1

Title

Prediction of inter-individual differences in hepatic functions and drug sensitivity by

using human iPS-derived hepatocytes

Authors

Kazuo Takayama, Yuta Morisaki, Shuichi Kuno, Yasuhito Nagamoto, Kazuo Harada,

Norihisa Furukawa, Manami Ohtaka, Ken Nishimura, Kazuo Imagawa, Fuminori

Sakurai, Masashi Tachibana, Ryo Sumazaki, Emiko Noguchi, Mahito Nakanishi,

Kazumasa Hirata, Kenji Kawabata, Hiroyuki Mizuguchi

Supporting Information (SI) Appendix

Contents

SI Appendix Fig. S1

SI Appendix Fig. S2

SI Appendix Fig. S3

SI Appendix Fig. S4

SI Appendix Fig. S5

SI Appendix Fig. S6

SI Appendix Fig. S7

SI Appendix Fig. S8

SI Appendix Table S1

SI Appendix Table S2

SI Appendix Table S3

SI Appendix Experimental Procedures

SI Appendix References

2

SI Appendix, Figures

Figure S1. Generation of Human iPSCs From PHHs. PHHs were Infected With

SeVdp-iPS Vectors. (A) The genome structure of a SeVdp-iPS vector is shown. The

SeV vector used in this study is designed to become extinct after iPSC generation since

it encodes a miR302a (strongly expressed in human iPSCs)-target sequence. (B) The

procedure for generation of human iPSCs from PHHs is presented schematically. A

phase-contrast micrograph of the PHHs, which were cultured for 48 hr, is shown (left).

Alkaline phosphatase staining was performed in PHH-iPSCs on day 23 (right). (C) The

gene expression levels of pluripotent markers (OCT3/4, SOX2, and NANOG) and

hepatic markers (ALB, CYP3A4, and αAT) were examined in PHHs, PHH-iPSCs, and

human ESCs (H9). On the y axis, the gene expression levels of pluripotent markers or

hepatic markers in human ESCs or PHHs, respectively, were taken as 1.0. Data

represent the mean ± SD from three independent experiments. Statistical significance

Sendai virus construction SeVdp-iPS vector (genome ; (-) ssRNA)A

KLF4 L

CN

P/V3’ 5’OCT3/4 SOX2 c-MYC

miR-302a

target

B

day0 1 2

SeVdp-iPS

vector

infection (5 MOI)

3

ALP stainingswitch to

iPSC medium

plate

PHHs

plate MEF

onto PHHs

17 - 23

virus Infection

HCM-iPS

iPSC generation

MEF

ReproStem

bFGF (10 ng/ml) + 3 compounds

20 mm 50 mm

C

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

PHHs PHH-iPSCs

101

100

10-1

10-2

10-3

10-4

rela

tive g

en

e e

xp

ressio

n

a

b b

a

b b

a

b b

hESCs

101

100

10-1

10-2

10-3

10-4

10-5

rela

tive

ge

ne

ex

pre

ss

ion

a

b b

a

b b

a

b b

3

was evaluated by ANOVA followed by Bonferroni post-hoc tests to compare all groups

(PHHs, PHH-iPSCs, and hESCs). Groups that do not share the same letter are

significantly different from each other (P<0.05).

4

Figure S2. PHH-iPSCs Could Differentiate into Three Germ Layer Cells and

Originated From PHHs. (A) The embryoid body (EB) structures were generated from

the PHH-iPSCs. A phase contrast image of a PHH-iPS-derived EB is shown. At 2 weeks

from EB formation, the gene expression levels of pluripotent markers (OCT3/4 and

NANOG), definitive endoderm genes (FOXA2 and SOX17), mesoderm genes (T and

TnTc), and ectoderm genes (MAP2 and PAX6) in the undifferentiated human ESCs (H9,

hESCs-day0), human ESC-derived EB (hESCs-EB), undifferentiated PHH-iPSCs

(PHH-iPS-day0), and PHH-iPSC-derived EB (PHH-iPS-EB) were examined by

A

20 mm

rela

tiv

e g

en

e e

xp

ressio

n pluripotent genes definitive endoderm genes mesoderm genes ectoderm genes

0

0.3

0.6

0.9

1.2

OCT3/4 NANOG

0

5

10

15

20

25

30

FOXA2 SOX17

0

10

20

30

40

50

60

T TnTc

0

20

40

60

80

MAP2 PAX6

hESCs-day0 hESCs-EB PHH-iPS-day0 PHH-iPS-EB

muscle neural tissue

B

** * *

* ** * *

*

**

* *

*

*

gut-like

epithelium cartilage adipose issue

C

TH01 D21S11

D5S818 D13S317 D7S820 D16S539 CSF1PO

AMEL vWA TPOX

TH01 D21S11

D5S818 D13S317 D7S820 D16S539 CSF1PO

AMEL vWA TPOX

PH

Hs (

do

no

r1)

PH

H-i

PS

Cs (

do

no

r1)

5

real-time RT-PCR. Data represent the mean ± SD from three independent experiments.

Student’s t test indicated that the gene expression levels in “EB” were significantly

different from those in “day 0” (P<0.01). (B) To perform the teratoma assay, the

PHH-iPSCs (1×106 cells/NOG mouse) were subcutaneously transplanted. At 6 weeks

from the transplantation, the tumors were harvested, and hematoxylin and eosin staining

was performed. (C) STR analysis was performed in PHH and PHH-iPSC-derived

hepatocyte-like cells (PHH-iPS-HLCs).

