Transplantation of hESC-derived hepatocytes protects mice from liver injury
Tolosa et al. Stem Cell Research & Therapy
Transplantation of hESC-derived hepatocytes protects mice from liver injury
Laia Tolosa 0 1 2 5
Jérôme Caron 0 2 5
Zara Hannoun 0 2 5
Marc Antoni 0 2 5
Silvia López 1
Deborah Burks 4
Jose Vicente Castell 1 3
Anne Weber 0 2 5
Maria-Jose Gomez-Lechon 1 3
Anne Dubart-Kupperschmitt 0 2 5
0 Univ Paris-Sud, UMR-S 1193 , Villejuif F-94800 , France
1 Unidad de Hepatología Experimental, IIS LA Fe , Valencia S-46026 , Spain
2 INSERM, U 1193, Hôpital Paul Brousse , Villejuif F-94807 , France
3 CIBERehd , FIS, Barcelona S-08036 , Spain
4 CIBERDEM, Centro de Investigacion Prıncipe Felipe , Valencia S-46012 , Spain
5 DHU Hepatinov , Villejuif F-94800 , France
Background: Hepatic cell therapy has become a viable alternative to liver transplantation for life-threatening liver diseases. However, the supply of human hepatocytes is limited due to the shortage of suitable donor organs required to isolate high-quality cells. Human pluripotent stem cells reflect a potential renewable source for generating functional hepatocytes. However, most differentiation protocols use undefined matrices or factors of animal origin; as such, the resulting hepatocytes are not Good Manufacturing Practice compliant. Moreover, the preclinical studies employed to assess safety and function of human embryonic stem cell (hESC)-derived hepatocytes are generally limited to immunodeficient mice. In the present study, we evaluate the generation of hepatocytes under defined conditions using a European hESC line (VAL9) which was derived under animal-free conditions. The function capacity of VAL9-derived hepatocytes was assessed by transplantation into mice with acetaminophen-induced acute liver failure, a clinically relevant model. Methods: We developed a protocol that successfully differentiates hESCs into bipotent hepatic progenitors under defined conditions, without the use of chromatin modifiers such as dimethyl sulphoxide. These progenitors can be cryopreserved and are able to generate both committed precursors of cholangiocytes and neonate-like hepatocytes. Results: Thirty days post-differentiation, hESCs expressed hepatocyte-specific markers such as asialoglycoprotein receptor and hepatic nuclear factors including HNF4α. The cells exhibited properties of mature hepatocytes such as urea secretion and UGT1A1 and cytochrome P450 activities. When transplanted into mice with acetaminophen-induced acute liver failure, a model of liver damage, the VAL9-derived hepatocytes efficiently engrafted and proliferated, repopulating up to 10 % of the liver. In these transplanted livers, we observed a significant decrease of liver transaminases and found no evidence of tumourigenicity. Thus, VAL9-derived hepatocytes were able to rescue hepatic function in acetaminophen-treated animals. Conclusions: Our study reveals an efficient protocol for differentiating VAL9 hESCs to neonatal hepatocytes which are then able to repopulate livers in vivo without tumour induction. The human hepatocytes are able to rescue liver function in mice with acetaminophen-induced acute toxicity. These results provide proof-of-concept that replacement therapies using hESC-derived hepatocytes are effective for treating liver diseases.
Human embryonic stem cells (hESCs); Directed differentiation; Hepatocytes; Transplantation; Liver injury; Preclinical animal model; Cell therapy
Hepatocyte transplantation has been proposed as an
alternative to orthotopic liver transplantation for
treatment of patients with acute liver failure (ALF) and
metabolic disorders. Various clinical trials using
hepatocyte transplantation have demonstrated partial
improvement of liver function. However, the transplanted
hepatocytes are unable to rescue patients due to the
inadequate levels of engraftment [
]. Moreover, there
is an increasing shortage of viable and functional
sources of human hepatocytes and the number of
patients who die (15 %) while on the liver transplant
waiting list has increased over the last few years. Recent
advances targeted towards the differentiation of human
embryonic stem cells (hESCs) or reprogrammed human
induced pluripotent stem cells (hiPSCs) to various cell
lineages offer significant promise for in vitro studies
and as a source of viable cells for use in therapy. In the
case of liver injuries or metabolic diseases, only a single
cell type, the hepatocyte, is required . Thus, the
generation of an unlimited supply of these cells from
pluripotent stem cells should be an important factor
when translating stem cell biology into the clinic.
Lastly, clinical data from patients with macular
degeneration treated with hESC-derived retinal cells have
demonstrated that hESCs may provide a potentially safe
renewable and reliable source of cells for the treatment
of various disorders [
]. Studies on liver development
in model organisms have identified genes and signalling
pathways vital for the formation of the hepatic lineage
] and, in recent years, a number of laboratories
have reported various protocols that can successfully
differentiate both hESCs and hiPSCs into
hepatocytelike cells by recapitulating liver development. The
differentiation process is based on the initial induction of
definitive endoderm [
], followed by hepatic
specification then differentiation into foetal hepatocyte-like
cells (HLCs) [
] and, finally, further maturation into
albumin-producing HLCs lacking nevertheless
important features of adult primary hepatocytes. However,
these approaches are based on culture media that
contain serum and or chromatin modifiers (such as
dimethyl sulphoxide or sodium butyrate), complex
matrices such as Matrigel and/or the use of mouse
embryonic fibroblasts as feeder cells. All of above are a
source of unknown factors that could obscure the
molecular mechanisms controlling human liver
development or render the resulting tissues incompatible with
future clinical applications. Over the last decade, our
team and others have developed approaches using fully
defined culture conditions required to generate HLCs
from hESCs and hiPSCs [
]. Pluripotent stem cells
were differentiated into a homogenous population of
endoderm cells, which were then induced to differentiate
further into hepatic bipotent progenitors, hepatoblasts,
and then into foetal hepatocytes. It should be noted
that all the approaches that have been developed on
hESC differentiation into HLCs, including ours, have
been conducted almost exclusively on a few US hESC
lines, H9 being the most popular for generating HLCs
In the liver, heterotypic cell interactions between
parenchymal cells and their non-parenchymal neighbours
result in the regulation of differentiation and tissue
proliferation in a three-dimensional microenvironment [
The in vitro differentiation protocol does not
reproduce this complex, three-dimensional, multicellular
environment of the native liver. In this context, the
engraftment and survival of HLCs in a native liver
parenchyma should promote further maturation and
long-term repopulation of transplanted cells. To date,
the engraftment of human stem cell-derived HLCs has
been described in a few models of immunodeficient
mice with transgene-induced [
13, 16, 23
] or chemically
] liver toxicity with low efficiencies.
