer sequences are shown in the supporting information (Supporting Table 1). Amplification and detection were carried out on an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific). The cycling parameters were 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 sec and 60°C for 1 min. The relative mRNA expression was determined by the ΔCt method. The ΔCt value was obtained by subtracting the Ct value of TATA-binding protein (TBP) mRNA (the product of a house-keeping gene) from the Ct value of the target mRNA, and the relative



expression was calculated as 2-ΔCt.

Permeability studies using iPS-BMECs The iPS cells were differentiated to BMECs from day -3 to day 7 as described above. On day 8, the cells were seeded in fibronectin/collagen IV-coated cell culture inserts and cultured in EM with hPDS, RA, and FGF2. On day 10, the assay buffer was added to the upper side (200 µL) or lower side (900 µL) of the inserts. Assay buffer containing a test drug was added to the other side to start the assay, and the system was incubated at 37°C for the designated time. When the effect of inhibitors was to be examined, the assay buffer containing the inhibitor was added to both the upper and lower sides, and pre-incubation of the cells was carried out. Following the incubation, 10% by volume of the buffer in the upper and lower chambers was collected and replaced with the same volumes of fresh assay buffer. The cumulative amount of drug transported across the membrane was plotted against time, and the slopes of the lines were used to calculate the permeability coefficient (Pe) of the iPS-BMECs monolayer as follows: PS = (dQ/dt) /D0 where PS, dQ/dt, and D0 are the permeability surface area product, the slope of the linear region of a plot of the cumulative amount of permeant in the receiver chamber versus


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time, and the starting concentration of the drug on the donor side, respectively. 1/PStotal = 1/PSe + 1/PSm Pe = PSe/S where PStotal and PSm are the permeability surface area product corresponding to the Transwell membrane with and without the iPS-BMECs monolayer, respectively, and S is the surface area of the membrane.

Uptake studies in iPS-BMECs The uptake study was performed as reported previously (16). Details of the method are given in the supporting information. The iPS cells were differentiated to BMECs from day -3 to day 7 as described above. On day 8, the cells were seeded on collagen I-coated 24-well plates and cultured in EM with hPDS, RA, and FGF2. The uptake was evaluated by adding drug solution (pH 7.4) for designed times at day 10. For the measurement of [14C]L-lactate uptake, the pH of the buffer was adjusted to 6.0. After incubation, the cellto-medium ratio (C/M, µL/mg protein) was calculated by dividing the uptake amount in the cells by the concentration of drug in the transport medium. In the inhibition study, the uptake was measured in the presence of test compounds at the designated concentration.



LC-MS/MS and UPLC Analysis Samples were deproteinized with acetonitrile, and centrifuged at 3000 rpm for 10 min. The supernatant or sample was diluted with mobile phase, and the drug concentration was quantified by LC-MS/MS, using an Accela HPLC system connected to a TSQ Quantum Ultra (Thermo Fisher Scientific) mass spectrometer, or a Nexera-XR (Shimazu, Kyoto, Japan) HPLC system connected to a Qtrap4500 (AB Sciex, Foster City, CA) mass spectrometer with an electrospray ionization interface. The methods are described in detail in the supporting information. Lucifer yellow was detected by ultra-performance liquid chromatography with a fluorescence detector composed of a Nexera system equipped with an RF-20 Axs (Shimadzu Corporation, Kyoto, Japan).

Determination of radioactivity Samples were mixed with 5 mL Hionic-Fluor (PerkinElmer). Radioactivity of 3H and 14C

in samples was measured using a liquid scintillation counter (Tri-Carb 3110TR;

PerkinElmer).

Statistical analysis


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1


All values are presented as average ± standard error.

Statistical analysis of the data

2

was performed with Student’s t-test and with one-way analysis of variance followed by

3

Dunnett’s test for single and multiple comparisons, respectively. Differences were

4

considered statistically significant at P < 0.05.



Results Protein expression of endothelial markers, TJ proteins and glucose transporter in hiPS-BMECs To confirm the differentiation from human iPS cells to hiPS-BMECs, immunohistochemical staining of endothelial markers and TJ proteins was performed. The endothelial markers platelet endothelial cell adhesion molecule (PECAM) 1 and vascular endothelial (VE)-cadherin were expressed in hiPS-BMECs (Supporting Figs. 1A and 1B).

In addition, protein expression of claudin-5, occludin, and zonula occludin

(ZO)-1, which are related to the formation of TJ, was also detected (Supporting Figs. 1C, 1D and 1E). Moreover, glucose transporter (GLUT) 1 was stained by the antibody (Supporting Fig. 1F). These properties were consistent with those reported in earlier work (8-10, 14)

Relative expression levels of transporter and receptor mRNAs in hiPS-BMECs As shown in Table 1, mRNA expression of 38 transporters and 4 receptors was determined.