6

Figure S3. Comparison of the Hepatic Functions Among Early- or Late-Passage

PHH-iPS-HLCs, PBMC#1-iPS-HLCs, HDF-iPS-HLCs, and HUVEC-iPS-HLCs. (A, B)

PHH-iPS (P10 or P40), PBMC-iPS (P30), HDF-iPS (P40), and HUVEC-iPS (P35) were

differentiated into the hepatocyte-like cells as shown in Fig. 2F. The TAT expression (A)

and ALB secretion levels (B) in the PHH-iPS-HLCs (P10 or P40), PBMC#1-iPS-HLCs

(P30), HDF-iPS-HLCs (P40), and HUVEC-iPS-HLCs (P35) were examined. On the y

axis, the gene expression level of TAT in PHHs was taken as 1.0 in SI Appendix, Fig.

S3A. Data represent the mean ± SD from three independent experiments. Statistical

significance was evaluated by ANOVA followed by Bonferroni post-hoc tests to

compare all five groups. Groups that do not share the same letter are significantly

different from each other (P<0.05). The TAT expression and ALB secretion levels in the

PHH-iPS-HLCs (P40) were similar to those in PBMC#1-iPS-HLCs (P30),

HDF-iPS-HLCs (P40), and HUVEC-iPS-HLCs (P35). Note that PHH-iPS-HLCs and

the other HLCs do not share the same genetic background.

A

B

AL

B s

ec

reti

on

(mg

/ml/

24

hr/

mg

pro

tein

)

0

0.25

0.5

0.75

0

10

20

30

a

b

b

b b b

b b b

a

0

0.25

0.50

0.75re

lati

ve

TA

T e

xp

res

sio

n

7

Figure S4. Highly Efficient Hepatocyte Differentiation From Human

Non-PHH-Derived iPSCs or ESCs Independent of Their Differentiation Tendency. (A,

B) PHH-iPS, PBMC#1-iPS, HDF-iPS, and human ES (KhES1) cells were differentiated

into the HLCs via the HBCs as described in Fig. 2A. On day 25 of differentiation, the

efficiency of hepatocyte differentiation was measured by estimating the percentage of

ASGR1- or ALB-positive cells using FACS analysis (A). The amount of ALB or urea

secretion was examined (B). (C, D) PHH-iPSCs were differentiated into the hepatic

lineage, and then PHH-iPS-HBCs were purified and maintained for 3 passages on

human LN111. Thereafter, expanded HBCs were differentiated into the HLCs as

described in Fig. 2F. The efficiency of hepatic differentiation from HBCs was measured

by estimating the percentage of ASGR1- or ALB-positive cells using FACS analysis (C).

The amount of ALB or urea secretion was examined (D). Data represent the mean ± SD.

These results indicated that the hepatic differentiation efficiency of these four lines

could be rendered uniform by performing hepatic maturation after the establishment of

self-renewing HBCs.

0

20

40

60

80

100

1

10

100

0

20

40

60

80

100

1

10

100

A

C

B

D

% o

f an

tig

en

-po

sit

ive c

ells

AL

B o

r u

rea s

ec

reti

on

le

ve

l

% o

f an

tig

en

-po

sit

ive c

ells

AL

B o

r u

rea s

ecre

tio

n level

ASGR1 ALB

ALB

UREA

(mg/ml/24hr/mg protein)

(mg/dl/24hr/mg protein)

ASGR1 ALB

ALB

UREA

(mg/ml/24hr/mg protein)

(mg/dl/24hr/mg protein)

101

100

102

101

100

102

8

Figure S5. Ratio of the CYP Activities in the PHH-iPS-HLCs to those in PHHs, and

Summary of the CYP2D6 Polymorphism Genotyping. (A) The ratios of the CYP1A2,

2C9, and 3A4 activity levels in the PHH-iPS-HLCs to those in the PHHs were

examined. (B) The CYP activity levels in the parent PHHs might be predicted from

those in the PHH-iPS-HLCs by using the formula shown here. “X” represents the CYP

activity levels in the PHH-iPS-HLCs. The predicted CYP activity levels in the parent

PHHs are shown here.

A B

0

0.2

0.4

0.6

0.8

1.0

1.2

CYP1A2 CYP2C9 CYP3A4

rati

o o

f C

YP

ac

tivit

y l

eve

l

(PH

H-i

PS

-HL

Cs

to

PH

Hs

)CYP1A2 CYP2C9 CYP3A4

predicted maximum X*0.87 Y*0.98 Z*0.88

predicted first quartile X*0.67 Y*0.71 Z*0.62

predicted median X*0.59 Y*0.6 Z*0.57

predicted third quartile X*0.45 Y*0.56 Z*0.43

predicted minimum X*0.38 Y*0.35 Z*0.23

9

Figure S6. Comparison of the CYP3A4 Activity Levels Among PHH-iPS-HLCs,

non-PHH-iPS-HLCs, and hES-HLCs. (A) Non-PHH-derived iPS cells (PBMC#1-iPS,

PBMC#2-iPS, HDF-iPS, MRC5-iPS, HUVEC#1-iPS, and HUVEC#2-iPS cells) and

human ES cells (H1, H9, KhES1-4) were differentiated into the hepatocyte-like cells

according to Fig. 2F, and then the CYP3A4 activity levels were measured by

LC-MS/MS analysis. (B) The CYP3A4 activity levels in PHH-iPS-HLCs (12 donor

average), non-PHH-iPS-HLCs (6 donor average), and hES-HLCs (6 donor average)

were shown in the graph. The average and variance of CYP3A4 activity levels were

both similar among these three groups.