These data suggest that the transplanted cells were
not responsive to the regenerative stimuli of host
mouse liver and, therefore, not functional in vivo.
However, the relationship between the level of HLC
differentiation from pluripotent cells and the
engraftment efficiency is not known. We previously
demonstrated that foetal hepatoblasts isolated from human
livers at an early stage of development (11–13 weeks
of gestation) were able to engraft and exhibit in vivo
mature functions such as CYP3A4 and
a-glutathioneS-transferase activity [
]. Taken together, these
data suggest that the transplanted stem cell-derived
hepatic cells lacked various functions involved in their
engraftment within the host parenchyma compared to
foetal cells .
Utilising a hESC line derived in Centro de
Investigacion Principe Felipe (CIPF) [
], we report a strategy
for the efficient generation of functional human
hepatocytes from VAL9 hESCs in animal-free conditions.
Using sequential modulation of the different signalling
pathways involved in the various developmental
stages, we were able to generate cells that mimic
functions of neonate hepatocytes (taken as reference) that
also demonstrate key features of hepatocytes including
the expression and activation of crucial cytochrome
P450 enzymes and UDP glucuronosyltransferase 1A1
(UGT1A1). Finally, we demonstrate, for the first time,
that the VAL9 hepatocytes (VAL9-HEP) were able to
engraft and repopulate up to 20 % of the liver and
rescue mice with acetaminophen-induced acute liver
injury post-transplantation. These findings emphasize
the potential value of these cells for use in liver cell
VAL9 hESCs were obtained from the Spanish National
Stem Cell Bank (http://www.isciii.es/ISCIII/es) after
approval of the InnovaLiv project by the following ethics
committees: Spanish “Comision Nacional Medicina
Regenerativa” on 21 May 2012 and French Agency of
Biomedicine on 25 June 2012. VAL9 hESCs were cultured
in feeder-free conditions on culture dishes pre-coated with
0.05 mg/ml Geltrex (Life technologies) in Nutristem
medium (Biological industries) supplemented with 8 ng/
ml fibroblast growth factor (FGF)2 (CellGenix) at 37 °C/
5 % CO2 in animal-free conditions [
The hiPSC line was already established in the
laboratory from human foreskin fibroblasts. hiPSCs were
maintained on MEF feeders in DMEM/F12 medium
supplemented with 20 % knockout serum replacement,
1 mM L-glutamine, 1 % non-essential amino acids,
0.1 mM β-mercaptoethanol and 4 ng/ml FGF2 at 37 °
C/5 % CO2. Prior to differentiation, hiPSCs were plated
on culture dishes pre-coated with Geltrex and
maintained in the same conditions as VAL9 hESCs for a few
passages before starting differentiation.
Hepatic differentiation of hESCs and hiPSCs was
performed following a multistep protocol adapted from
Hannan et al. [
] up to the hepatoblast stage. First,
Val9 hESCs at 80–90 % confluence are subjected to a
1day treatment with 100 ng/ml Activin A (CellGenix),
100 ng/ml basic (b)FGF (CellGenix), 10 ng/ml BMP4
(R&D Systems), 10 μM LY294002 (Cayman Chemical
Company) and 3 μM CHIR99021 (Miltenyi Biotec). On
day 2, cells are exposed to the same cytokines in the
absence of CHIR, and finally, on day 3, cells are exposed to
100 ng/ml Activin A, 100 ng/ml bFGF to induce
definitive endoderm (DE). The efficiency of induction of DE
was then assessed by immunofluorescence for SOX17,
FOXA2 and GATA binding protein 4 (GATA4) and flow
cytometry for CXCR4. Only cell preparations with above
85 % of CXCR4-positive cells were used for further
differentiation. Cells were then exposed to 50 ng/ml
Activin A for 3 days and 4 additional days to 10 ng/ml
BMP4 and 10 ng/ml FGF10 (Biovalley) to promote
hepatic specification. The hepatoblast stage was then
assessed by immunofluorescence of hepatocyte nuclear
factor (HNF)4α, CK19 and alpha foetoprotein (AFP) and
flow cytometry for epithelial cell adhesion molecule
(EpCAM). Confluent hepatoblast cells were then passed
1 to 2 in the presence of hepatocyte growth factor
(HGF) 50 ng/ml to collagen-coated dishes and cultured
in hepatocyte culture medium (HCM; Lonza) in the
presence of HGF (Peprotech) and oncostatin M (OSM;
Peprotech) for 3 days. Finally, cells were maintained in
HCM in the presence of 20 ng/ml HGF up to day 30. The
percentage of asialoglycoprotein receptor (ASGR)-positive
cells was used to validate the efficiency of differentiation.
The differentiation was also determined by
immunofluorescence of hepatocyte markers such as albumin (ALB) and
Cells were fixed with 4 % paraformaldehyde for 15 minutes
at room temperature and permeabilized with 0.5 % Triton
X-100 for 15 minutes. They were then incubated in 3 %
bovine serum albumin (BSA)-phosphate-buffered saline
(PBS) for 30 minutes at room temperature. Primary
antibodies were diluted in 1 % BSA-PBS, and incubated
overnight at 4 °C. Secondary antibodies were diluted in
1 % BSA-PBS and incubated for 1 hour at room
temperature. (See primary and secondary antibody
dilutions and information in Additional file 1: Table S1).
All the photographs were taken with a Leica HMR
microscope (Leica Microsystems).