The mRNA expression levels of energy transport system components

GLUT1, GLUT3 and MCT1 were 17, 129 and 8.3 times, respectively, greater than that of TBP. Among the organic cation transporters, CTL2 showed the highest mRNA


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expression level, followed by CTL1, OCTN2, and OSCP1, while MATE1, OCTN1, and OCT1-3 were much more weakly expressed.

The mRNA expression levels of organic

anion transporters, OATPs and OATs, were much lower than that of TBP, except for OATP2A1.

In contrast, the mRNA expression levels of amino acid transporters GLAST,

CAT1 and LAT1 was 2.0, 2.0 and 33 times greater, respectively, than that of TBP. Among ABC transporters, MRP1, MRP4 and BCRP showed higher mRNA expression than TBP, whereas P-gp and MRP5 were detected at relatively low levels. When compared with hCMEC/D3 cells, hiPS-BMECs showed over 200 times lower expression of P-gp (Table 1 and Supporting Fig. 3). Similarly, the mRNA expression level of P-gp in hiPS-BMECs was much lower than that of freshly isolated human brain microvessels reported by Geier et al. (17) (Supporting Fig. 4).

In contrast, the mRNA

expression level of BCRP in hiPS-BMECs was 13 times greater than that in hCMEC/D3 cells.

Although OATP1A2 mRNA was detected in hiPS-BMECs, in contrast to

hCMEC/D3 cells (Table 1), its level was lower than that in freshly isolated human brain microvessels (Supporting Fig. 4).

Table 1 Relative mRNA expression levels of ABC, SLC transporters and receptors in hiPS-BMECs and hCMEC/D3cells.



Gene (Protein name)

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mRNA expression (Target mRNA/TBP mRNA) hiPS-BMECs hCMEC/D3 cells

ABCB1 (P-gp) ABCC1 (MRP1) ABCC4 (MRP4) ABCC5 (MRP5) ABCG2 (BCRP) SLC1A3 (GLAST) SLC2A1 (GLUT1) SLC2A3 (GLUT3) SLC3A2 (4F2hc) SLC5A7 (CHT1) SLC7A1 (CAT1) SLC7A5 (LAT1) SLC16A1 (MCT1) SLC19A1 (RFC1) SLCO1A2 (OATP1A2) SLCO1B1 (OATP1B1) SLCO1C1 (OATP1C1) SLCO2A1 (OATP2A1) SLCO2B1 (OATP2B1) SLC22A1 (OCT1) SLC22A2 (OCT2) SLC22A3 (OCT3)

0.0100 1.82 4.00 0.253 2.03 2.04 16.9 129 8.19 0.241 1.98 32.9 8.28 0.934 0.00687

2.73 ± 3.65 ± 0.688 ± 0.832 ± 0.141 ± 0.0223 ± 8.79 ± 0.503 ± 4.53 ± 0.0261 ± 3.45 ± 5.50 ± 4.09 ± 1.53 ± ULQ 0.0010 ± ULQ 0.435 ± 0.00903 ± 0.00180 ± ULQ 0.00733 ±

0.13 0.26 0.022 0.039 0.013 0.0051 0.52 0.065 0.82 0.0035 0.14 0.31 0.21 0.04

0.00465 4.07 0.0445 0.00500 0.00140 0.00753

± 0.0031 ± 0.04 ± 0.07 ± 0.009 ± 0.03 ± 0.02 ± 0.5 ± 3 ± 0.18 ± 0.021 ± 0.04 ± 0.8 ± 0.28 ± 0.025 ± 0.00081 ULQ ± 0.00191 ± 0.05 ± 0.0092 ± 0.00136 ± 0.00036 ± 0.00223

SLC22A4 (OCTN1) SLC22A5 (OCTN2) SLC22A6 (OAT1) SLC22A7 (OAT2) SLC22A8 (OAT3) SLC29A1 (ENT1) SLC29A2 (ENT2) SLC29A4 (PMAT) SLC35F2 SLC44A1 (CTL1) SLC44A2 (CTL2) SLC47A1 (MATE1)

0.00998 0.513 0.00236 0.120 0.00242 2.02 2.16 0.253 3.93 0.920 15.9 0.00321

± ± ± ± ± ± ± ± ± ± ± ±

0.0273 ± 0.251 ± ULQ ULQ 0.00215 ± 4.19 ± 0.110 ± 0.00337 ± 1.41 ± 3.75 ± 4.12 ± ULQ

0.0055 0.021

0.00129 0.005 0.00056 0.011 0.00131 0.02 0.08 0.005 0.11 0.034 0.3 0.00184


0.0005 0.013 0.00334 0.00081 0.00106

0.00057 0.18 0.015 0.00081 0.51 0.19 0.05

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SLC47A2 (MATE2) OSCP1 MFSD2A AQP4 INSR LRP1 TFRC RAGE