0

20

40

60

80

100

0

15

30

45

60

75

PHH-iPS-HLCs (12donors)

non-PHH-iPS-HLCs (6donors)

hES-HLCs (6 donors)

A

B

ac

tivit

y l

eve

l

CYP3A4a

cti

vit

y l

eve

l

CYP3A4

Non-PHH-iPS-HLCs

(6 donors)

hES-HLCs

(6 donors)

10

Figure S7. CYP Induction Capacity and Gene Expression Levels of Transporters in

PHHs and PHH-iPS-HLCs. (A-C) To examine CYP1A2 (A), 2B6 (B), and 3A4 (C)

induction potency, the cells were treated with 50 μM omeprazole for 24 hr, 500 μM

phenobarbital for 48 hr, or 20 μM rifampicin for 48 hr; these agents are known to induce

CYP1A2, 2B6, and 3A4, respectively. The gene expression levels of CYPs were

measured by real-time RT-PCR. Controls were treated with DMSO (final concentration

0.1%). The R-squared values are indicated in each figure. (D, E) The gene expression

profile of CYPs (D) and transporters (E) is shown.

1

10

100

1000

1 2 3 4 5 6 7 8 9 101112

1

10

100

1 2 3 4 5 6 7 8 9 101112

1

10

100

1 2 3 4 5 6 7 8 9 101112

R² = 0.7491

0

100

200

300

400

500

0 10 20

R² = 0.6164

0

10

20

30

40

50

60

70

0 10 20

R² = 0.6353

0

5

10

15

20

25

30

0 5 10

A

C

B

E

101

100

102

103

donor number

donor number

donor number

101

100

102

101

100

102

fold

in

du

cti

on

fold

in

du

cti

on

fold

in

du

cti

on

fold

in

du

cti

on

of

PH

Hs

fold induction of HLCs

fold induction of HLCs

fold induction of HLCs

fold

in

du

cti

on

of

PH

Hs

fold

in

du

cti

on

of

PH

Hs

CYP1A2

CYP2B6

CYP3A4

10+ 1.910- 1.8

D

10+ 2.210- 2.2

11

Figure S8. Tamoxifen-Induced Breast Cancer Cell (T-47D) Toxicity and

Perhexiline-Induced Hepatotoxicity were Examined. (A) The pharmacological activity

of tamoxifen-dependent conversion to its metabolite, endoxifen, by the CYP2D6. The

co-culture system of breast cancer cells (T-47D cells) and the PHH-iPS-HLCs is

illustrated. (B) The cell viability of T-47D cells was assessed after 72 hr exposure to

A B

0

20

40

60

80

100

120

0 1 2 3 4 5

0

20

40

60

80

100

120

0 1 2 3 4 5

0

20

40

60

80

100

0

20

40

60

80

100

0

25

50

75

100

125

0

25

50

75

100

tamoxifen

active form of

tamoxifen, endoxifen

CYP2D6

breast cancer cells, T-47D

PHH-iPS-HLCs or PHHs

perhexilin

CYP2D6

hydroxy-metabolites of drug

hepatotoxic

E

non-hepatotoxic

F

PHH-WT

PHH-NUL

HLC-WT

HLC-NUL

cell v

iab

ilit

ycell v

iab

ilit

y

concentration of tamoxifen

0 50 500 5,000 (nM)

concentration of perhexilin

0 0.5 5 50 (mM)

G

cell v

iab

ilit

y

control quinidine

*

control quinidine

cell v

iab

ilit

y

*

*

*

C D

NO

NE

Ad

-LacZ

Ad

-CY

P2D

6

HL

C-W

T

NO

NE

Ad

-La

cZ

Ad

-CY

P2D

6

PH

H-W

T

PHH-NUL HLC-NUL

PH

H-W

T

PH

H-N

UL

HL

C-W

T

HL

C-N

UL

PH

H-W

T

PH

H-N

UL

HL

C-W

T

HL

C-N

UL

NO

NE

Ad

-La

cZ

Ad

-CY

P2D

6

HL

C-W

T

NO

NE

Ad

-La

cZ

Ad

-CY

P2D

6

PH

H-W

T

PHH-NUL HLC-NUL

cell v

iab

ilit

ycell v

iab

ilit

y

Hd d

aa

b bc c

aa

aa

b b

c c

12

different concentrations of tamoxifen. (C) The cell viability of T-47D cells, which were

co-cultured with PHH-WT, PHH-NUL, HLC-WT, and HLC-NUL, was assessed after

72 hr exposure to 500 nM of tamoxifen in the presence or absence of 3 nM quinidine (a

CYP2D6 inhibitor). (D) The cell viability of T-47D cells, which were co-cultured with

Ad-CYP2D6-transduced PHH-NUL and HLC-NUL, was examined after 72 hr exposure

to 500 nM of tamoxifen. (E) The detoxification of perhexiline-dependent conversion to

its conjugated form by the CYP2D6. (F) The cell viabilities of PHH-WT, PHH-NUL,

HLC-WT, and HLC-NUL were assessed after 24 hr exposure to different concentrations

of perhexiline. (G) The cell viabilities of the PHH-WT and HLC-WT were assessed

after 24 hr exposure to 5 μM of perhexiline in the presence or absence of 5 μM

quinidine (CYP2D6 inhibitor). (H) The cell viabilities of the Ad-CYP2D6-transduced

PHH-NUL and HLC-NUL were examined after 24 hr exposure to 5 μM of perhexiline.

The cell viabilities are expressed as a percentage of that in the cells treated with only

solvent. Data represent the mean ± SD from three independent experiments. In SI

Appendix, Fig. S8C and S8G, student’s t test indicated that the cell viability in “control”

were significantly higher than that in “quinidine” (P<0.01). In SI Appendix, Fig. S8D

and S8H, statistical significance was evaluated by ANOVA followed by Bonferroni

post-hoc tests to compare all groups. Groups that do not share the same letter are

significantly different from each other (P<0.05).

