New Approaches in the Differentiation of Human Embryonic
Stem Cells and Induced Pluripotent Stem Cells toward
Iman Saramipoor Behbahan & Yuyou Duan &
Alexander Lam & Shiva Khoobyari & Xiaocui Ma &
Tijess P. Ahuja & Mark A. Zern
Published online: 19 February 2011
# The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Orthotropic liver transplantation is the only
established treatment for end-stage liver diseases. Utiliza-
tion of hepatocyte transplantation and bio-artificial liver
devices as alternative therapeutic approaches requires an
unlimited source of hepatocytes. Stem cells, especially
embryonic stem cells, possessing the ability to produce
functional hepatocytes for clinical applications and drug
development, may provide the answer to this problem. New
discoveries in the mechanisms of liver development and the
emergence of induced pluripotent stem cells in 2006 have
provided novel insights into hepatocyte differentiation and
the use of stem cells for therapeutic applications. This
review is aimed towards providing scientists and physicians
with the latest advancements in this rapidly progressing
Keywords Hepatocyte.Hepatocyte differentiation.
Hepatocyte transplantation.Human embryonic stem cells.
Induced pluripotent stem cells
Orthotropic liver transplantation is the only established
treatment for end-stage liver disease. However, because of
the shortage of viable livers available for transplant, many
patients die while remaining on the extensive waiting list
and many more are never added to the list. Utilization of
hepatocyte transplantation and bio-artificial liver devices
have been proposed as alternative therapeutic approaches to
this problem [1, 2]. These two approaches, however,
require an unlimited source of hepatocytes, and human
primary hepatocytes provide the most desirable solution for
cell therapies. Yet, the utilization of primary hepatocytes in
therapy has been hindered by their slow growth, loss of
function and de-differentiation in vitro . Stem cells,
possessing the ability to produce functional hepatocytes for
clinical applications and drug development, may provide
the answer to this problem. As yet, it is not clear which
stem cell type will be the most effective in forming lines
that will be effective in regenerative medicine.
Hepatocytes-like cells have previously been derived from
embryonic stem cells (ESC), bone marrow stem cells,
adipose tissue, and mesenchymal cells, as well as multi-
potent progenitor cells in the human umbilical cord [4–10].
New discoveries in the mechanisms of liver development
and the emergence of induced pluriplotent stem cells in
2006 have provided novel insights into hepatocyte differ-
entiation and the use of stem cells for therapeutic
applications. This review is aimed towards providing
scientists and physicians with the latest advancements in
this rapidly progressing field.
Early Liver Development
During embryogenesis, the differentiation of progenitor
cells into fully mature hepatocytes depends on the initiation
of complex pathways by numerous signals released from
adjacent cells. Variations in timing and concentration of the
cell signals are both necessary for the regulation of specific
I. S. Behbahan:Y. Duan:A. Lam:S. Khoobyari:X. Ma:
T. P. Ahuja:M. A. Zern (*)
Transplant Research Program, Department of Internal Medicine,
University of California Davis Medical Center,
4635 2nd Ave. Suite 1001,
Sacramento, CA 95817, USA
Stem Cell Rev and Rep (2011) 7:748–759
transcription factors that ultimately orchestrate this transi-
tion into mature cells. With the elucidation of the intricate
mechanisms of liver development through recent discover-
ies in mouse, zebrafish and chicken embryos (reviewed by
Si-Tayeb K et al. 2010 , Lemaigre FP 2009 ), new
protocols of hepatocyte differentiation have begun to mimic
the development of hepatocytes in vivo.
In a portion of the ventral endoderm located adjacent to
the developing heart, the cells expressing albumin, trans-
thyretin, and α-fetoprotein (AFP) are the first molecular
evidence for liver development [13, 14]. At approximately
3 weeks of human gestational age, liver and pancreas
progenitor cells in three separate regions of the endoderm,
begin to differentiate: this is known as “specification”.