0.223 0.265 0.00881 0.0579 0.806 4.13 0.00981 0.365

± ± ± ± ± ± ± ±

0.015 0.014 0.00188 0.0024 0.017 0.23 0.00245 0.021

0.0413 0.0589 0.0320 0.0136 1.18 0.108 0.0667 0.111

± ± ± ± ± ± ± ±

0.0088 0.0053 0.0064 0.0035 0.03 0.010 0.0055 0.017

mRNA levels of target genes in cells were normalized by that of TATA-binding protein (TBP). Each value represents the mean ± S.E. from three determinations. ULQ, under the limit of quantification.

Tight junction-forming ability of hiPS-BMECs monolayer hiPS-BMECs monolayers cultured on Transwell membranes showed high TEER values (> 2000 Ω・cm2).

The transcellular transport of lucifer yellow (a paracellular

marker) was much less than the transport across the Transwell membrane (< 0.5% of total amount) up to 4 hr (Fig. 1A).

These results suggest strong tight-junction formation in

hiPS-BMECs monolayers.



Figure 1 ---- Accumulation of test compounds in the upper (luminal side, A) and lower (abluminal side, B) chambers across hiPS-BMEC monolayers. (A) Transport of lucifer yellow (a paracellular marker, 10 µM) across the insert membrane with (■) or without (□) hiPS-BMECs was examined. (B-J) Directional transport of prazosin and dantrolene (BCRP substrates, 10 µM and 2 µM, respectively), rhodamine 123 and quinidine (P-gp substrates, 10 µM and 0.5 µM, respectively), gabapentin (a LAT1 substrate, 10 µM), fexofenadine (an OATP1A2 substrate, 10 µM), [14C]L-lactate (a MCT1 substrate, 1 µCi/mL), [3H]L-arginine (a CAT1 substrate, 3 µCi/mL), and [3H]L-glutamate (a GLAST substrate, 2 µCi/mL) across hiPS-BMECs was measured. Open and closed circles show the accumulations in the A-to-B and B-to-A directions, respectively.

Each value represents the mean ± S.E. of three determinations.

Transcellular transport across a hiPS-BMECs monolayer


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The transcellular transport rates in the luminal (upper chamber)-to-abluminal (lower chamber) direction (A-to-B) and abluminal-to-luminal (B-to-A) direction were compared. Transport of prazosin, dantrolene, and rhodamine 123 was greater in the B-to-A direction than in the A-to-B direction (Figs. 1B-D), and the efflux ratio values were 4.6, 5.5, and 4.1, respectively. On the other hand, the transport of quinidine showed no significant difference between the A-to-B and B-to-A directions (Fig. 1E). Transcellular transport rates of gabapentin (a LAT1 substrate), and fexofenadine (an OATP1A2 substrate) also showed no significant difference between the A-to-B and B-to-A directions (Fig. 1F and 1G). [14C]L-Lactate transport from the luminal side at pH 6.0 was greater than that from the basal side at pH 7.4 (Fig. 1H).

In addition, [3H]L-arginine transport in the A-to-B

direction was greater than that in the B-to-A direction (Fig. 1I). Moreover, [3H]Lglutamate showed a higher transport rate in the A-to-B direction compared to the opposite direction (Fig. 1J). The permeability coefficients of all compounds tested in the transcellular transport experiment are listed in Table 2.

The permeability coefficients of diphenhydramine

(79.0 × 10-6 cm/sec) and varenicline (57.0 × 10-6 cm/sec) in the A-to-B direction were higher than that of antipyrine (a passive diffusion marker). The permeability coefficient of fexofenadine was the lowest (0.21 × 10-6 cm/sec) among the clinically used drugs tested.



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Table 2 Permeability coefficient of transporter substrates across hiPS-BMECs. Drug Diphenhydramine Varenicline Antipyrine [14C]L-Lactate [3H]L-Arginine Quinidine Prazosin Gabapentin [3H]L-Glutamate Dantrolene Fexofenadine Lucifer yellow Rhodamine 123

Permeability coefficient (×10-6 cm/sec) A-to-B 79.0 ± 57.0 ± 38.9 ± 34.3 ± 20.0 ± 10.4 ± 8.65 ± 6.14 ± 7.08 ± 2.26 ± 0.208 ± 0.0849 ± 0.0839 ±

B-to-A 0.1 0.3 0.2 1.1†, § 2.7 0.1§ 0.85 0.16 1.11 0.07§ 0.0004 0.0020 0.0020

4.10 5.71 8.77 40.0 5.29 1.94 12.5 0.204 0.343

― ― ― ± ± ± ± ± ± ± ± ― ±

0.31 0.26 0.33§ 0.1 0.26 0.11 0.51§ 0.002 0.035

The permeability coefficients of transporter substrates across hiPS-BMECs in the A-toB and B-to-A directions were measured.