13

SI Appendix, Tables

Table S1. The Primary Antibodies Used in This Study

antigen type company

AFP (IHC) rabbit DAKO

AFP (FACS) mouse Cell Signaling

ALB mouse Abcam

CYP2D6 mouse BD Biosciences

ASGR1 goat Santa Cruz Biotechnology

αAT chicken Abcam

NANOG mouse Santa Cruz Biotechnology

OCT3/4 mouse Santa Cruz Biotechnology

SSEA4 mouse Abcam

SOX2 goat Santa Cruz Biotechnology

Tra1-81 mouse Santa Cruz Biotechnology

KLF4 rabbit Santa Cruz Biotechnology

EpCAM mouse Milltenyi Biotech

CD133 mouse Milltenyi Biotech

b-actin mouse Sigma

control IgG rabbit Santa Cruz Biotechnology

control IgG mouse Santa Cruz Biotechnology

control IgG goat Santa Cruz Biotechnology

14

Table S2. The Secondary Antibodies Used in This Study

antigen fluorescent dye company

rabbit IgG alexa fluor 594 Molecular Probes

rabbit IgG alexa fluor 488 Molecular Probes

mouse IgG alexa fluor 594 Molecular Probes

mouse IgG alexa fluor 488 Molecular Probes

goat IgG alexa fluor 594 Molecular Probes

goat IgG alexa fluor 488 Molecular Probes

chicken IgG alexa fluor 488 Molecular Probes

15

Table S3. The Summary of CYP2D6 Polymorphisms (CYP2D6*3, *4, *5, *6, *7, *8,

*16, and *21) Genotyping is Shown.

sample

CYP2D6 SNPs

*3, *4, *6, *7,

*8 *5 *16 *21

PHH1 *4/wt wt/wt wt/wt wt/wt

PHH2 wt/wt wt/wt wt/wt wt/wt

PHH3 *4/wt wt/wt wt/wt wt/wt

PHH4 wt/wt wt/wt wt/wt wt/wt

PHH5 wt/wt wt/wt wt/wt wt/wt

PHH6 wt/wt wt/wt wt/wt wt/wt

PHH6 wt/wt wt/wt wt/wt wt/wt

PHH8 *4/*4 wt/wt wt/wt wt/wt

PHH9 wt/wt wt/wt wt/wt wt/wt

PHH10 wt/wt wt/wt wt/wt wt/wt

PHH11 *4/*4 wt/wt wt/wt wt/wt

PHH12 wt/wt wt/wt wt/wt wt/wt

16

SI Appendix, Experimental Procedures

Human ESCs/iPSCs Culture

The human ESC lines, H1, H9 (WiCell Research Institute), and KhES1-4

(provided by Dr. N. Nakatsuji, Kyoto University), were maintained on a feeder layer of

mitomycin C-treated EmbryoMax Primary Mouse Embryo Fibroblasts (MEF, Merck

Millipore) with ReproStem medium (ReproCELL) supplemented with 5 ng/ml

fibroblast growth factor 2 (FGF2, KATAYAMA CHEMICAL INDUSTRIES). Human

ESCs were used following the Guidelines for Derivation and Utilization of Human

Embryonic Stem Cells of the Ministry of Education, Culture, Sports, Science and

Technology of Japan and furthermore, and the study was approved by the Independent

Ethics Committee. The human iPSC line, Tic (provided by Dr. A. Umezawa, National

Center for Child Health and Development), PBMC#1-iPSCs, PBMC#2-iPSCs,

and PHH-iPSCs were maintained on a feeder layer of mitomycin C-treated MEF with

ReproStem medium supplemented with 10 ng/ml FGF2. Human iPSC lines generated

from Human Dermal Fibroblasts (HDFs, HDF-iPS) and Human Umbilical Vein

Endothelial Cells (HUVECs, HUVEC#1-iPS and HUVEC#2-iPS) were maintained on a

feeder layer of mitomycin C-treated MEF with ReproStem medium supplemented with

5 ng/ml FGF2.

In Vitro Hepatocyte Differentiation

For each human iPSC line used in hepatocyte differentiation, all differentiated

cells were constantly removed by manual collection with a pipette. Before the initiation

of hepatocyte differentiation, human iPSCs were dissociated into clumps by using

dispase (Roche) and plated onto BD Matrigel Basement Membrane Matrix Growth

Factor Reduced (BD Biosciences). These cells were cultured in the MEF-conditioned

medium for 3-4 days. The differentiation protocol for the induction of definitive

endoderm cells, hepatoblasts, and hepatocytes was based on our previous reports with

some modifications (1-4). Briefly, in the definitive endoderm differentiation, human

iPSCs were cultured for 4 days in L-Wnt3A-expressing cell (ATCC,

CRL2647)-conditioned RPMI1640 medium (Sigma) which contains 100 ng/ml Activin

A (R&D Systems), 4 mM L-Glutamine, 0.2% FBS (PAA Laboratories), and 1×B27

Supplement Minus Vitamin A (Life Technologies). For the induction of HBCs, the

definitive endoderm cells were cultured for 5 days in RPMI1640 medium (Sigma)

17

which contains 30 ng/ml bone morphogenetic protein 4 (BMP4) (R&D Systems) and 20

ng/ml FGF4 (R&D Systems), 4 mM L-Glutamine, and 1×B27 Supplement Minus

Vitamin A. The protocol for HBC proliferation using human recombinant laminin-111