Flow cytometry analysis
Cells were dissociated with cell dissociation buffer, and
suspended in 3 % BSA-PBS. They were then incubated
with SSEA4-PE, CXCR4-APC or EpCAM-FITC
conjugated antibodies or in control isotypes for 30 minutes at
4 °C in the dark. Cells were then washed with PBS,
centrifuged, and suspended in PBS-BSA 1 % for analysis.
Cells were detected in FL2 and FL4 channels with an
Accuri C6 flow cytometer (BD biosciences). Dead cells
were eliminated with 7AAD staining (Beckman coulter
A07704). For ASGR analysis, cells were incubated with
anti-ASGR antibody for 30 minutes at 4 °C and then
with the secondary antibody Alexafluor 488 in the dark
for 30 minutes at room temperature. Cells were then
washed twice and suspended in PBS-BSA 1 % for
analysis in the Accuri C6 flow cytometer. Quantification of
cell death after thawing was analyzed by 7AAD staining.
RNA extraction and real-time quantitative PCR
Total RNA was extracted from hepatocytes using a
commercial kit (Qiagen) following the manufacturer’s
recommendations. The amount of isolated RNA was estimated
by ribogreen fluorescence and its purity was assessed by
the absorbance ratio 260/280 nm. Total RNA (1 μg) was
reverse-transcribed and real-time quantified using SYBR
Green I Master and the appropriate primers (Additional
file 2: Table S2) in a LightCycler 480 instrument. In
parallel, the mRNA concentration of human housekeeping
βactin was always analysed as an internal normalization
control. The real-time monitoring of the polymerase chain
reaction (PCR) and the precise quantification of the
products in the exponential phase of the amplification were
performed with the LightCycler Relative Quantification
Analysis software (Roche Applied Sciences) in
accordance with the manufacturer’s recommendations.
Moreover, a positive sample with a stable ratio of target and
reference cDNA (a calibrator) was included in each
PCR run to normalize all the samples within one run
and to provide a constant calibration point among
several amplification runs.
Lentivector production and transduction of VAL9-derived cells
The EF1α–green fluorescent protein (GFP) and
CYP3A4GFP lentivectors were constructed and produced by
Vectalys. The apolipoprotein A-II (APOA-II)–GFP
lentivector was constructed in the laboratory and produced by
On day 13 of hepatic differentiation, cells were washed
once with PBS, and fresh HamF12/Williams (HPM)
supplemented with HGF (20 ng/ml final concentration) was
added. The lentivectors were used at a multiplicity of
infection (MOI) of 10 and were incubated with cells overnight.
The cells were then cultured following the normal protocol.
For transplantation experiments, the VAL9-HEP were
transduced at day 28 of differentiation and subsequently
injected 2 days later.
Functional characterization of differentiated cells
Ureogenesis was assessed in the thawed cells by
measuring the formation of urea from NH4+, according to
]. The periodic acid-Schiff (PAS) staining system was
purchased from Sigma-Aldrich. In order to assess the
response to hormones, VAL9-HEP were incubated with
insulin (10−7 M) and/or glucagon (10−6 M) for 24 hours
prior to the assay. Culture dishes containing cells were
fixed in 4 % paraformaldehyde. Further assay was under
the manufacturer’s instruction.
The indocyanine green (ICG) uptake test was assayed
by incubating differentiated cells in medium containing
1 mg/ml ICG for 60 minutes at 37 °C. Cells were then
washed three times with media and fresh HCM is added.
ICG release was evaluated 24 hours later.
Cytochrome P450 activities were assayed by
differentiated cells with a cocktail mixture of substrates for five
individual P450 enzymes: 10 μM phenacetin (CYP1A2),
10 μM diclofenac (CYP2C9), 10 μM bufuralol (CYP2D6),
50 μM chlorzoxazone (CYP2E1) and 5 μM midazolam
(CYP3A4). After 24 hours of incubation at 37 °C, cell
media were recovered and stored at −80 °C until analysis.
Formation of the corresponding metabolites was
quantified by high-performance liquid chromatography tandem
mass spectrometry (HPLC/MS; Waters) as previously
UGT1A1 activity was assayed by incubating
differentiated cells in medium containing 15 μM β-estradiol for
24 hours. Cell media were recovered and stored at −80 °C
until analysis, The formation of the corresponding
metabolite was measured by HPLC/MS, as previously
Animals and induction of acute liver failure (ALF)
Animals were housed at the animal facilities of the
Instituto de Investigación Sanitaria La Fe. All animals had
free access to food and water in a
temperaturecontrolled room with a 12-hour dark/light cycle. All the
animals received human care and all the experimental
protocols were approved by the Institutional Animal
Ethics Committee (Comite Etico de Bienestar Animal) of
the La Fe Hospital and performed in accordance with
Spanish national and institutional regulations. Male
NOD/SCID mice (4–6 weeks) were treated with 300 mg
acetaminophen (APAP)/kg to induce ALF 3 hours prior
to cell transplantation. ALF was evaluated by means of
histological staining and determination of transaminases
in the sera of treated animals.
Transplantation of VAL9-HEP into mice with ALF
At day 30 of differentiation, VAL9-HEP were collected
and injected into the spleen of NOD/SCID mice with
ALF. Three hours after the injection of APAP, mice were
anaesthetized with a sevoflurane/O2 mixture and the
lower pole of the spleen was exposed. Animals received
an intrasplenic injection of 1 × 106 VAL9-HEP in 200 μl
infusion medium within seconds. The control mice,
which had also received APAP treatment, received an
intrasplenic injection of only the infusion medium [
At different time points, mice were sacrificed under
anaesthesia (sevoflurane/O2 mixture). Blood was collected
and serum aliquots were protected from light and stored
at −80 °C until analysis. Liver and spleen were collected
and stored at −80 °C until the histological analyses.
From each tissue specimen, serial sections (7 μm) were
cut with a cryostat (Micron HM 505 N) for fluorescent
Evaluation of tumourigenicity of differentiated cells
In order to assess the tumourigenic potential of
VAL9HEP, different tissues were histologically analysed. For
this purpose, hematoxylin-eosin staining was performed
and samples were examined by a pathologist in search of
any sign of tumourigenicity.