Each value represents the mean ± S.E. of three

determinations. †The

permeability coefficient of [14C]L-lactate from A to B was determined with the A

side at pH 6.0 and the B side at pH 7.4, because MCT1 is a proton-coupled transporter. §The

value was calculated from PStotal/S, since PSe exceeded PSm.

Effects of various compounds and Na+ on transcellular transport across the hiPSBMECs monolayer 20 ACS Paragon Plus Environment

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Next, the effects of selected compounds and Na+ on the directional transport shown in Fig. 1 were assessed (Table 3). Prazosin transport in the B-to-A direction was decreased by elacridar, which inhibits BCRP with an IC50 of 0.15 µM (18). In addition, the permeability coefficient of dantrolene in the A-to-B direction was increased 4-fold, whereas that in the B-to-A direction was decreased in the presence of Ko-143 (a potent inhibitor of BCRP), and the efflux ratio was decreased from 5.53 to 1.02 (Table 3). These results suggest the functional expression of BCRP.

The permeability of

rhodamine 123 in the A-to-B direction was increased 4.7- to 11-fold in the presence of cyclosporin A, verapamil, and PSC-833 (P-gp inhibitors), while the efflux ratio of rhodamine 123 was decreased from 4.0 to near 1. On the other hand, Ko-143 had only a moderate effect on the permeability of rhodamine 123. These results are consistent with reported findings (Table 3) (8, 10, 13, 14). But, in marked contrast, these P-gp inhibitors had little effect on quinidine transport across the hiPS-BMECs monolayer.

[14C]L-

Lactate and [3H]L-arginine transport in the A-to-B direction was inhibited by 10 and 1 mM unlabeled L-lactate and L-arginine, respectively, suggesting the involvement of a carrier-mediated transport process.

[3H]L-Glutamate transport in the A-to-B direction

was clearly decreased in the presence of 1 mM L-glutamate and in the absence of Na+ (Table 3).

Additionally, the B-to-A transport of [3H]L-glutamate was inhibited by 1 mM



Page 22 of 46

1

glutamate and in the absence of Na+ (Table 3). These results suggest that a carrier-

2

mediated, Na+-dependent transport process is involved in bidirectional transport of [3H]L-

3

glutamate.

4 5

Table 3 Effects of selected compounds and Na+ on directional transport across hiPS-

6

BMECs. Drug Prazosin

Dantrolene

Rhodamine 123

Permeability coefficient (× 10-6 cm/sec)

　

B-to-A

Control + 5 µM Elacridar

8.65 ± 0.85 －

40.0 ± 0.1 15.0 ± 0.2

4.62

Control + 1 µM Ko-143

2.26 ± 0.07 § 8.89 ± 0.03 §

12.5 ± 0.5 § 9.06 ± 0.51 §

5.53 1.02

Control + 10 µM Cyclosporin A + 100 µM Verapamil + 10 µM PSC-833 + 1 µM Ko-143

Quinidine

Efflux ratio

A-to-B

　