(BioLamina) was based on our previous report with some modifications (3). Briefly, the

hPSC-derived HBCs were first purified from the hPSC-derived cells (day 9) by

selecting attached cells on a human recombinant LN111-coated dish (the final coating

concentration was 1.0 μg/cm2) at 15 min after plating. The hPSC-derived HBCs were

cultured on a human LN111-coated dish (2.5 × 104 cells/cm

2) in maintenance

DMEM/F12 medium (DMEM/F12 medium (Invitrogen) was supplemented with 10%

FBS (PAA laboratories), 1×insulin/transferrin/selenium, 10 mM nicotinamide, 10-7

M

Dexamethasone (DEX) (Sigma), 20 mM HEPES, 25 mM NaHCO3, 1×GlutaMAX, 40

ng/ml hepatocyte growth factor (HGF) (R&D Systems) and 20 ng/ml epidermal growth

factors (EGF) (R&D Systems)). The medium was refreshed every day. The HBCs were

dissociated with Accutase (Millipore) into single cells, and subcultured every 6 or 7

days. To perform the hepatocyte differentiation, the HBCs were cultured for 5 days in

RPMI1640 medium (Sigma) which contains 20 ng/ml HGF, 4 mM L-Glutamine, and

1×B27 Supplement Minus Vitamin A. And then, the cells were cultured for 11 days in

Hepatocyte Culture Medium (HCM, Lonza) without EGF but with 20 ng/ml oncostatin

M (OsM). Unlike the method used in our previous reports (1-6), the current

differentiation method could omit the use of adenovirus-mediated overexpression of

hepatic transcription factors.

CYP Induction

Total RNA was isolated from human iPSCs and their derivatives using an RNeasy

Mini Kit (Qiagen). To measure CYP1A2, 2B6, and 3A4 induction potencies, the gene

expression levels of CYPs were measured by real-time RT-PCR. Real-time RT-PCR

was performed with TaqMan Gene Expression Assays (Applied Biosystems). The

assay IDs for CYP1A2, 2B6, and 3A4 are Hs00167927_m1, Hs04183483_g1, and

Hs00430021_m1 (all from Applied Biosystems), respectively. The cells were treated

with 50 μM omeprazole for 24 hr, 500 μM phenobarbital for 48 hr, or 20 μM rifampicin

for 48 hr (all from Wako); these agents are known to induce CYP1A2, 2B6, and 3A4,

respectively. Controls were treated with DMSO (final concentration 0.1%, Wako).

Inducer compounds were replaced daily. Relative quantification was performed against

a standard curve and the values were normalized against the input determined for the

housekeeping gene, GAPDH and beta-actin (ACTB). The assay IDs for GAPDH and

18

ACTB are Hs02758991_g1 and Hs01060665_g1 (all from Applied Biosystems),

respectively.

UPLC-MS/MS Analyses

All human iPSCs, human iPS-HLCs, and PHHs were cultured with medium

containing 10 μM phenacetin (PHE, Cambridge Isotope Laboratories), 10 μM

diclofenac (DIC, Wako), 1 μM bufuralol (BF, Santa Cruz Biotechnology), or 100 μM

testosterone (TS, Wako). The metabolites of each substrate are acetaminophen (APAP),

4'-hydroxy diclofenac (OHDIC), 1'-hydroxybufuralol (OHBF), and

6β-hydroxytestosterone (OHTS). After the treatment with substrates, the supernatant

was collected at 2 hr, and then immediately mixed with two volumes of acetonitrile

(Wako). Samples were filtrated with AcroPrep Advance 96-Well Filter Plates (Pall

Corporation) for 5 min at 1,750 g, and then the supernatant was analyzed by

UPLC-MS/MS to measure the concentration of metabolite according to each standard

curve. UPLC analysis was performed using an Acquity UPLC (Waters) and MS/MS was

performed on a Q-Premier XE (Waters). The mass spectrometer was set to the

multiple-reaction monitoring (MRM) mode and was operated with the electrospray

ionization source in positive ion mode. MRM transitions (m/z of precursor ion / m/z of

product ion) for APAP, OHDIC, OHBF, and OHTS were 152.0/110, 312.2/166.8,

278.4/186.1, and 305.4/287.5, respectively. For each transition, the cone voltage and

collision energy were set at 28 V, 28 eV (APAP), 24 V, 60 eV (OHDIC), 26 V, 18 eV

(OHBF), and 38 V, 14 eV (OHTS). The dwell time for each MRM transition was set

at 100 milliseconds. LC separations were carried out at 40oC with an Acquity UPLC

BEH C18 column, 1.7 μm, 2.1 X 50 mm (Waters). The mobile phase was delivered at a

flow rate of 0.5 ml/min using a gradient elution profile consisting of solvent A (0.02%

formic acid/distilled water) and solvent B (0.02% formic acid/acetonitrile). The initial

composition of the binary solvent was 10% B from 0 to 0.5 min. Solvent B was

increased from 10 to 100% over 2.0 min. The composition of solvent remained for 1.0

min at 100% B. 10 μl of sample solution was injected into the column. The

concentrations of each metabolite were calculated according to each standard followed

by normalization to the protein content per well.

Generation of Human iPSCs from PHHs

To generate human iPSCs from PHHs, Sendai virus (SeV) vectors carrying

19

OCT3/4, SOX2, KLF4, and c-MYC (SeVdp-iPS vector) were used. SeV vectors were

produced as described previously (7). At 24 hr from the PHHs plating (6.12×104

cells/cm2), PHHs were infected with the SeVdp-iPS vector at 5 multiplicity of infection

(MOI) and incubated with HCM-iPS (HCM-iPS consists of HCM and the rock inhibitor

(Y27632 (10 μM, Millipore) or Thiazovivin (0.5μM, Santa Cruz Biotechnology))).

