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ORIGINAL
Role of Nrf2 signaling in development of hepatocyte-like cells
Chie Takasu MD, PhD, Shuhai Chen MD, Luping Gao MD, PhD, Yu Saito MD, PhD, Yuji Morine MD, PhD,
Tetsuya Ikemoto MD, PhD, Shinichiro Yamada MD, PhD, and Mitsuo Shimada MD, PhD
Department of Surgery, Institute of Health Biosciences, Tokushima University, Tokushima, Japan

Abstract : Generation of hepatocytes from human adipose-derived mesenchymal stem cells (hADSCs) could be
a promising alternative source of human hepatocytes. However, mechanisms to differentiate hepatocytes fromhADSCs are not fully elucidated. We have previously demonstrated that our three-step differentiation protocol
with glycogen synthase kinase (GSK) 3 inhibitor was effective to improve hepatocyte functions. In this study,
we investigated the activation of the nuclear factor erythroid-2 related factor 2 (Nrf2) on hADSCs undergoing
differentiation to HLC (hepatocyte-like cells). Our three-step differentiation protocol was applied for 21 days
(Step 1 : day 1-6, Step2 : day 6-11, Step3 : day 11-21). Our results show that significant nuclear translocation of Nrf2
occurred from day 11 until the end of HLC differentiation. Nuclear translocation of Nrf2 and CYP3A4 activity
in the GSK3 inhibitor-treated group was obviously higher than that in Activin A-treated groups at day 11. The
maturation of HLCs was delayed in Nrf2-siRNA group compared to control group. Furthermore, CYP3A4 activity
in Nrf2-siRNA group was decreased at the almost same level in Activin A-treated group. Nrf2 translocation mightenhance the function of HLC and be a target for developing highly functional HLC. J. Med. Invest. 70 : 343-349,
August, 2023
Keywords : HLC, ADSC, differentiation

INTRODUCTION
Globally, more than 1 million people die of liver disease every
year (1, 2). For acute or chronic liver failure associated with endstage liver dysfunction, liver transplantation is regarded as the
only available therapy. However, considering the lack of healthy
donors, hepatocyte transplantation shows potential as an alternative treatment – except for liver transplantation (3-5). The
demand for liver transplantation has significantly outstripped
the supply of donor organs. Consequently, multiple attempts to
expand the availability of donor organs have been employed,
including opt-out organ donation programs, use of suboptimal
donor organs [e.g. from deceased cardiac donors or steatotic(fatty) livers], split-donor transplantation, and living-donor livertransplantation (6, 7). Fortunately, unlike most other organs, theintroduction of exogenous hepatocytes into the liver parenchyma
is a relatively simple undertaking, suggesting that the liver ishighly amenable to tissue therapy using induced pluripotent
stem cell–derived hepatocytes (8, 9). Several studies showed thathuman mesenchymal stem cells (MSCs) derived from adipose
tissue, bone marrow, or umbilical cord blood are capable of differentiating into hepatocyte-like cells (HLCs) in specialized in vitro
culture conditions (10-14). Recent studies also demonstrated that
hepatocytes derived from human adipose stem cells (hADSCs)
are a potentially scalable and applicable alternative to humanhepatocytes (13, 15-18). However, a precise mechanism to generate mature HLCs from hADSCs remains unclear.
The nuclear factor erythroid-2 related factor 2 (Nrf2) / Kelchlike ECH-associated protein 1 (Keap1) pathway protects cells
against oxidative stress. In response to oxidative stress, Nrf2
dissociates from Keap1 and enters the nucleus, whereby it
Received for publication August 1, 2022 ; accepted March 26, 2023.
Address correspondence and reprint requests to Chie Takasu, MD,
PhD, Department of Surgery, The University of Tokushima, 3-18-15
Kuramoto-cho, Tokushima, 770-8503, Japan and Fax : +81-88-6319698. E-mail : takasu.chie@tokushima-u.ac.jp

regulates transcription of hemeoxygenase-1 and other target
genes encoding proteins for detoxification, energy metabolism,or other cytoprotective functions (19-23). For example, activatedNrf2 signaling reportedly plays a protective role in liver injury
associated with nonalcoholic steatohepatitis, and overexpressionof hepatic Nrf2 can functionally restore liver injury in mousemodels (24). Furthermore, the Nrf2 / farnesoid X receptor / liver X
receptor pathway was found to regulate lipogenesis and CYP3A4
expression in murine hepatocytes (25). Here, we investigated the
involvement of Nrf2 in differentiation of HLCs from hADSCs.