0.0839 0.908 0.411 0.398

± ± ± ±

0.0020 0.054 0.017 0.026

0.14 ± 0.01 0.1 § 0.09 § 0.07 § 0.08 §

0.343 0.819 0.414 0.460

± ± ± ±

0.035 0.052 0.027 0.057

0.29 ± 0.03

10.4 6.52 9.17 7.56

[14C]L-Lactate

Control + 10 mM L-Lactate

34.3 ± 1.1†, § 5.27 ± 0.24

4.10 ± 0.31 －

[3H]L-Arginine

Control

20.0 ± 2.7 1.27 ± 0.08

5.71 ± 0.26

+ 1 mM L-Arginine

± ± ± ±


8.77 5.27 7.26 6.10

0.33 § 0.13 § 0.24 § 0.25 §

Control + 10 µM Cyclosporin A + 100 µM Verapamil + 10 µM PSC-833

± ± ± ±

－

－

4.09 0.902 1.01 1.16 2.07 0.843 0.808 0.792 0.807

Page 23 of 46


1 2 3 4 5 6 [3H]L-Glutamate Control 7.08 ± 1.11 1.94 ± 0.11 7 + 1 mM L-Glutamate 0.663 ± 0.066 0.572 ± 0.007 8 9 Na+-free 0.810 ± 0.079 0.941 ± 0.040 10 11 1 Permeability coefficients of selected transporter substrates across hiPS-BMECs in the A12 13 14 2 to-B and B-to-A directions were measured under various conditions. Each value 15 16 17 3 represents the mean ± S.E. of three determinations. 18 19 20 †The permeability coefficient of [14C]L-lactate from A to B was determined at an A side 4 21 22 23 5 pH of 6.0 and a B side pH of 7.4, because MCT1 is a proton-coupled transporter. 24 25 26 §The value was calculated from PS 6 total/S, since PSe exceeded PSm. 27 28 29 7 30 31 32 8 Carrier-mediated transport of BBB transporter substrates in hiPS-BMECs 33 34 35 9 To further examine the function of LAT1, OATP1A2, OCTN2, CAT1, GLAST, 36 37 38 10 MCT1, and H+/OC antiporter, we carried out uptake and inhibition studies of selected 39 40 41 11 substrates: gabapentin (LAT1), fexofenadine (OATP1A2), d3-L-carnitine (OCTN2), 42 43 44 12 [14C]L-lactate (MCT1), [3H]L-arginine (CAT1), [3H]L-glutamate (GLAST), and 45 46 47 13 diphenhydramine (H+/OC antiporter). 48 49 50 14 The cell-to-medium (C/M) ratio of each substrate increased in a time-dependent 51 52 53 15 manner (Fig. 2), indicating uptake of the compound into the cells, and was significantly 54 55 56 16 decreased at 4°C (Table 4). The initial uptake rates of gabapentin, fexofenadine, d3-L57 58 59 60



Page 24 of 46

carnitine, [3H]L-arginine, [3H]L-glutamate, [14C]L-lactate, and diphenhydramine were saturable, and the Km values were estimated to be 185 µM, 18.4 µM, 4.08 µM, 45.3 µM, 39.0 µM, 7.45 mM, 48.5 µM, respectively (Fig. 3 and Table 5). These results suggest that a carrier-mediated transport process is involved in each case.

Fig. 2 ---- Time-dependent uptakes of gabapentin (A), fexofenadine (B), d3-Lcarnitine (C), [14C]L-lactate (D), [3H]L-arginine (E), [3H]L-glutamate (F) and diphenhydramine (G) into hiPS-BMECs. hiPS-BMECs were incubated with buffer containing a test compound at 37ºC for the designated time. The buffer pH was adjusted to 7.4, except for the buffer containing [14C]L-lactate (pH 6.0). Cellular uptake is expressed as cell-to-medium ratio.


Each

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value represents the mean ± S.E. of three determinations.

Fig. 3 ---- Concentration-dependence of the initial uptake velocity of gabapentin (A), fexofenadine (B), d3-L-carnitine (C), [14C]L-lactate (D), [3H]L-arginine (E), [3H]Lglutamate (F), and diphenhydramine (G) into hiPS-BMECs. Initial uptakes of drug by hiPS-BMECs were measured at 37ºC. Solid, dotted, and dashed lines represent estimated total, saturable, and nonsaturable uptakes, respectively. Each value is the mean ± S.E. of three determinations.


Molecular Pharmaceutics 1 2 3 4 5 1 6 7 8 2 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 46

Table 4 Effect of selected inhibitors, temperature, and Na+ on uptake of each transporter substrate into hiPS-BMECs. Concentration (mM) Gabapentin (LAT1) Control + L-dopa + L-Phenylalanine + Leucine + BCH + L-Carnitine 4°C Fexofenadine (OATP1A2) Control + Naringine + E1S + PAH 4°C d3-L-Carnitine (OCTN2) Control + Carnitine + L-Arginine + PAH Na+-free 4°C [14C]L-Lactate (MCT1) Control + Valproate + Benzoate + Citrate + L-Phenylalanine 4°C 3 [ H]L-Arginine (CAT1) Control + L-Ornithine

1 1 1 1 1

1 1 1

1 1 1

10 10 10 10

1


Uptake (% of control) 100 13.8 12.4 7.87 7.85

± ± ± ± ±

14 1.1* 14.8* 11.04* 4.28*

97.4 8.30

± ±

22.7 9.54*

100 73.0 72.4 70.7 14.9

± ± ± ± ±

9 5.8* 4.1* 5.0* 2.3*

100 15.0 136 113

± ± ± ± ULQ 20.0 ±

28 2.7* 2 28

100 27.4 23.1 161 115 15.4

± ± ± ± ± ±

9 3.2* 3.7* 2* 6* 2.5*

100

±

12

22.8

±

0.8*

3.6*

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+ L-Leucine 1 + L-Leucine 5 + L-Phenylalanine 1 Na+-free 4°C [3H]L-Glutamate (GLAST) Control + L-Aspartate 1 + UCPH101 0.05 + L-Cystine 1 + L-Alanine 1 Na+-free 4°C Diphenhydramine (proton-coupled organic cation antiporter) Control + Clonidine 1 + Quinidine 1 + Pyrilamine 1 + L-Carnitine 1 + TEA 1 4°C