Then, two lateral progenitor regions move ventral-medially
to form the hepatic endoderm  (Fig. 1a). At this stage,
repression of mesodermal Wnt and Fibroblast Growth
Factor (FGF) 4 are initially required for hepatic induction
[16, 17]. Retinoic acid signaling also helps determine the
position of endodermal organs along the anterior-posterior
position and also appears to help liver development from
gut endoderm [18, 19]. Moreover, low concentrations of
FGFs through activation of the mitogen-activated protein
kinase (MAPK) pathway from cardiac mesoderm are
necessary for liver programming. As a result, hepatic
endoderm cells move away from the cardiac mesoderm
cells to keep a lower concentration of FGFs [20, 21]. The
interaction of FGFs with bone morphogenetic protein
(BMP)-2 and BMP-4 from the septum transversum
appears to be essential to induce hepatic gene expression
[19, 22]. The ventral and lateral regions of the endoderm
corresponding to specific hepatic and pancreatic regions
have diverse responses to varying levels of FGFs and
BMPs. Wandzioch and Zaret reported that during specifi-
cation hepatic domains initially inhabit a region of high
BMP activity at the ventral midline and high FGF activity
at the lateral endoderm. Pancreatic progenitors initially
develop in a region of low BMP activity; however, a
region of high BMP activity is required to initiate the
ventral pancreatic bud at the midline. The timing of BMP
signaling for generating hepatic progenitor cells may be
less important than it is for the pancreatic programming
The newly specified bipotent hepatic cells in embryos
are known as hepatoblasts which express albumin and
transthyretin, and are capable of becoming either hepato-
cyte or biliary epithelial cells  (Fig. 1b). After
specification, the liver diverticulum forms from the prim-
itive gut at day 22 of human embryonic age. Proliferation
of these hepatoblasts then leads to the production of a
pseudostratified epithelium-like tissue, called the hepatic
bud [12, 26]. Afterwards, the columnar hepatoblasts go
through an epithelial-mesenchymal transition while invad-
ing into the septum transversum  (Fig. 1a). A complex
transcriptional network consisting of factors such as GATA-
6 , prospero homeobox protein 1 (Prox1) , T-box
Fig. 1 Fetal liver development (adopted and modified from Ref. [12, 19, 32, 45]). a. Early liver development and liver bud formation; b.
Transcription factors network involving in liver development; c. Some of the known markers suggested for each step
Stem Cell Rev and Rep (2011) 7:748–759 749
transcription factor 3 (Tbx3) , hepatocyte nuclear factor
6 (HNF-6)  and HNF-4  control these events .
Fetal Hepatic Lineage Commitment and Hepatocyte
The development of the embryonic liver results in three
distinct cell populations, which differ in their cell markers
and induction signals. Hepatocyte-committed cells are
recognized by extensive expression of albumin and AFP.
Cholangiocyte-committed cells are distinguished by their
cytokeratin (CK)-19 expression. The cell population
expressing both hepatic and biliary markers is the bipotent
hepatoblast [11, 12, 32]. Oncostatin M (OCM) which is
expressed in hematopoietic cells in the fetal liver  along
with glucocorticoids [32, 34] suppress the differentiation
toward the hematopoietic lineage and promote the differ-
entiation of the liver progenitor cells towards hepatocytes in
vitro [11, 32, 35]. Meanwhile, some mitogenic factors from
sinusoidal endothelial cells such as hepatocyte growth
factor (HGF) and interleukin 16 (IL-16) acting via the
vascular endothelial growth factor (VEGF) receptors
enhance hepatocyte proliferation . HGF inhibits the
progression of the cells into the cholangiocytic lineage by
the blocking of notch signaling. This enhancement of
hepatocyte specification appears to be stimulated by
insulin. Gradually, during fetal hepatic maturation the
number of bipotent hepatoblasts decreases and the number
of mature cells expressing specific hepatic or biliary
markers increases . During this process, AFP is highly
expressed in early embryonic and fetal liver development,
then the production of AFP decreases later in maturation
and is markedly lower in adulthood [32, 36]. Finally, whole
liver maturation occurs after birth by a combination of
growth factors secreted by surrounding non-parenchymal
liver cells, such as HGF, FGF4, and epidermal growth
factor (EGF) .