Enzyme-linked immunosorbent assay (ELISA) analysis
The human ALB values secreted into the medium of VAL9
cells (prior to or after differentiation) and secreted into the
sera of transplanted animals were determined by the
Human Albumin ELISA Quantitation kit (Bethyl; http://
www.bethyl.com) following manufacturer’s instructions.
Evaluation of engraftment of VAL9-HEP
Several sections from different lobes were used for the
evaluation of the engrafted cells. The number of
GFPhepatocytes around portal and centrolobular veins was
counted and referred to the total number of hepatocytes.
Cholangiocyte differentiation and biliary cyst formation
hESC were differentiated into cholangiocytes as described
by Dianat et al. [
The biliary cysts were generated as follows: day 18 cells
were detached with trypsin, centrifuged and resuspended
in biliary differentiation media (BDM; William’s E/Ham
F12 1:1, 10−5 M linoleic acid-albumin (Sigma L9530), 5 ×
10−8 M 3,3′,5-triiodo-L-thyronine (Sigma T2752), 0.2 IU
insulin, 6.10−4 M vitamin C (Boyer), 6 × 10−4 M human
apo-transferrin (Sigma T5391), 1 mM sodium pyruvate
(Gibco)). The cells were then added to a mixture of
rattail type I collagen (BD Biosciences), Matrigel 40 % (BD
Biosciences), HEPES (0.02 M) and NaHCO3 (2.35 mg/ml).
A cell suspension containing 5000 cells was added to
24well inserts (BD biosciences 353104) and incubated at
37 °C for 3 hours. BDM medium (1 ml) supplemented
with 20 ng/ml HGF and 10 ng/ml epidermal growth factor
(EGF) was added on top of the insert as well as in the well,
and incubated at 37 °C/5 % CO2 up to 2 weeks. The cells
were fed every other day and, after 2 weeks, the cells were
fixed in 3 % paraformaldehyde and stained.
The differentiation of VAL9 hESCs into hepatic progenitors
At day 0 of the differentiation protocol, the VAL9-hESC
colonies were positive for the pluripotency markers
octamer-binding transcription factor 4 (OCT4),
homeobox transcription factor (NANOG), tumour resistance
antigen 1–60 (TRA1-60), SSEA4 and SSEA3. The cells
also exhibited a normal karyotype (Additional file 3:
We assessed the ability of a previously published
protocol to generate hepatoblasts using the VAL9 cell line [
The VAL9 cells were subjected to a multistep
differentiation protocol outlined in Fig. 1a. Clusters of hESC line
were induced into definitive endoderm, followed by 6 days
of hepatic specification, where the cells differentiated into
hepatic bipotent progenitors. At day 5 more than 98 % of
the cells expressed CXCR4 (Fig. 1c), a marker of DE.
These cells were also found to be significantly positive for
Sox17 and HNF3β, also markers of DE. Furthermore, less
than 0.1 % of the cells expressed pluripotency markers
(Fig. 1b). These data suggest that the VAL9 hESCs can be
successfully differentiated into a homogenous population
of endoderm cells. At day 11, the majority of the cells
expressed CK19, GATA4, HNF3β, HNF6 and HNF4α, a
master regulator of hepatic differentiation (Fig. 1d) and
97 % of the cells expressed EpCAM, a hepatoblast marker
Transcriptional analysis of the in vitro differentiated
progenitors demonstrated that these cells also expressed
hepatic markers such as HNF4α, AFP, FOXM1B, and
LDLR (Fig. 3f ).
Cryopreservation and thawing procedures have been
reported to have detrimental effects on the viability and
function of primary human hepatocytes when
compared to freshly isolated cells [
]. The successful
cryopreservation of human hepatic progenitors that retain
high viability, as well as the ability to be cultured and
further differentiated, would allow for long-term
banking of the cells required for subsequent research and
clinical applications. We therefore assessed the ability
of VAL9-derived progenitors to be thawed and cultured
post-cryopreservation. As shown in Additional file 4
(Figure S2A–D) the hepatic progenitors maintained
their cuboid morphology and were able to proliferate
and express hepatic-specific markers such as AFP,
HNF4α, FOXA2, CK19, EpCAM and AFP.
Interestingly, the thawed cells also expressed claudin 1
(CLDN1), a co-receptor for hepatitis C virus (HCV)
and a significant proportion of the cells also expressed
CD81, another co-receptor for HCV. They also
maintained a good viability (>80 %) along the post-thawing
differentiation (Additional file 4: Figure S2D).
As the hepatoblasts are bipotent progenitors, they
are able to give rise not only to hepatocytes but also to
cholangiocytes; we therefore investigated the capacity
of VAL9-hepatoblasts to differentiate into committed
cholangiocyte precursors. Treatment of hepatoblasts
with GH/EGF, then interleukin-6 allowed the cells to
reach confluence at around day 17 (Fig. 2a). This
population of proliferating biliary-committed cells expressed
osteopontin (OPN), a downstream target of NOTCH
during normal liver development, HNF6 and HNF1β,
whereas HNF4α expression was not detected, as shown by
co-staining experiments with CK7/HNF4α (Fig. 2b).
When grown in three dimensions these cells were able to
generate ducts and tubules which showed polarity as
demonstrated with F-actin and β-catenin staining (Fig. 2c).
Differentiation of VAL9 hepatic progenitors into VAL9-HEP
Confluent hepatic progenitors were passaged at a ratio of
1:2 onto collagen 1-coated plates and allowed to further
differentiate in the presence of HGF in medium supplemented
with 10 ng/ml oncostatin for 3 days in addition to several
other hepatic maturation factors (see Methods section)
(Fig. 3a). At the end of this protocol, the differentiated cells
exhibited characteristic hepatic morphology presenting a
polygonal shape and round single or double nuclei (Fig. 3b).