64.9 54.7 93.5 53.6 1.21

± ± ± ± ±

7.7* 4.5* 8.3 5.3* 0.21*

100 11.5 11.6 110 94.6 5.77 1.70

± ± ± ± ± ± ±

4 0.7* 1.4* 16 7.7 0.54* 0.50*

100 28.0 6.61 5.31 134 109 33.5

± ± ± ± ± ± ±

3 0.9* 1.21* 0.96* 6 9 3.2*

1

Uptake of drugs was measured at 37ºC in the absence or presence of each inhibitor and/or

2

substrate of transporters.

3

value represents the mean ± S.E. from three determinations.

4

difference, *P < 0.05 vs control. ULQ, under the limit of quantification.

Cellular uptake is presented as percent of control.

Each

Symbols show a significant

5 6

Table 5 Kinetic parameters for uptake of transporter substrate into hiPS-BMECs. Kinetic parameters

Drugs Gabapentin

Km 185

±

48

Vmax µM

5.06


±

0.65

nmol/mg protein/min

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 15 16 17 2 18 19 20 3 21 22 23 4 24 25 26 5 27 28 29 6 30 31 32 7 33 34 35 8 36 37 38 9 39 40 41 10 42 43 44 11 45 46 47 12 48 49 50 13 51 52 53 14 54 55 56 15 57 58 59 60

Fexofenadine d3-L-Carnitine [14C]L-Lactate [3H]L-Arginine [3H]L-Glutamate Diphenhydramine

18.4 4.08 7.45 45.3 39.0 48.5

± ± ± ± ± ±

8.2 2.61 1.11 8.2 12.0 19.0

µM µM mM µM µM µM

0.358 0.797 61.1 0.843 0.274 5.08

Page 28 of 46

± ± ± ± ± ±

0.119 0.256 7.4 0.111 0.116 1.05

nmol/mg protein/min pmol/mg protein/min nmol/mg protein/min nmol/mg protein/min nmol/mg protein/min nmol/mg protein/min

In order to estimate the kinetic parameters, the initial uptake velocity of each compound at various concentrations was analyzed.

Michaelis constant (Km) and maximum uptake

rates (Vmax) were calculated using the Michaelis-Menten equation.

Effects of test compounds and Na+ on uptake of drugs by hiPS-BMECs To characterize the uptake of drugs by hiPS-BMECs, we examined the effects of selected compounds and Na+ (Table 4). L-Dopa, L-phenylalanine, L-leucine, and BCH, which are LAT1 substrates and/or inhibitors, all significantly inhibited gabapentin uptake to less than 20% of the control, whereas L-carnitine had little effect. d3-L-Carnitine uptake was significantly inhibited to less than 20% by L-carnitine, but was not inhibited by L-arginine or PAH. L-Lactate uptake was decreased in the presence of valproate and benzoate (MCT1 substrates), in contrast to citrate and L-phenylalanine. L-Arginine uptake was moderately decreased under Na+-free conditions and was strongly inhibited by L-ornithine (a CATs substrate), whereas L-leucine (system y+L and system b0,

+

substrate) and L-phenylalanine (system B0, + substrate) showed moderate inhibition and


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no effect, respectively. L-Glutamate uptake was significantly decreased to below 20% in the absence of Na+ and in the presence of L-aspartate (an EAATs substrate) and UCPH101 (a GLAST inhibitor), but was not inhibited by L-cystine (an xCT substrate) or L-alanine (an ASCTs substrate). Diphenhydramine uptake was significantly inhibited by clonidine, quinidine and pyrilamine (H+/OC antiporter substrates). These results suggest that LAT1, OCTN2, CAT1, GLAST, MCT1, and H+/OC antiporter are functionally expressed in hiPS-BMECs. Fexofenadine uptake was inhibited by naringin and E1S (OATPs substrates and/or inhibitors), but these effects were small, as was that of PAH (an OATs substrate), suggesting that OATP1A2 is not a major contributor to fexofenadine uptake.