Liver Stem Cells
In the injured liver, a heterogeneous population of
progenitor cells in or adjacent to the canal of Hering
become activated . Farber first described them as “small
oval cells with scant lightly basophilic cytoplasm and pale
blue-staining nuclei” with a high nuclear/cytoplasmic ratio
. “Oval cells” are recognized as bipotent hepatoblasts
possessing the ability to differentiate into either hepatocytes
or biliary epithelial cells and express both hepatocyte (AFP,
albumin) and cholangiocyte (CK 7, CK 19, OV-6) markers
. In addition, activation of stem cell genes such as c-kit
, CD34 , flt 3 receptor  and leukemia inhibitory
factor (LIF)  has been reported during oval cell
propagation. It is suggested that oval cells produce duct-
like structures in the periportal regions and expand into the
liver acinus; after several days, clusters of small basophilic
hepatocytes and mature bile ducts form from these duct-like
Previously, bipotent hepatoblasts were recognized as
the only human hepatic stem cell. Recently, adult hepatic
stem cells were characterized by their multipotency and
self-renewal capacity. Hepatic stem cells and hepatoblasts
are now considered the multipotent progenitor popula-
tions in human liver. The ductal plates of fetal and
neonatal livers and the canals of Hering in pediatric and
adult livers are proposed to be their niche [45, 46]. Both
cell populations express albumin, CK8, CK18, and CK19,
Sonic and Indian Hedgehog proteins, telomerase, and
CD133, and they are negative for hematopoietic markers
(CD45, CD38, CD14, and glycophorin A), hepatic stellate
cell markers (desmin, α-smooth muscle actin, and
CD146), and endothelial cell markers (VEGFr, CD31,
and van Willebrand’s factor) [45–47]. The hepatic stem
cell population which is thought to be the precursors of
hepatoblasts express epithelial cell adhesion molecule
(EpCAM), neural cell adhesion molecule (NCAM),
CK19, as well as ± albumin, and are also negative for
AFP. Hepatoblasts are characterized by the expression of
the combination of EpCAM, intercellular cell adhesion
molecule (ICAM-1), CK19, albumin++, and AFP++ [45,
47] (Fig. 1c).
Human Embryonic Stem Cells Differentiate Toward
ESC are continuously growing stem cell lines of embryonic
origin first isolated from the inner cell mass of blastocysts
from the developing mouse embryo. Later, hESCs were
successfully derived from in vitro fertilized human embryos
. It has been shown that hESCs possess a normal
karyotype  with high levels of telomerase activity 
and express specific markers. They also show continuous
renewal capability . Hundreds of hESC lines have been
created with more than a hundred in the National Institutes
of Health registry (http://stemcells.nih.gov/research/registry/)
. These cells have been shown to differentiate into nearly
all human cell types [51, 54]. Abe et al. in 1996, as well as
Levinson-Dushnik and Benvenistry in 1997, demonstrated
the ability of mouse ESC (mESC) to differentiate into
endodermal cells [55, 56]. Duncan et al. not only showed
that mESC cells were capable of differentiating into visceral
endoderm cells, but also established the capacity of the cells
to produce liver-specific proteins . Later, Hamsaki et al.
750Stem Cell Rev and Rep (2011) 7:748–759
reported the use of specific growth factors to drive mESC
into hepatocyte-like cells . The differentiation of hESC
into hepatocytes was first demonstrated by Rambhatla et al.
in 2003. Since then, many studies have focused on
enhancing the culture conditions to obtain a more homoge-
neous cell population
Differentiation via Embryoid Bodies (EB)
without Activin A
Human ESCs undergo spontaneous differentiation into the
three germ layers, among them endodermal cells expressing
AFP and albumin [60, 61]. Lavon et al. demonstrated that
hESC differentiated into hepatic-like cells through the
intermediates step of EB formation. They showed that
albumin-expressing cells isolated by fluorescence activated
cell sorting were capable of growing in vitro for a few
weeks [62, 63].