MATERIALS AND METHODS

Culture and differentiation of hADSCs
For this study, we applied our established three-step differentiation protocol, as previously reported (26). Briefly, hADSCs(StemPro Human Adipose-Derived Stem Cells, R7788-115)
purchased from Invitrogen (Waltham, MA, USA) and cultured
in MesenPRO RS medium (Thermo Fisher Scientific, USA).
hADSCs were passaged three times before use in our three-step
differentiation protocol.
In Step 1, definitive endoderm differentiation was induced
by incubating cells with Dulbecco’s Modified Eagle’s Medium
with F-12 Supplement containing 0.5 mg / mL bovine serum
albumin fraction V (BSA ; Sigma-Aldrich, Tokyo, Japan) and2 μM CHIR99021 [glycogen synthase kinase 3 (GSK3) inhibitor ; Selleck Chemicals, Houston, TX, USA) for 24 h from day 3.
The following day, 1% insulin-transferrin-selenium (ITS, Sigma-Aldrich) was added to the medium and cells were cultured
for 2 days. In Step 2, subsequent hepatoblast differentiation
was promoted by changing the medium to Minimum Essential
Medium with non-essential amino acids (Thermo Scientific) supplemented with 0.5 mg / mL BSA, 1% ITS, 20 ng / mL bone morphogenic protein 2 (PeproTech, Rocky Hill, NJ, USA), and 30
ng / mL fibroblast growth factor 4 (FGF4, PeproTech) for 5 days.
In Step 3, hepatocyte differentiation is stimulated by treating
cells with 20 ng / mL hepatocyte growth factor (HGF, PeproTech)

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for 5 days, followed by 20 ng / mL HGF, 10 ng / mL oncostatin M(PeproTech), and 1 × 10-6 M dexamethasone (Sigma-Aldrich) for
another 5 days.
In the Nrf2-knockdown group, hADSCs were transfectedwith an Nrf2 siRNA (10 nmol / L, S9492 ; Applied Biosystems,
Waltham, MA, USA) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s
instructions.

CYP3A4 activity assay

Immunofluorescence

Culture supernatants of HLCs from each step were isolated
and measured using a urea nitrogen kit (BioAssay Systems, San
Francisco, CA, USA) to quantify urea production. Optical density values of sample plate wells were detected with a plate reader.

We cultured the cells in chamber slides (Matsunami, Lot
No. 191029) for immunofluorescence. Next, we removed thecovers of chamber slides, washed the cells in the bottom withphosphate-buffered saline (PBS, 4°C), and fixed slides with 4%paraformaldehyde. After washing with PBS, slides were treatedwith Triton X-100 (Lot No. 308T1683 ; Kanto Chemical Co.,Tokyo, Japan), washed again, and incubated with an anti-Nrf2
primary antibody (ab62352 ; Abcam, Cambridge, UK) overnightat 4°C. The next day, slides were washed and incubated witha fluorophore-conjugated secondary antibody (A21428 ; Life
Technologies, Waltham, MA, USA), and sealed with ProLong
Gold Antifade Reagent with DAPI (P36931 ; Thermo Fisher
Scientific, MA, USA). Finally, slides were observed and imaged
with a fluorescence microscope (Keyence Corporation, Itasca,
IL, USA). Nrf2 translocation was defined when the color showed
yellow (blue ; nucleus, red ; Nrf2, yellow : merged red and green)(27-29). The translocation ratio of Nrf2 was obtained from thenumber of Nrf2 nuclear stained cells (yellow) divided by the
number of DAPI stained cells (DAPI) in five independent ×40
magnification.

RNA isolation and real-time PCR (RT-PCR)
Total RNA was isolated with a RNeasy Mini Kit (Qiagen,Hilden, Germany) and subsequently synthesized into complementary DNA with a reverse transcription kit (Applied Biosystems). RT-PCR was performed in triplicate using a StepOnePlus
Real-Time PCR System (Applied Biosystems). Human GAPDH(4352339E, Applied Biosystems) was used as an internal control.