Discussion hiPS-BMECs are thought to have great potential to facilitate CNS drug development as an innovative human BBB model, but their functions, especially transport functions, remain to be fully characterized. In the present study, we evaluated the gene expression and transport function of various ABC and SLC transporters in hiPS-BMECs. We first confirmed that the brain capillary endothelial cells had differentiated from the human iPS cell line (IMR90-C4) according to the method reported previously (8-10). Cells differentiated from IMR90-C4 stained positive for PECAM-1, VE-cadherin, claudin-5, occludin, ZO-1, and GLUT1, as shown in Supporting Fig. 1. In addition, cultures of these cells on Transwell membranes showed high TEER values ( > 2000 Ω・ cm2). The permeability coefficient of lucifer yellow (a paracellular marker) was approximately 0.2% of that of antipyrine (Table 2), supporting the idea that paracellular transport is greatly restricted by the formation of strong tight junctions. As shown in Table 1, various ABC and SLC transporters and representative receptors were expressed in hiPS-BMECs.

Among the ABC transporters detected in this study,

BCRP showed 13-fold higher expression in hiPS-BMECs than in hCMEC/D3 cells (Table 1). In addition, BCRP was detected by Western blot analysis, as shown in Supporting Fig. 2. This result indicates that efflux transport via BCRP would be


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substantial in hiPS-BMECs. Indeed, the efflux ratios of prazosin and dantrolene in hiPSBMECs, which were calculated as the ratio of permeability coefficients (B-to-A / A-toB), were more than 4.6, supporting functional expression of BCRP in hiPS-BMECs (Table 2). Furthermore, the efflux ratio was decreased from 5.53 to 1.02 in the presence of Ko-143, suggesting that the Ko-143 concentration used was sufficient to inhibit BCRP. A previous study using PET demonstrated that P-gp is functional at the human BBB, even though its protein expression is lower than in rodents (19). The P-gp gene in hiPSBMECs is expressed at a relatively low level compared to that in hCMEC/D3 cells. Delsing’s group reported similar results (20). In addition, protein expression of P-gp was not detected in hiPS-BMECs, whereas it was detected in hCMEC/D3 cells by Western blot analysis (Supporting Fig. 2). On the other hand, the efflux ratio of rhodamine 123 was 4.1, and it was decreased by cyclosporin A, verapamil, and PSC-833 (representative P-gp inhibitors) (Table 2 and 3). Again, these results are consistent with previous reports (8, 10, 13, and 14). In contrast, however, we found that the transcellular transport of quinidine (a P-gp substrate) showed no significant difference between the A-to-B and Bto-A directions, and the efflux ratio was hardly changed in the presence of cyclosporin A, verapamil, or PSC-833, or Ko-143 (Tables 2 and 3). It has been reported that the efflux ratio of quinidine is comparable to that of rhodamine 123 in human P-gp-overexpressing



MDCK cells and Caco-2 cells (21). It is unclear why there is a discrepancy in the directional transport of rhodamine 123 and quinidine in hiPS-BMECs, but possible explanations include: 1) some other transporter, which shows similar responses to inhibitors to P-gp, mediates directional transport of rhodamine 123, or 2) since quinidine is known to be a substrate of not only P-gp, but also H+/OC antiporter, an active influx transporter (22), transport mediated by another system, possibly H+/OC antiporter, may mask the role of P-gp in quinidine transport in hiPS-BMECs. Further studies will be needed to resolve this. As for SLC transporters at the rodent BBB, it has been demonstrated that MCT1 and CAT1 mediate blood-to-brain influx transport of monocarboxylates and cationic amino acids, respectively (23). We found that the permeability of [14C]L-lactate, a MCT1 substrate, from the luminal side (pH 6.0) to the abluminal side (pH 7.4) is 8-fold faster than that in the opposite direction (pH 7.4 to pH 6.0) (Table 2). In addition, [14C]L-lactate uptake was saturable, with a Km value close to that of human MCT1 expressed in Xenopus laevis oocytes (Km = 6.0 mM) (24), and it was inhibited by the MCT1 substrates valproate and benzoic acid (Table 4). These results strongly suggest that MCT1, an H+-coupled transporter, functions in the transport of not only L-lactate, but also monocarboxylic drugs such as salicylic acid in hiPS-BMECs. Transport of [3H]L-arginine, a CAT substrate, in


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the A-to-B direction was faster than that in the B-to-A direction, and was inhibited by 1 mM L-arginine (Tables 2 and 3). Moreover, hiPS-BMECs expressed CAT1 mRNA (Table 1), and L-arginine uptake was strongly inhibited by L-ornithine (a CATs substrate) (Table 4), suggesting involvement of CAT1 in L-arginine transport from the luminal side to the abluminal side across hiPS-BMECs. On the other hand, L-arginine uptake was partially decreased under Na+-free conditions, and L-leucine (system y+L and system b0,+ substrate) showed a moderate inhibitory effect (Table 4). It is reported that system y+L transports neutral amino acids in a Na+-dependent manner (25). Although it is not clear whether system y+L is involved in L-arginine transport, the above results suggest that a Na+-dependent transport system plays a role, at least in part, in cationic amino acid transport in hiPS-BMECs. In contrast to influx transporters, it is reported that GLAST is involved in brain-toblood efflux transport of L-glutamate at the rodent BBB (23). In hiPS-BMECs, [3H]Lglutamate uptake was Na+-dependent and saturable (Table 4 and Fig. 3), with a similar Km value to that reported for human GLAST expressed in COS-7 cells (Km = 48 µM) (26). In addition, it was inhibited by L-aspartate and by a selective GLAST inhibitor, UCPH101, while L-cystine and L-alanine showed no inhibitory effect (Table 4), suggesting that GLAST is the main contributor to L-glutamate uptake by hiPS-BMECs.