Immunostaining of the differentiated VAL9-HEP showed
that the cells were positive for alpha-1-antitrypsin (A1AT)
and ALB and the hepatic transcription factors HNF4α and
HNF3β. Notably, ALB-expressing cells also expressed
CYP3A4 (Fig. 3c). VAL9-HEP also expressed the entry
cellular factors necessary for productive HCV infection, such
as CD81 and CLDN1 (Fig. 3c). Fluorescence-activated cell
sorting analysis revealed that 85 % of the cell population
expressed ASGR, a cell surface receptor specifically
expressed in the normal hepatocyte membrane (Fig. 3d).
The expression of liver-specific genes was assessed by
quantitative reverse transcription PCR (Fig. 3e). The
results showed the appearance of HNF4α, as expected,
as early as day 11, and ALB, MRP, UGT1A1, CYP1A2,
and CYP2C9, whereas expression of OCT4 was
abolished (Fig. 3e). The cells also expressed ALB, LDLR and
the gene encoding the transcription factor FOXM1B as
shown by RT-PCR (Fig. 3f ).
Characterization of VAL9-HEP functions in vitro
VAL9-HEP demonstrated the ability to accumulate
glycogen detected by PAS staining and these PAS-positive cells
had the corresponding hepatocyte morphology. Moreover,
the VAL9-HEP were responsive to hormones. Addition of
insulin and glucose resulted in an increase in glycogen
storage; by contrast, addition of glucagon to the cells
resulted in a significant depletion of glycogen content
(Fig. 4a). We also examined the cellular uptake and
excretion of ICG, an organic dye that is taken up and
subsequently eliminated specifically by hepatocytes.
The cellular uptake was observed in VAL9-HEP in a
very high percentage of cells and the majority of the
ICG was excreted within a few hours and almost
completely disappeared 24 hours later, indicating that a
functional biotransforming system was generated in
our VAL9-HEP (Fig. 4b).
An important function of hepatocytes is the ability to
synthesize urea from ammonia and excrete it as urea.
Therefore, we analysed the VAL9-HEP for urea excretion
and compared the values to that of neonates [
found that the VAL9-HEP values represented 28 % of
mean neonatal values: 0.36 nmol/min/106 cells and 1.30
± 0.47 (0.76–2.19) nmol/min/106 cells, respectively
In contrast to foetal hepatocytes, the VAL9-HEP
displayed UGT1A1 activity (1 pmol/mg/min) representing
10 % of that of newborn hepatocytes (Fig. 4e). In order
to assess the specificity of the assay, the expression of
UGT1A1 was also assessed by western blot. The
VAL9HEP expressed UGT1A1 when compared to
undifferentiated VAL9, where the protein was not detected (Fig. 4f ).
Another important function displayed by mature
hepatocytes is serum protein production. ALB secretion was
assessed using ELISA, and confirmed that post 20 and
30 days of differentiation ALB-secreting VAL9-HEP
were generated, indicating their successful maturation
The drug detoxification capability of VAL9-HEP was
assessed by measuring the activities of the major
cytochrome P450 enzymes responsible for the oxidative
metabolism of drugs in the human liver (CYP1A2,
CYP2C9, CYP2D6, CYP2E1 and CYP3A4).
Differentiated cells displayed significant cytochrome P450
activity (Fig. 4f ). An important property of hepatocyte
function is the ability to respond to compounds able to
induce the biosynthesis of an isozyme. The activity of
CYP3A4 was significantly increased (100 %) when cells
were treated with rifampicin when compared with
untreated VAL9-HEP (Fig. 4h).
Differentiation of hiPSCs
hiPSCs were differentiated using the same protocol as
described above for VAL9 hESCs. As in VAL9
differentiation, hiPSC gave rise to a homogeneous population of
endoderm cells expressing GATA4 and FOXA2 at day 5,
then to bipotent progenitors expressing HNF4α and
CK19 at day 11. They further differentiated into
hiPSCHEP expressing AFP and ALB (Additional file 5: Figure
S3A–C). After 26 days of differentiation, hiPSC-HEP
expressed hepatocytes markers, although the levels of
gene expression were lower than in VAL9-HEP
(Additional file 3: Figure S3E).
VAL9-HEP rescue acetaminophen-induced ALF
A major challenge for stem cell-derived HLCs is their
limited ability to mimic their in vivo counterparts. To
assess the therapeutic potential of VAL9-HEP in vivo,
we used the well-defined model of acetaminophen
toxicity (APAP) in immunocompromised mice, which
mimics ALF. This model was chosen because consistent
hepatotoxicity has been shown in murine models and
hepatocyte damage occurs at doses similar to those
reported to provoke damage in human liver [
]. ALF is a
multistep process that involves apoptosis followed by
necrosis of hepatocytes in humans.
A dose of acetaminophen at 300 mg/kg body weight
resulted in lethality in 50 % of the control animals
2 weeks after the administration of APAP.
Histological analysis indicated the presence of massive
necrosis in the liver which became apparent as soon as
3 hours after APAP injection (Fig. 5b) and was
concomitant with the release of alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) in the circulation
To genetically trace the transplanted cells in vivo,
VAL9-HEP were transduced in vitro with a
GFPexpressing lentiviral vector under the control of the
hepatic specific promoter of the human A1AT gene.
Thus, GFP was expected to be expressed in the
differentiated VAL9 cells only. As shown in Fig. 5a, 75 % of
the transduced VAL9-HEP expressed GFP, suggesting
that the majority of the transduced cells were
VAL9-HEP transplantation resulted in a three- and
fivefold reduction in AST and ALT values, respectively,
when compared to control animals (Fig. 5f ).
Fifty percent of the untreated control animals with
ALF died within 2 weeks of transplantation, whereas all
the animals which were transplanted with VAL9-HEP
survived, indicating a survival advantage for those
animals receiving cell therapy (Fig. 5c). Thus, the
VAL9HEP display sufficient detoxifying enzyme activity
required to rescue the animals. Transplanted mice were
sacrificed at three time points (2, 4 and 8 weeks) after
transplantation. To verify that the transplanted
hepatocytes homed towards the liver without migrating to
other organs, we analysed the transplanted cell
distribution in other organs such as the spleen and lungs
8 weeks after transplantation. No human cells were
detected in any of the analysed organs as assessed by
immunohistochemistry (IHC) against GFP (data not
shown). IHC revealed that the liver displayed a normal
histology with no sign of tumours (data not shown).