CYP3A4 enzyme activity was assessed by a P450-Glo assay(Promega, Madison, Wisconsin) in accordance with the manufacturer’s instructions. Luminescence was measured with aluminometer (2104-0010 ; Envision, Waltham, Massachusetts).

Urea Assay

Statistical analysis
Data analysis was performed with GraphPad Prism 5 software, which was used to conduct one-way ANOVA and Mann–
Whitney U tests. All data are presented as mean ± standard
deviation. Values of P < 0.05 were considered statisticallysignificant.

RESULTS

Differentiation of HLCs from hADSCs
First, we differentiated hADSCs to HLCs according to ourestablished three-step protocol (26) (Fig. 1A). The morphology
of HLCs changed during each step ; eventually, HLCs adopted
a round shape compared with the long, spindle-like shape ofhADSCs (Fig. 1B). We next evaluated expression of relevant
genes to confirm the HLC differentiation. Our results confirmed
significant decreases of stemness markers such as SOX (SRYBox Transcription Factor) 2 (p < 0.001, Fig. 2A) and OCT4 (octamer-binding transcription factor 4) (p < 0.01 Fig. 2B), as wellas significant increases of the expression of endoderm-specificgenes such as SOX17 (p < 0.001, Fig. 2C) and CXCR4 (C-X-C
Chemokine Receptor Type 4) (p < 0.001, Fig. 2D) at day 6 (endof Step 1). Next, the elevation of hepatocyte-related gene AFP,a hepatoblast marker, was confirmed during Step 2 (Fig. 2C).
Finally, expression of the hepatocyte gene ALB, a marker ofhepatocyte maturation, was confirmed during Step 3 (Fig. 2D).

Figure 1. Differentiation of HLCs from hADSCs.
(A) Three-step protocol for differentiation of HLCs. (B) Morphology of HLCs at day 6, day 11, and day 21.

The Journal of Medical Investigation Vol. 70 August 2023
Gene expression and hepatocyte functions
Expression of genes indicative of the ammonium clearancefunction of hepatocytes, namely CPS1 (Carbamoyl phosphatesynthase 1) (p < 0.001, Fig. 3A), SLC25A1 (Fig. 3B), and AAT(alpha-1 antitrypsin) (p < 0.05, Fig. 3C), were notably increasedin HLCs compared with hADSCs, although there was still a

345

great distance to reach levels observed in primary hepatocytes.
Similar results were observed for CYP3A4 activity, as indicatedby significantly increased expression of Luciferin-IPA in HLCs
at day 21 (p < 0.001, Fig. 3D). Urea synthesis was also higher in
HLCs at Day 21 compared with hADSCs (p < 0.001, Fig. 3E) but
lower than primary hepatocytes.

Figure 2. Hepatic-specific gene expression during three-step differentiation of hADSCs into HLCs.
Step 1 : mRNA expression of SOX2 (A), OCT4 (B), SOX17 (C), and CXCR4 (D) in HLCs at day 6.
Step 2 : mRNA expression of AFP, a hepatoblast marker, in HLCs at day 11 (E).
Step 3 : mRNA expression of ALB, a hepatocyte maturation marker, in HLCs day 21 (F).
* P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3. Expression of hepatic-specific genes in HLCs.

mRNA expression of (A) CPS1, (B) SLC25A1, and (C) AAT in HLCs at day 21. (D) CYP3A4 activity at day 21.
(E) Urea Assay of HLCs at day 21. * P < 0.05, ** P < 0.01, *** P < 0.001.

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C. Takasu, et al. Role of Nrf2 in HLC

Nuclear translocation of Nrf2 increased during differentiation of
HLCs
We assessed the distribution of Nrf2 (cytoplasmic or nuclear)
during the 21 days of differentiation by immunofluorescence(Fig. 4A). Nuclear translocation of Nrf2 was not observed atday 6 (end of Step 1) during definitive endoderm differentiation.
However, nuclear translocation of Nrf2 was notably increased atduring hepatoblast differentiation compared to Step 1 (Fig. 4B,
P < 0.001). The rate of nuclear translocation of Nrf2 was still
highly observed at day21 (Step 3) but there was no difference
between Step 2 and Step 3.