Three hours after the infection, the medium was replaced with fresh HCM-iPS. The next

day, mitomycin C-treated MEF were plated onto SeV-infected PHHs at a density of

1×104 cells/cm

2 with HCM-iPS, and then cultured for 24 hr. The PHHs were cultured

with ReproStem-iPS (ReproStem-iPS consists of ReproStem medium, 10 ng/ml FGF2,

ALK inhibitor SB431542 (2 μM, Wako), MEK inhibitor PD0325901 (0.5 μM, Wako),

and rock inhibitor (Y27632 (10 μM) or Thiazovivin (0.5μM)) for 2-3 weeks. There was

no difference between Y27632- and Thiazovivin-treated cells in terms of

reprogramming efficiency. The reprogramming efficiency of the primary human

hepatocytes (PHH) was approximately 0.005%. Note that no iPS colonies could be

obtained from PHHs without using SB431542, PB0325901, and rock inhibitor. Then,

the human iPS colonies were manually picked up using a pipette and plated onto MEF

and cultured with ReproStem medium containing 10 ng/ml FGF2 in the absence of

SB431542, PD0325901, and rock inhibitor.

Generation of Human iPSCs from HDFs, HUVECs, and PBMCs

To generate human iPSCs from HDFs and HUVECs (donor #1 and #2),

SeVdp-iPS vectors were used. The HDFs (Cell Applications) were cultured with

DMEM (Sigma) containing 10% FBS (GIBCO), penicillin/streptomycin. The HUVECs

(Lonza) were cultured with EGM-2 (Lonza). At 24 hr after the plating of HDFs or

HUVECs (1.25×104 cells/cm

2), HDFs or HUVECs were infected with the SeVdp-iPS

vector at 5 MOI. Three hours after the infection, the medium was replaced with fresh

DMEM or EGM-2. The next day, SeV-infected HDFs or HUVECs were plated onto

MEF. The HDFs or HUVECs were cultured with ReproStem medium containing 5

ng/ml FGF2 for 2-3 weeks. The three compounds (SB431542, PB0325901, and rock

inhibitor), which were used for generation of PHH-iPS cells, were not required to

generate human iPSCs from HDFs, HUVECs, and PBMCs.

To generate human iPSCs from PBMCs (donor #1 and #2), Yamanaka

factor-expressing SeV vectors (CytoTune-iPS For Blood Cells; DNAVEC) were used.

The PBMCs were cultured with X-VIVO 10 Chemically Defined, Serum-free

Hematopoietic Cell Medium (Lonza). At 24 hr after the plating of PBMCs (1.87×105

20

cells/cm2), PBMCs were infected with the SeV vector at 20 MOI. Twenty-four hours

after the infection, the medium was replaced with fresh X-VIVO 10 Chemically

Defined, Serum-free Hematopoietic Cell Medium. The next day, SeV-infected PBMCs

were plated onto MEF. The PBMCs were cultured with ReproStem medium

containing 5 ng/ml FGF2 for 2-3 weeks.

Benzbromarone-Induced Hepatotoxicity

Cell viability was assessed by using a Cell Counting Kit-8 (DOJINDO

LABORATORIES) according to the manufacturer’s instructions. In Fig. 3E, the cell

viabilities of PHH5/6/9, PHH1/2/12, PHH5/6/9-iPS-HLC, or PHH1/2/12-iPS-HLC were

measured after 24 hr exposure to the different concentrations of benzbromarone (Wako).

The control refers to incubations in the absence of test compounds and was considered

as 100% viability value. Controls were treated with DMSO (final concentration 0.1%).

In Fig. 3F, the percentage of cells with energized mitochondria in PHH5/6/9,

PHH1/2/12, PHH5/6/9-iPS-HLC, or PHH1/2/12-iPS-HLC was examined after 24 hr

exposure to benzbromarone (25 μM) by FACS analysis. The mitochondrial membrane

potential was assessed by using a Mito-ID Membrane potential cytotoxicity kit (Enzo

Life Sciences) according to the manufacturer’s instructions. In energized cells, the

Mito-ID Membrane Potential dye exists as a green-fluorescent monomer in the cytosol,

and also accumulates as orange-fluorescent aggregates in the mitochondria. However, in

apoptotic and necrotic cells, this dye exits mitochondria and exists primarily as

green-fluorescent monomers in the cytosol. The “PHH5/6/9” represents for average

value of cell viability or mitochondrial membrane potential in PHH5, PHH6, and PHH9.

The “PHH1/2/12” represents for average value of cell viability or mitochondrial

membrane potential in PHH1, PHH2, and PHH12. PHH5, PHH6, and PHH9 are top

three for their CYP2C9 activity levels, while PHH1, PHH2, and PHH12 are bottom

three for their CYP2C9 activity levels.

Tamoxifen-Induced Toxicity in the Breast Cancer Cells

The breast cancer cells, MCF7 cells, were cultured with Minimum Essential

Media (MEM, Invitrogen) containing 10% FBS, penicillin/streptomycin, 0.1 mM MEM

Non-Essential Amino Acids Solution (NEAA, Invitrogen), 1 mM sodium pyruvate

(Invitrogen), and 10 μM human insulin (Sigma). The other breast cancer cells, T-47D,

were cultured with the RPMI1640 medium (Sigma) containing 10% FBS,

21

penicillin/streptomycin, and 10 μM human insulin. The hepatocytes (PHHs or

PHH-iPS-HLCs) and breast cancer cells were co-cultured by using a Millicell Cell

Culture Insert & Plates (Millipore) in the presence of various concentrations of

tamoxifen (Sigma). The breast cancer cells were cultured on the plate, while the

hepatocytes were cultured on the insert. After 72 hr in co-culture, the insert was

removed, and then the cell viability of MCF7 and T-47D cells was measured by using a

Cell Counting Kit- 8 (Figs. 4D and SI Appendix, Fig. S8, respectively). The control

refers to incubations in the absence of test compounds and was considered as 100%

viability value. Controls were treated with DMSO (final concentration 0.1%). In the

CYP2D6 inhibition assays, the hepatocytes and breast cancer cells were co-cultured

with the medium containing 500 nM tamoxifen in the presence or absent of 3 μM

quinidine (Sigma) (Figs. 4E and SI Appendix, Fig. S8). In the CYP2D6 overexpression

experiments, the PHH-NUL and HLC-NUL were transduced with 75 vector particles

(VPs)/cell and 125 VPs/cell, respectively, of Ad-LacZ or Ad-CYP2D6 for 90 min and

cultured for 48 hr. Thereafter, the hepatocytes and breast cancer cells were co-cultured

with the medium containing 500 nM tamoxifen (Figs. 4F and SI Appendix, Fig. S8).