The spleen, lung and kidneys were also normal. No
signs of adenocarcinomas were visible in the
peritoneum (data not shown).
Engraftment of VAL9-HEP in APAP-treated mice
In order to investigate whether the transplanted cells
were engrafted within the livers of the recipient mice,
we first used an antibody against GFP to detect the
presence of human VAL9-HEP. Human cells were visible
throughout the liver parenchyma in the form of clusters.
This indicates that, in response to liver failure, the
transplanted cells have not only engrafted but also
proliferated (Fig. 6a). GFP-expressing VAL9-HEP were found in
all the mice analysed (n = 8). By counting several
sections from each mouse and different lobes we calculated
that the percentage of liver repopulation ranged from
0.6 to 10.2 % of the liver parenchyma. Since 75 % of
transplanted cells expressed GFP, the proportion of
engrafted cells is underestimated.
Human ALB was then measured using ELISA in the
sera of transplanted mice and control non-transplanted
mice for 2 and 4 weeks post-engraftment (Fig. 5e).
Human ALB was detected in every injected mouse,
confirming the successful engraftment of VAL9-HEP.
In addition, the hepatic functions of the engrafted
VAL9-HEP were identified using IHC; namely the
detection of human ALB expression. A large number of
positive cells co-expressing ALB and GFP were
detected in the parenchyma at 4 weeks after
transplantation confirming that VAL9-HEP were integrated and
functional post-transplantation. We also performed
coimmunostaining on sections of engrafted livers at day 30
and compared them to the VAL9-HEP used for the
transplantation. Before transplantation a significant proportion
of VAL9-HEP expressed AFP (Fig. 3). However, 4 weeks
after transplantation all engrafted ALB-positive cells
were negative for human AFP expression,
demonstrating that it was downregulated as in endogenous
hepatocytes (Fig. 6c). Non-transplanted control liver tissues
were negative for human proteins (data not shown).
Together, these results demonstrate that engrafted
VAL9-HEP underwent maturation in situ.
We report the development of a new strategy to
generate a homogenous population of hepatocytes. We used
a new cohort of hESC, VAL9 cells, which were established
in traceable conditions easily transposable to Good
Manufacturing Practice (GMP) compatible conditions.
We demonstrate that VAL9 hESCs can be efficiently
differentiated, recapitulating the key stages of liver development,
into viable hepatic cells with various hepatocyte-specific
functions both in vitro and in vivo, where the hepatocytes
were able to rescue mice with ALF. This was
accomplished by the stepwise addition of defined factors,
without the addition of mesenchymal or endothelial cells or
any other liver cells that normally accompany hepatocyte
development, nor serum or complex matrixes. However,
although recombinant proteins such as fibronectin can be
used (unpublished data), we utilized gelatin for cell
differentiation up to hepatoblast stage and then collagen which
is the matrix currently used for primary cell culture.
Although in our experiments both of these matrix
components were from animal origin, it is noteworthy that
GMP-compatible, recombinant human collagen is now
available as well as GMP-grade gelatin, suggesting that in
the near future differentiation of pluripotent stem cells
into hepatocytes will be possible under conditions
appropriate for clinical applications. hiPSCs were also
differentiated into hepatocyte-like cells following the same protocol
used with VAL9-hESCs, highlighting the therapeutic
potential of our approach.
At the progenitor stage, hepatoblasts could be
cryopreserved and further differentiated into hepatocytes. They
were also successfully induced to differentiate along the
cholangiocyte lineage as previously reported [
although conditions for further differentiation need to be
At the hepatocyte stage, VAL9-HEP retain some
characteristics of foetal hepatocytes, such as expression of AFP.
However, the differentiated cells reproduce key features of
mature hepatocytes, such as ICG metabolism. Due to the
importance of maintaining blood glucose levels, the
synthesis and degradation of hepatic glycogen, the storage
form of glucose in the liver, are tightly regulated and the
binding of hormones, such as glucagon, to cell receptors
signals the need for glycogen to be degraded. Although
the signalling pathways were not investigated, our data
show that, upon the addition of glucagon, the amount of
glycogen storage decreased in VAL9-HEP. It should be
noted that fresh and thawed VAL9-HEP also exhibited the
expression of receptors known to be involved in HCV
Different strategies have been used to improve the
differentiation of the hepatocyte-like cells (HLCs) in vitro,
such as the use of special matrices, co-culture with
stromal cells or purification of hepatoblasts [
20, 38, 39
However, to date, a protocol has yet to be developed
resulting in the maturation of the HLCs at a comparable
level to that of primary adult hepatocytes. This was
reported by Baxter and colleagues after the extensive
characterization of several hESC and iPSC lines
including the evaluation of CYP activities [
]. To date, the
expression of cytochrome P450 enzymes was mostly
studied at the transcriptional level and CYP activities,
when measured, were assessed by luminometry, which
was misleading [
Drug-metabolizing enzymes are expressed at negligible
or very low levels in the foetus. Recently, we have
demonstrated the importance of both gestational and
postnatal development for the maturation of CYPs in
neonatal hepatocytes [
]. CYP2D6 and CYP1A2 activity
was not detected in the younger neonatal hepatocytes
]. Although CYP3A4 activity was low in VAL9-HEP,
CYP2D6 and CYP1A2 activities were in the range of that
detected in new-born hepatocytes, suggesting that the
“adult” levels could be reached in transplanted cells after
in vivo maturation.
Urea is formed within the urea cycle and represents the
major end-product of ammonia detoxification in the liver.
It is a good indicator of the degree of hepatocyte
mitochondria preservation. VAL9-HEP were able to synthesize
urea from ammonia at rates representing one-quarter to
one-half of neonate values, indicating that this pathway is
active in our cells [
]. The cells also displayed UGT1A1
activity, representing one-tenth of neonate values. In
humans, UDP-glucuronosyltransferases (UGTs) are an
important group of Phase II (conjugative) metabolizing
enzymes that play a critical role in human health and
disease. UGTs are involved in the metabolism and
detoxification of numerous endogenous compounds and
xenobiotic chemicals including therapeutic agents such as
]. The activity of UGT1A1, the major
enzyme responsible for bilirubin glucuronidation, is not
detected in the foetal liver [
]; it is induced after birth,
which accounts for the onset of hyperbilirubinemia.