Nuclear translocation of Nrf2 in the presence of Activin A or GSK3
inhibitor
Our protocol used CHIR99021 (GSK3 inhibitor) to induce
definitive endoderm differentiation during Step 1. We also investigated the effect of Activin A, a multifunctional cytokine, thatplays important roles in the microenvironment of hepatocyte
phenotypes and regional endoderm specification (1). Activin
A was generally used for definitive endoderm differentiation
during Step 1. Nuclear translocation of Nrf2 was investigatedamong the three groups : untreated hADSCs, GSK3 inhibitor-treated hADSCs, Activin A-treated hADSCs. No differencesin nuclear translocation of Nrf2 were observed between thethree groups at day 6 (end of Step 1 ; Fig. 5A). Although nuclear
translocation of Nrf2 was observed in Activin A-treated cells, it
was significantly higher in the GSK3 inhibitor-treated groupcompared to untreated hADSCs (p < 0.001) and Activin A-treated hADSCs (p < 0.001) at day 11 (end of Step 2 ; Fig. 5B and 5D).
Nuclear translocation of Nrf2 in the GSK3 inhibitor-treatedgroup was still significantly higher than the other three groups(Fig. 5C and 5E, p < 0.001).

Effect of knockdown of Nrf2 in differentiation of HLC
We next knocked down Nrf2 expression during Step 1 withsiRNA. The results of morphological analyses showed that cells
in the siRNA-treated group at day 21 were not completely differentiated into HLCs (Fig. 6A). Nrf2-siRNA treated hADSCs
at day 21 did not have a spindle shape and had spindle form.
Functional comparison of HLCs with a CYP3A4 activity assay(Luciferin-IPA) revealed that the GSK3 inhibitor-treated group
had significantly higher activity than the other three groups (pFigure 4. Nuclear translocation of Nrf2 in each step.

< 0.05) (Fig. 6B). However, this result was cancelled by the addition of siRNA Nrf2. CYP3A4 activity in Nrf2-siRNA treatedhADSCs was almost same in that in Activin A-treated hADSCs.

DISCUSSION
In the present study, we established human HLCs derived
from hADSCs according to a three-step protocol (15). Nrf2 wasactivated during the procedure, and the maturation of HLCs
was delayed in Nrf2-siRNA group. Thus, Nrf2 might be a target
for developing highly functional human HLCs.
Generation of HLCs derived from hADSCs could be a promising alternative source of human hepatocytes. Of the donor
sources of hepatocytes proposed, including induced pluripotent
stem cells, embryonic stem cells, and MSCs, MSCs may be best
cell source because they can overcome problems associated withgenetic damage, rejection, and ethics. In particular, ADSCs – atype of MSC derived from fat tissues – can be collected frompatients with minimal invasiveness. We previously reported theeffectiveness of a three-dimensional culture system for HLCs
generated from hADSCs with an recombinant peptide μ-piece(26). However, mechanisms underlying the differentiation ofhepatocytes from hADSCs are not well elucidated.
We previously reported that Nrf2, a transcription factor that
regulates cellular defenses against oxidative stress (30), protects
against liver ischemia / reperfusion injury (31) and calcineurin
inhibitor-induced nephrotoxicity (32) by inhibiting oxidative
stress, inflammation, and apoptosis. In addition, Nrf2 maintains
optimal levels of intracellular reactive oxygens species to regulate adipocyte differentiation (33). Moreover, Nrf2 overexpression in MSCs induce stem-cell marker expression and enhancesosteoblastic differentiation by preventing apoptosis under oxidative stress (34). Considering this perspective, we anticipated arole for Nrf2 in regulating hADSC differentiation.
Wnt signaling is key for the differentiation of MSCs (35).
Wnt / β-catenin signaling regulates foregut endoderm fate, proliferation, and morphogenesis (36). Previous studies revealed
that Wnt signaling is required to specify definitive endoderm
from hADSCs, and manipulations of Wnt signaling through useof GSK3 inhibitors have been applied to direct differentiationof definitive endoderm and hepatocytes (37). GSK3 inhibitors
upregulate transcription factors involved in specification of(A) Immunofluorescence staining of Nrf2 expression in HLCs at each step. (B) Quantification and graphicalpresentation of the Nrf2 translocation rates. *** P < 0.001.