Desipramine- and Perhexiline-Induced Hepatotoxicity

Cell viability was assessed by using a Cell Counting Kit-8 according to the

manufacturer’s instructions. In Figs. 4J or SI Appendix, Fig. S8, the cell viabilities of

the PHH-WT, PHH-NUL, HLC-WT, or HLC-NUL were measured after 24 hr exposure

to different concentrations of desipramine or perhexiline (both from Sigma). The control

refers to incubations in the absence of test compounds and was considered as 100%

viability value. Controls were treated with DMSO (final concentration 0.1%). In the

CYP2D6 inhibition assays, the PHH-WT, PHH-NUL, HLC-WT, or HLC-NUL were

cultured with medium containing 5 μM desipramine or perhexiline in the presence or

absence of 3 μM quinidine (Sigma) (Figs. 4K and SI Appendix, Fig. S8, respectively).

In the CYP2D6 overexpression experiments, the PHH-NUL and HLC-NUL were

transduced with 75 VPs/cells and 125 VPs/cell, respectively, of Ad-LacZ or

Ad-CYP2D6 for 90 min, and cultured for 48 hr. Thereafter, the hepatocytes were

cultured with medium containing 5 μM desipramine or perhexiline (Figs. 4L and SI

Appendix, Fig. S8, respectively).

Ad Vectors

22

Ad vectors were constructed by an improved in vitro ligation method (8, 9). The

human CYP2D6 gene (accession number NM_000106.5) was amplified by PCR using

primers: CYP2D6 Fwd 5’- gtTCTAGAggtatggggctagaagcactg-3’ and CYP2D6 Rev 5’-

gtGCGGCCGCctagcggggcacagcac -3’. The human CYP2D6 gene was inserted into

pHMEF5 (10), which contains the human elongation factor-1α (EF-1α) promoter,

resulting in pHMEF-CYP2D6. The pHMEF-CYP2D6 was digested with I-CeuI/PI-SceI

and ligated into I-CeuI/PI-SceI-digested pAdHM41-K7, resulting in pAd-CYP2D6. The

human EF-1α promoter-driven LacZ-expressing Ad vectors (Ad-LacZ) were

constructed previously (11). All of Ad vectors contain a stretch of lysine residue (K7)

peptides in the C-terminal region of the fiber knob for more efficient transduction of

human iPSCs and its derivatives, in which transfection efficiency was almost 100%, and

purified as described previously (1, 5, 12). The VP titer was determined by using a

spectrophotometric method (13).

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from human ESCs/iPSCs and their derivatives using

ISOGENE (NIPPON GENE). cDNA was synthesized using 500 ng of total RNA with a

Superscript VILO cDNA synthesis kit (Invitrogen). Real-time RT-PCR was performed

with SYBR Green PCR Master Mix (Applied Biosystems) using a StepOnePlus

real-time PCR system (Applied Biosystems). Relative quantification was performed

against a standard curve and the values were normalized against the input determined

for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR

primers sequences were obtained from qPrimerDepot (http://primerdepot.nci.nih.gov/).

Alkaline Phosphatase (ALP) Staining and Immunohistochemistry

ALP staining was performed using an ALP detection kit (Chemicon) according to

the manufacturer’s instructions. To perform the immunohistochemistry, the cells were

fixed with 4% PFA in PBS for 20 min. After incubation with 0.1% Triton X-100 (Wako)

in PBS for 10 min, the cells were blocked with PBS containing 2% FBS and 2% bovine

serum albumin (BSA) for 50 min, the cells were incubated with a primary antibody

(described in SI Appendix, Table S1) at 4°C overnight, and finally, incubated with a

secondary antibody (described in SI Appendix, Table S2) at room temperature for 1 hr.

ELISA

23

The culture supernatants, which were incubated for 24 hr after fresh medium was

added, were collected and analyzed for the amount of ALB secretion by ELISA. Human

Albumin ELISA Quantitation Set was purchased from Bethyl Laboratories. ELISA was

performed according to the manufacturer’s instructions. The amount of ALB secretion

was calculated according to each standard followed by normalization to the protein

content per well.

Urea Secretion

The culture supernatants, which were incubated for 24 hr after fresh medium was

added, were collected and analyzed for the amount of urea secretion. QuantiChrom Urea

Assay Kit was purchased from BioAssay Systems. The experiment was performed

according to the manufacturer’s instructions. The amount of urea secretion was

calculated according to each standard followed by normalization to the protein content

per well.

PHHs Culture

Platable cryopreserved human hepatocytes were purchased from VERITAS (lot

AKB, BEB, DOO, FCL, IZT, OHO, QOQ, YEM, and YOW), CellzDirect (Hu8072),

XenoTech (HC2-14), and Lonza (7F3063). The vials of hepatocytes were rapidly

thawed in a shaking water bath at 37°C, and then the contents of the vial were emptied

into prewarmed Cryopreserved Hepatocyte Recovery Medium (CHRM, Life

Technologies) and the suspension was centrifuged at 750 rpm for 10 min at room

temperature. The hepatocytes were seeded at 1.25x105 cells/cm

2 in HCM containing

10% FBS (Life Technologies) onto Cellmatrix Type I-A acid-soluble type I collagen

(Nitta Gelatin)-coated plates. The medium was replaced at 6 hr after seeding. The

hepatocytes, which were cultured 48 hr after plating the cells, were used in the

experiments.