Interestingly, type 1 Crigler-Najjar syndrome, a genetic
deficiency in hepatic UGT1A1, is a metabolic disorder treated
by hepatocyte transplantation.
Since transplantation of foetal stem or progenitor cells
into livers of immunodeficient mice resulted in cell
expansion and maturation [
27, 28, 42, 43
], we evaluated these
properties after VAL9-HEP transplantation. It was
reported that immature iPSC-derived HLCs (with a few cells
weakly positive for CYP3A4) could engraft in Mu-uPA
SCID transgenic mice expressing urokinase in the liver
. This model has been widely employed to produce an
ideal chronic liver injury model used for transplanting
primary hepatocytes and HLCs. Several studies suggest that
uPA facilitates the engraftment and proliferation of
transplanted hepatocytes. In liver regeneration, uPA activates
plasminogen, which degrades the extracellular matrix to
promote reorganization of the hepatic architecture [
In a model of CCL4-intoxicated animals, subpopulations
of HLCs were transplanted after laser microdissection and
pressure capturing, which selected for ICG high cells. This
resulted in 10 % of ALB-expressing cells [
these models cannot be transposed to clinical situations,
the data suggest that the maturity level of the pluripotent
stem cell-derived hepatocyte-like cells plays a vital role in
the efficiency of engraftment.
To assess the function of VAL9-HEP, we chose the
mouse model of APAP-induced hepatotoxicity due to its
clinical relevance. APAP overdose accounts for the
majority of cases of drug liver injury resulting in fatal ALF
]. It has become the most common cause of ALF in
the United Kingdom and accounts for approximately
half of ALF cases in the United States [
Our data show significant engraftment of VAL9-HEP
in the liver parenchyma for at least 1 month. The
efficiency of repopulation was approximately 15 %. Given
that a mean of 75 % of hepatocytes expressed GFP prior
to transplantation, efficiency of repopulation is
underestimated and should more likely represent 18–20 %.
Importantly, the engrafted cells also demonstrated in vivo
Moreover, it is imperative that therapeutically
advantageous hepatocyte-like cells are safe (i.e. non
tumourigenic), express hepatic-specific genes comparable to
mature hepatocytes, and contribute to liver function in
vivo as demonstrated by VAL9-HEP. The potential of
hESCs and their differentiated progeny to generate
spontaneous tumours is of particular concern with regards to
their use in clinical applications. Several reports show
tumour formation post-transplantation of hESC-derived
cells despite pre-differentiation [
demonstrate that the transplanted cells contain a number of
undifferentiated hESCs . On the other hand, additional
studies have demonstrated that the transplantation of
highly differentiated cells did not result in tumour
], thus suggesting that directing hESCs
to an appropriate state is an important step for their
safe and effective use in cell therapies. To this end,
well-defined methods should be established to reduce
the tumourigenicity of transplanted cells and a strict
elimination of undifferentiated hESCs from
transplanted cells [
]. In our VAL9-HEP engrafted mice,
we investigated the appearance of tumours at the time
of sacrifice. No sign of tumour formation was evident
in grafted livers or in other major organs. However,
additional long-term studies are required to confirm
the ultimate safety of VAL9-HEP.
We demonstrate in this study, for the first time, that
engrafted human VAL9-HEP are able to rescue mice
with ALF. This was evaluated by the significant decrease
in AST and ALT and by the rescue of transplanted
animals. The data suggest that the VAL9-HEP expressed
sufficient levels of detoxification enzymes at the time of
transplantation. Different models show a correlation
between the number of infused cells and the percentage of
repopulation; up to 2 to 7 M HLCs were infused per
25, 26, 44
]. We infused a significantly lower
amount of cells, 1 million, which corresponds to 1–1.5 %
of the mouse hepatocyte mass, which was enough to
rescue half of the animals. Interestingly, hepatocytes derived
from the HepaRG cell line were able to rescue
CCL4treated animals only when the cells were transduced with
In humans, transplanting no more than 1–2 % of liver
mass per cell infusion is recommended in order to avoid
portal hypertension [
]. The scarce supply of donated
cadaveric livers combined with the fact that mature
hepatocytes display short-term survival, poor in vitro
proliferation and tolerance to cryopreservation, results in limited
transplantation options for patients, as well as use in other
applications such as high-throughput drug screening.
Clinical trials have been ongoing for years to assess the
effects of foetal liver cells transplanted into patients with
various liver diseases, in particular cirrhosis or inborn
metabolic diseases [
]. Recently, a clinical trial was
performed with freshly isolated biliary tree stem cells to treat
patients with advanced cirrhosis. This procedure resulted
in a 6- to 12-month improvement in both biochemical
and clinical features [
Immunosuppression is an important issue in cell
therapy strategies for liver diseases, but optimal
regimens for inducing tolerance to transplanted liver cells
are not well established. Even if hESC-hepatocytes
could be antigen-matched to the recipient,
immunosuppression would still be required, since hESCs
express low levels of HLA class I antigens [
] and they
are still subjected to immune system targeting.
Therefore, this question will have to be addressed, but in a
specific study using embryonic stem cells and an animal
model from the same species.
The strategy that we describe here may address the
problem of cell limitation, as it utilizes a renewable cell
source. It offers the advantage of immediate availability
and unlimited supply of functional donor hepatocytes
for emergency treatments required by patients with ALF
when an organ is not immediately available. In addition,
unrestricted availability of donor hepatocytes could allow
programmed and repeated treatment of patients with
debilitating, liver-based metabolic disorders, which are not
now considered candidates for organ transplantation,
such as familial hypercholesterolemia and partial urea
cycle disorders. The generation of hepatocytes also
provides a potentially useful step toward the generation of
hepatic organs. Tissue engineering of a hepatic organ
will require the incorporation of hepatic niche cells,
such as mesenchymal, stellate, endothelial cells and
cholangiocytes, into cultures of stem cell-derived
In summary, our strategy allows the stepwise
differentiation in defined conditions of the new VAL9 hESC line
into bipotent progenitors that are able to give rise to
cholangiocyte precursors, then into neonate-like hepatocytes
with detoxification activities. VAL9-HEP were able to
successfully engraft and proliferate into mice livers suffering
from acute failure in a clinically relevant model, resulting
in a decrease of transaminases to control levels and the
rescue of transplanted mice. Taken together, our data
suggest that cell therapy using hESC-derived hepatocytes may
be an effective treatment for liver diseases.