GLAST is predominantly expressed at the abluminal side of the bovine BBB (27), and is involved in the brain-to-blood transport of L-glutamate in bovine and rodents (28, 29). However, the present study showed that L-glutamate transport in the A-to-B direction across hiPS-BMECs was predominant over that in the B-to-A direction (Table 2). These results indicate that there may be a species difference in L-glutamate transport across the BBB between humans and other species. Species difference of BBB transporters of thyroid hormone has been reported (30). The apparent inconsistency may also be due to a difference of GLAST expression in the luminal and abluminal membranes and/or the involvement of a human-specific transport system. Further work will be needed to address this question. The uptake studies of gabapentin (a substrate of LAT1), d3-L-carnitine (a substrate of OCTN2), and diphenhydramine (a substrate of H+/OC antiporter) showed that these substrates are taken up into hiPS-BMECs time-, temperature-, and concentrationdependently (Figs. 2 and 3, Table 4); this is important, because these transporters have been suggested as potential candidates for drug delivery to the brain. The Km values of gabapentin, d3-L-carnitine, and diphenhydramine uptakes were similar to reported values (31-33), and substrates and/or inhibitors of LAT1, OCTN2, and H+/OC antiporter decreased the respective uptakes (Table 4). These results suggest the functional


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expression of LAT1, OCTN2, and H+/OC antiporter in hiPS-BMECs. On the other hand, although hiPS-BMECs expressed OATP1A2 mRNA at a higher level than did hCMEC/D3 cells (Table 1), the uptake of fexofenadine did not show appreciable concentration-dependency and was not inhibited by E1S (a potent inhibitor of OATP1A2) (Fig. 3 and Table 4). It appears that the contribution of OATP1A2 to drug transport in hiPS-BMEC is minor. If hiPS-BMECs are to be used as an in vitro model to evaluate the BBB permeability and transport mechanisms of candidate drugs, we need to know how closely they mimic the human BBB. In this connection, transcellular transport studies enabled us to determine the permeability coefficients of small molecules with a wide range of lipophilicities (4.08 to 2.4 as log D) (34, 35). Further, directional transport through hiPS-BMCEs could be analyzed using the present system, because hiPS-BMECs form strong tight junctions. In particular, the contributions of individual ABC transporters such as BCRP could be successfully evaluated by the present model. Furthermore, in evaluating the BBB permeability of drugs with hiPS-BMECs, it will be important to consider the genetic polymorphisms of transporters. However, the method of differentiation to hiPS-BMEC presented here is limited to one type of hiPS cell line. Therefore, it will be important to develop the method to enable differentiation from hiPS cells with diverse backgrounds in



the future. In conclusion, hiPS-BMECs developed strong tight junctions, and expressed multiple ABC and SLC transporters. In particular, the nutrient and drug transporters LAT1, OCTN2, CAT1, GLAST, MCT1, H+/OC antiporter and BCRP were functionally expressed in hiPS-BMECs. Although further studies are necessary to characterize hiPSBMECs in comparison with other BBB models, our findings here should contribute to the development of high-fidelity in vitro models of the BBB, and should facilitate effective early screening of drug candidates targeting the brain.

Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research and by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, provided by The Ministry of Education, Culture, Sports, Science and Technology.

Supporting Information The supporting information includes detailed experiment methods and supplemental results. Supporting Figure 1: Immunohistochemical staining of endothelial cell markers,


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tight-junction proteins and GLUT1. Supporting Figure 2: Western blot analysis for Pgp and BCRP in hiPS-BMECs and hCMEC/D3 cells. Supporting Figure 3: Quantitative comparison of mRNA expression levels between hiPS-BMECs and hCMEC/D3 cells. Supporting Figure 4: Relationship between log-mRNA levels in hiPS-BMECs and those in freshly prepared human brain capillaries. Supporting Table 1: Sequences of sense and antisense primers used for qPCR. Supporting Table 2: MRM transitions of test compounds.



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Footnotes The authors declare that there is no conflict of interest.

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