Embryoid Body Formation and Teratoma Assay

The generation of embryoid bodies and in vitro differentiation was performed as

described elsewhere (14). For the teratoma assay, subcutaneous transplantation of the

PHH-iPSCs (1×106 cells/NOG mouse) was performed. At 6 weeks from the

24

transplantation, the tumors were harvested and fixed in 4% PFA. Hematoxylin and

eosin staining was carried out at the Applied Medical Research Laboratories, Inc. All

animal experiments were conducted in accordance with institutional guidelines.

STR Analysis

Total DNA was isolated from the PHHs and PHH-iPSCs using a DNeasy Blood &

Tissue Kit (Qiagen). STR analysis was carried out at Takara Bio, Inc. As shown in SI

Appendix, Fig. S2C, the PHH1-iPSCs indeed originated from PHH1. All other

PHH-iPSCs also originated from PHHs.

Determination of CYP2D6 SNP

Total DNA was isolated from the PHHs and PHH-iPSCs using a DNeasy Blood &

Tissue Kit (Qiagen). SNP analysis was carried out at the Falco SD Holdings Co., Ltd. A

summary of the genotyping of CYP2D6 polymorphisms (CYP2D6*3, *4, *5, *6, *7, *8,

*16, and *21) is shown in SI Appendix, Table S3. Although the CYP2D6

polymorphism genotype was examined in more than 50 lots of PHHs, there were only

two PHH donors who had the two null alleles for CYP2D6 (*4/*4). The PHH-WT used

in Figure 4 were randomly chosen, and then purchased from the companies described

above in the “PHHs Culture” section.

Western Blotting Analysis

The human iPSC-derivatives and PHHs were homogenized with lysis buffer (20

mM HEPES, 2 mM EDTA, 10% glycerol, and 1% Triton X-100) containing a protease

inhibitor mixture (Sigma). After being frozen and thawed, the homogenates were

centrifuged at 15,000 g at 4°C for 10 minutes, and the supernatants were collected.

The lysates were subjected to SDS-PAGE on 7.5% polyacrylamide gel, and then

transferred onto polyvinylidene fluoride membranes (Millipore). After the reaction was

blocked with 1% skim milk in TBS containing 0.1% Tween 20 at room temperature for

1 hr, the membranes were incubated with anti-human CYP2D6 or β-actin antibodies at

4°C overnight, followed by reaction with horseradish peroxidaseconjugated anti-mouse

IgG antibodies at room temperature for 1 hr. The band was visualized by ECL Plus

Western blotting detection reagents (GE Healthcare) and the signals were read using an

LAS-4000 imaging system (Fuji Film). All the antibodies are listed in SI Appendix,

25

Table S1.

SI Appendix, References

1. Takayama K, et al. (2012) Efficient generation of functional hepatocytes from

human embryonic stem cells and induced pluripotent stem cells by HNF4alpha

transduction. Mol Ther 20(1):127-137.

2. Takayama K, et al. (2012) Generation of metabolically functioning hepatocytes from

human pluripotent stem cells by FOXA2 and HNF1alpha transduction. J Hepatol

57(3):628-636.

3. Takayama K, et al. (2013) Long-Term Self-Renewal of Human ES/iPS-Derived

Hepatoblast-like Cells on Human Laminin 111-Coated Dishes. Stem Cell Reports

1(4):322-335.

4. Inamura M, et al. (2011) Efficient generation of hepatoblasts from human ES cells

and iPS cells by transient overexpression of homeobox gene HEX. Mol Ther

19(2):400-407.

5. Takayama K, et al. (2011) Efficient and directive generation of two distinct

endoderm lineages from human ESCs and iPSCs by differentiation stage-specific

SOX17 transduction. PLoS One 6(7):e21780.

6. Takayama K, et al. (2014) CCAAT/enhancer binding protein-mediated regulation of

TGFbeta receptor 2 expression determines the hepatoblast fate decision.

Development 141(1):91-100.

7. Nishimura K, et al. (2011) Development of defective and persistent Sendai virus

vector: a unique gene delivery/expression system ideal for cell reprogramming. J

Biol Chem 286(6):4760-4771.

8. Mizuguchi H & Kay MA (1998) Efficient construction of a recombinant adenovirus

vector by an improved in vitro ligation method. Hum Gene Ther 9(17):2577-2583.

9. Mizuguchi H & Kay MA (1999) A simple method for constructing E1- and

E1/E4-deleted recombinant adenoviral vectors. Hum Gene Ther 10(12):2013-2017.

10. Kawabata K, Sakurai F, Yamaguchi T, Hayakawa T, & Mizuguchi H (2005) Efficient

gene transfer into mouse embryonic stem cells with adenovirus vectors. Mol Ther

12(3):547-554.

11. Tashiro K, et al. (2008) Efficient adenovirus vector-mediated PPAR gamma gene

transfer into mouse embryoid bodies promotes adipocyte differentiation. J Gene Med

10(5):498-507.

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12. Tashiro K, et al. (2010) Adenovirus vector-mediated efficient transduction into

human embryonic and induced pluripotent stem cells. Cell Reprogram

12(5):501-507.

13. Maizel JV, Jr., White DO, & Scharff MD (1968) The polypeptides of adenovirus. I.

Evidence for multiple protein components in the virion and a comparison of types 2,

7A, and 12. Virology 36(1):115-125.

14. Takahashi K, et al. (2007) Induction of pluripotent stem cells from adult human

fibroblasts by defined factors. Cell 131(5):861-872.