Additional file 1: Table S1. Antibodies used in this study. (PDF 48.5 KB)
Additional file 2: Table S2. Primers used for RT-PCR of Human mRNAs.
(PDF 79.6 KB)
Additional file 3: Figure S1. Pluripotency of VAL9 hESCs. (A)
Phasecontrast image of a representative VAL9 hESC colony. (B) Representative
immunofluorescence staining for human OCT-4, NANOG and TRA1-60. (C)
Fluorescence-activated cell sorting (FACS) analysis of the expression of stem
cell specific surface markers: TRA1-81, SSEA-3, SSEA-4. (D) Karyotype analysis
of VAL9 hESCs. (TIF 4911 kb)
Additional file 4: Figure S2. Cryopreservation of VAL9-hepatoblasts. (A)
Phase-contrast image of a representative VAL9-hepatoblasts at day 15 of
differentiation before (fresh) and after cryopreservation (4 days after
thawing). (B) Phase-contrast image of a representative VAL9-hepatoblasts
at day 20 of differentiation (9 days after thawing). (C) Representative
immunofluorescence staining for human cytokeratin (CK)19, hepatic
nuclear factor (HNF)1α, alpha foetoprotein (AFP), FOXA2 ; and co-staining
for human hepatic nuclear factor (HNF)4α/epithelial cell adhesion molecule
(EpCAM), and for human claudin (CLDN)1/CD81. (D) Representative
fluorescence-activated cell sorting (FACS) analysis for cell viability of
thawed VAL9 cells at different time points after thawing as detected
by 7AAD staining (TIF 5667 kb)
Additional file 5: Figure S3. Differentiation of hiPSCs into hepatocytes.
(A) Phase contrast microscopy images of cell morphology at different key
steps of the differentiation protocol. (B) Representative immunofluorescence
staining of definitive endoderm. Cells express GATA binding protein
(GATA)4, and hepatic nuclear factor (HNF)3β (FOXA2). (C) Representative
immunofluorescence staining of hepatic progenitors. Cells express
HNF4α, cytokeratin (CK)19, FOXA2 and GATA4. (D) Representative
immunofluorescence staining of hepatic progenitors. Cells express HNF4α,
AFP and ALB. (E) Quantitative RT-PCR analysis at day 0,4,11 and 30 of
differentiation of hiPSCs. Data are expressed as a percentage of the value
obtained for differentiated VAL9 cells. (TIF 6373 kb)
A1AT: Alpha-1-anti-trypsin; AFP: Alpha foetoprotein; ALB: Albumin; ALF: Acute
liver failure; ALT: Alanine aminotransferase; APAP: Acetaminophen;
ASGR: Asialoglycoprotein receptor; AST: Aspartate aminotransferase;
BDM: Biliary differentiation media; bFGF: Basic fibroblast growth factor;
BSA: Bovine serum albumin; CD81: Cluster of differentiation 81 (tetraspanin
28); CHIR99021: Glycogen synthase kinase 3 inhibitor; CK: Cytokeratin;
CLDN1: Claudin 1; CYP3A4: Cytochrome P450 3A4; DE: Definitive endoderm;
EGF: Epidermal growth factor; ELISA: Enzyme-linked immunosorbent assay;
EpCAM: Epithelial cell adhesion molecule; FGF: Fibroblast growth factor;
GATA4: GATA binding protein 4; GFP: Green fluorescent protein; GMP: Good
Manufacturing Practice; HCM: Hepatocyte culture medium; HCV: Hepatitis C
virus; hESC: Human embryonic stem cell; HGF: Hepatocyte growth factor;
HLC: Hepatocyte-like cell; ICG: Indocyanine green; hiPSC: Human induced
pluripotent stem cell; HLC: Hepatocyte-like cell; HNF: Hepatocyte nuclear
factor; HPLC/MS: High-performance liquid chromatography tandem mass
spectrometry; HPM: HamF12/Williams; IHC: Immunohistochemistry;
LY294002: Phosphoinositide-3-kinase inhibitor; MOI: Multiplicity of infection;
NANOG: Homeobox transcription factor; OCT4: Octamer-binding
transcription factor 4; OPN: Osteopontin; PAS: Periodic acid-Schiff;
PBS: Phosphate-buffered saline; PCT: Polymerase chain reaction; SOX: Sex
determining region box; TRA-1-60: Tumour resistance antigen 1–60;
UGT: UDP glucuronosyltransferase; VAL9-HEP: VAL9 hepatocytes.
The authors declare that they have no competing interests.
LT was involved in conception and design, data collection, analysis and
interpretation, and manuscript writing. JC and ZH were involved in data
collection, analysis and interpretation and manuscript writing. MA and SL
were involved in data collection and in drafting the manuscript. DB made
critical contributions to conception, design and implementation and
manuscript writing. JVC was involved in analysis and interpretation and
critical reading of the manuscript. AW, MJGL and ADK conceived the
project, interpreted the results, supervised the work and wrote the
manuscript. All authors read and approved the final manuscript.
This work was supported by the European Commission’s Seventh
Framework Programme FP7-HEALTH.2011.1.4.2 under grant agreement No.
278152 “InnovaLiv”, and from French National Research agency (ANR,
2010RFCS-004 « Liv-iPS »). LT was a recipient of a Sara Borrell Contract from the
“Instituto de Salud Carlos III” of the Spanish Ministry of Economy and
Competitiveness. JC has a fellowship from the French Ministry of Research.
ZH and MA were supported by the InnovaLiv grant.
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