The Journal of Medical Investigation Vol. 70 August 2023

Figure 5. Nuclear translocation of Nrf2 in each group.

Immunofluorescence staining of Nrf2 expression following treatment with GSK3 inhibitor, Activin A, or GSK3
inhibitor with Nrf2 siRNA at day 6 (A), day 11 (B), and day 21 (C). Quantification and graphical presentationof the Nrf2 translocation rates at day 11 (D) and day 21 (E). *** P < 0.001.

Figure 6. Morphology of HLCs in siRNA-Nrf2.

(A) Morphology of HLCs at day 21 (B) CYP3A4 activity was highest in the GSK3 inhibitor-treated group.
However, this effect was cancelled by Nrf2 siRNA. * P < 0.05, ** P value < 0.01, *** P < 0.001.

347

348

C. Takasu, et al. Role of Nrf2 in HLCdefinitive endoderm, namely GATA4 (GATA Binding Protein4), FOXA2 (Forkhead Box A2), SOX17, and CXCR4 (15). In thisstudy, we used the GSK3 inhibitor (CHIR99021) in Step 1 andsuccessfully acquired homogenous hepatic progenitor cells. Notably, GSK3β is a negative regulator of Nrf2 (38). Exposure of cells
to inducers of antioxidant response elements (AREs), such as oxidative stress, leads to the dissociation of Nrf2 from its cytosolic
inhibitor Keap1 in a process mediated by protein kinase C (38).
After nuclear translocation, Nrf2 promotes the AREs of manycytoprotective genes in combination with other transcription
factors, such as small musculoaponeurotic fibrosarcoma, activation transcription factor 4, and polyamine-modulated factor 1.
Nuclear export of Nrf2 is regulated by the tyrosine kinase Fyn.
Moreover, GSK3β acts upstream of Fyn kinase to control nuclear export and subsequent proteasomal degradation of Nrf2. Because a previous report showed an equal ability of CHIR99021
and Activin A to induce hepatic progenitor cells (15), the roleof Nrf2 seems to be not vital for endoderm differentiation. Inline with this notion, application of the GSK3 inhibitor did not
significantly enhance Nrf2 nuclear translocation during Step 1
in the present study compared with Activin A. However, knockdown of Nrf2 during Step 1 resulted in a longer time for HLCs
to mature in the present study, implicating the importance of theNrf2 pathway in promoting HLC differentiation. Moreover, ratesof Nrf2 nuclear translocation were higher in the presence of the
GSK3 inhibitor compared with Activin A in Step 2. Thus, Nrf2
may play an important role in hepatoblast differentiation not in
definitive endoderm differentiation.
Another possible mechanism is FGFR4-GSK3β signaling,which promotes translocation of Nrf2 to the nucleus. FGFR4
receptors on the surface of hepatocellular carcinoma cells wereactivated by FGF19 through inactivation of GSK3β in the tumor
microenvironment (39). Although it the role of the FGFR4GSK3β-Nrf2 pathway under basal conditions is unknown, thehomodimer of FGFR4 binds with FGF subfamily members
including FGF4 or FGF19. We used FGF4 for hepatoblast differentiation in Step 2. FGF4 is the key cytokine required for
differentiation of MSCs into a hepatic lineage (40). Nuclear
translocation of Nrf2 mainly occurred during Step 2. Taken
together, the FGFR4-GSK3β-Nrf2 pathway may play a key rolein hepatoblast differentiation, although further investigation isneeded.
As a limitation of the present study, we did not investigate
other molecules in the Wnt signaling pathway or GFR4-GSK3βNrf2 pathway. Second, we did not investigate the effect of a
specific Nrf2 inducer, such as dimethyl fumarate. Furthermore,
only single siRNA was used in this study. Third, we did not perform transplantation of HLCs in this study to verify their actual
function in vivo.
In conclusion, we demonstrated that Nrf2 was activatedduring differentiation of HLCs, especially during the hepatoblast differentiation step, whereas knockdown of Nrf2 might
delay the maturation and impaired specific functions of HLCs.
Our findings suggest that Nrf2 is a target for developing highly
functional human HLCs.

CONFLICT OF INTEREST STATEMENT
The authors declare no competing interests.

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