Supplementary Content

ER stress reliever enhances functionalities of in vitro cultured

hepatocytes

Jeong Seong Kim, Seon In Hwang, Jung Lim Ryu, Hee Su Hong, Ji-Min Lee, Sang Min Lee, Xiong Jin, Choongseong Han, Jong-Hoon Kim, Jaeseok Han, Man-Ryul Lee, and

Dong-Hun Woo

Supplementary figuresFigure S1, Figure S2, Figure S3, Figure S4, Figure S5

Supplementary tableTable S1, Table S2

Supplemental information

Supplementary Figure S1

Fig S1. Expression of ER stress-related genes in various cell sources. Relative

expression analysis of ATF3, ATF4, ATF6, PERK, and XBP1 in undifferentiated ESCs

(1), fetal liver (2), ESC-derived hepatocytes differentiated from Hay’s protocol (3), ESC-

derived hepatocytes differentiated from Si-tayeb’s protocol (4), present study (5),

HepG2 (6), and adult liver (7).

Supplementary Figure S2

Fig S2. Selection of ER stress reliever to increase the maturation of hESC-

derived hepatocytes. (A) Schematic diagram depicting the treatment of ER stress

reliever at hepatic maturation stage 1. (B) Relative gene expression analysis of

Albumin after treatment of ER stress reliever TUDCA and 4PBA with the concentration

of 10, 20 and 50 μM at hepatic maturation stage. Expression levels were normalized to

those of non-treated (control) sample. Error bars indicate the standard deviation of

triplicate values. *P<0.05, **P<0.01.

Supplementary Figure S3

Fig S3. Treatment of ER stress reliever consistently reduces UPR genes in

differentiating hepatocytes and increased hepatic gene expression after

differentiation. (A) Relative gene expression analysis of hepatic genes (ALB, AAT,

TAT, HNF4A, and TTR) in the differentiated hepatocytes from Hay’s protocol and Si-

tayeb’s protocol with or without TUDCA treatment in hepatic induction stage. Error bars

indicate the standard deviation of triplicate values. *P<0.05, **P<0.01. (B) Relative

gene expression pattern of ER-stress related genes at three different batches (#1, #62,

and #64) of differentiated hepatocytes with or without treatment of 50 μM TUDCA.

Expression levels were normalized to each of non-treated (control) samples. Error bars

indicate the standard deviation of triplicate values. *P<0.05, **P<0.01.

Supplementary Figure S4

Fig S4. Gene Set Enrichment Analysis (GESA) of UPR genes in 3 different human

induced pluripotent stem cells (iPSCs), iPSC-derived hepatoblast and iPSC-

derived hepatic organoids. (A) Comparison of UPR gene expression in

undifferentiated iPSCs (16 samples) and iPSC-derived hepatoblast (HB, 136 samples).

P < 0.01. (B) Comparison of UPR gene expression in iPSC-derived hapatoblasts and

hepatic organoids (phLO, 170 samples). P > 0.05.

Supplementary Figure S5

Fig S5. Functional analysis of hESC-derived hepatocytes, HepG2, and human

primary hepatocytes. (A) Comparison of albumin secretion in differentiated

hepatocytes from hESCs with or without transient treatment of TUDCA in hepatic

induction stage. ***P < 0.001 versus control. (B) Comparison of CYP3A4 activity in

differentiated hepatocytes from hESCs with or without transient treatment of TUDCA in

hepatic induction stage. “+” indicates 50 μM of TUDCA and “++” indicates 100 μM of

TUDCA. (C) Comparison of CYP3A4 activity in differentiated hepatocytes from hESCs

with or without TUDCA and 50 μM of Rifampicin. “+” indicates 50 μM of TUDCA and “+

+” indicates 100 μM of TUDCA. **P < 0.01 versus no treated. (D) The amount of

human serum Albumin was measured in cultured HepG2 and primary hepatocytes after

treatment of TUDCA. n.s. indicates no significance.

Supplementary Table S1. Primers used for qPCR.

Gene Name Primer sequence

ALB 5’- CACGCCTTTGGCACAATGAA-3’5’- ATCTCGACGAAACACACCCC-3’

HNF4α 5’- ACTACATCAACGACCGCCAGT-3’5’- ATCTGCTCGATCATCTGCCAG-3’

AAT 5’- CCTCCGTACCCTAAACCAGC-3’5’- TTCTTGGCCTCTTCGTGATCC-3’

TAT 5’- GTGGCTGCACGGCAGTGTGTCCCCA-3’5’- AGCCAGCGCTTGGCCAGCCCTCCA-3’

TTR 5’- CCACTCATTCTTGGCAGGAT-3’5’- AGGTGTCATCAGCAGCCTTT-3’

CYP1A2 5’- GGAGCAGGATTTGACACAGTCA-3’5’- GCTCCTTCTGGATCTTCCTCTGTA-3’

CYP2B6 5’- AGCTTCATGACCGAGCCAAA-3’5’- CAGGATTGAAGGCGTCTGGT-3’

CYP2D6 5’- GTGTCCAACAGGAGATCGACG-3’5’- CACCTCATGAATCACGGCAGT-3’

CYP3A4 5’- GGTGGTGAATGAAACGCTCAG-3’5’- CACCCCTTTCCCAATGAACA-3’

ATF3 5’- CCTCTGCGCTGGAATCAGTC-3’5’- TTCTTTCTCGTCGCCTCTTTT-3’

ATF4 5’- ATGACCGAAATGAGCTTCCT-3’5’- GCTGGAGAACCCATGAGGT-3’

ATF6 5’- TCCTCGGTCAGTGGACTCTTA-3’5’- CTTGGGCTGAATTGAAGGTTT-3’

BIP 5’- CATCACGCCGTCCTATGTCG-3’5’- CGTCAAAGACCGTGTTCTCG-3’

GADD34 5’- ATGATGGCATGTATGGTGAG-3’5’- AACCTTGCAGTGTCCTTATCA-3’

sXBP1 5’- GGTCTGCTGAGTCCGCAGCA-3’5’- GGGCTTGGTATATATGTGG-3’

tXBP1 5’- CCCTCCAGAACATCTCCCCAT-3’5’- ACATGACTGGGTCCAAGTTGT-3’

CHOP 5’- GGAAACAGAGTGGTCATTCCC-3’5’- CTGCTTGAGCCGTTCATTCTC-3’

PERK 5’- ATCTGTTCAGCTCTGGGTTG-3’5’- GAGGTCCGACAGCTCTAACA-3’

GAPDH 5’- CACTCCACCTTTGACGC-3’5’- GGTCCAGGGGTCTTACTCC-3’

Supplementary Table S2. List of antibodies used for immunofluorescence

and western-blot analysis

Antibody Dilution Source

Mouse anti-ALBUMIN 1:500 Sigma (A6684)

Rabbit anti-HNF4α 1:500 Abcam (ab92378)

Mouse anti-ASGPR1 1:50 Thermo Scientific (MA1-40244)

Goat anti-NTCP 1:100 Santacruz (sc-107030)

eIF2α 1:2000 Abcam (ab32157)

p-eIF2α 1:2000 Cell Signaling technology (#9722)

HSP90α/β 1:2000 Santacruz (sc-13119)