Our group, among others, extended the original finding
by optimizing growth factors, ECM, and medium selection
to enhance the purity of the differentiation process using
EBs [64–68]. In addition, it was suggested by Baharvand et
al. that plating EB in a 3D collagen type I scaffold with the
sequential addition of growth factors is more efficient than
applying 2D cultures. They also reported the migration of
cells out of aggregates and the formation of multilayer
cord-like structures in a 3D culture system . However,
hepatocyte differentiation through EBs is no longer consid-
ered the most effective approach. The major problem is the
relatively low differentiation rate and non-homogeneous
population of cells after differentiation.
Differentiation toward Definitive Endoderm Employing
During gastrulation, epiblast migration inward from the
primitive streak forms the definitive endoderm germ layer.
Later, the primitive buds of the liver, lung, thyroid and
ventral rudiment of the pancreas will develop from the
anterior-ventral domain of the foregut endoderm. Nodal
signaling, a member of the transforming growth factor-β
(TGF-β) superfamily, plays a critical role in the activation
of transcription factors which initiate the development of
endoderm. Activin A, an agonist for two types of cell
surface transmembrane receptors, initiates this nodal path-
way by binding to the type II receptor. This then elicits a
cascade response via the recruitment and phosphorylation
of various type I Activin receptors, eventually resulting in
the phosphorylation of SMAD2 and SMAD3. SMAD2 and
SMAD3 then translocate to the nucleus, complex with
SMAD4, and initiate the activation of gene-specific
transcription factors [69–71]. Hepatocytes differentiate
from the endoderm during embryonic development .
The seminal study was accomplished by D’Amour and
colleagues who reported differentiation of hESC to defin-
itive endoderm with more than 80% purity using Activin A
and low serum . The most effective hepatocyte
differentiation protocols have employed this approach of
definitive endoderm formation as the first step.
Ishii et al. compared endodermal differentiation through
EB or without forming EBs on different ECMs. They found
the best result by employing Matrigel-coated dishes
with100 ng/ml Activin A without employing EBs. In
addition, they showed that HGF, BMP4, and FGF4 have
no significant effect on endodermal differentiation, and,
they suggested an inhibitory effect of all-trans-retinoic acid
on endodermal differentiation. The presence of BMP4 and/
or FGF4 reduced the effects of Activin A and HGF on
endodermal differentiation .
Brolén et al. showed that using Activin A and FGF2
together enhanced the expression of endodermal markers
( Sox17, HNF3β and CXCR4 ) in comparison with using
Activin A alone .
Hay and colleagues examined Wnt3a expression in liver
at different times during human liver development. They
reported that in the first trimester Wnt3a activity developed
in the portal system with no parenchymal expression. In
contrast, in the second trimester Wnt3a expression was
observed both in the portal region and in the liver
parenchyma suggesting a potential role in early hepato-
genesis. Furthermore they found enhanced endodermal
differentiation of hESC using ActivinA plus Wnt3a in
comparison with Activin A plus sodium butyrate. They
postulated that Activin A plus Wnt3a induced a rapid and
more homogeneous hepatic endoderm production and led to
more functional hepatocyte-like cells .
As mentioned above, FGFs and BMPs appear to play
significant roles in liver specification. Jun Cai et al.
reported that FGF4 or BMP2 alone in the culture medium
had little effect on liver specification from definitive
endoderm of hESC. However, their combination lead to a
significant increase in albumin expression in vitro, and this
effect was confirmed by the FGFR1 inhibitor Su5402 or the
BMP inhibitor Noggin. Also, they showed definitive
endoderm induction is essential for the effectiveness of
FGF4 and BMP2 on hepatic induction. They reported
similar results for the combinations of aFGF and BMP4,
bFGF and BMP4, or FGF4 and BMP4 . DMSO, which
modifies histone acetylation, appears to have an inductive
effect toward hepatocyte lineage specification and has been
utilized in some protocols [4, 76, 78].
Stem Cell Rev and Rep (2011) 7:748–759751
Brolén et al. used the combination of BMP2/4 and
FGF1/2/4 for hepatic induction. They observed multiple
EpCAM-expressing cell clusters with adjacent groups of
CK7, CK19 positive cells and some CD54 positive areas. In
the control group which was not induced by BMPs and
FGFs, although EpCAM, α1-antitrypsin, AFP, CK7/8/18/
19 and CD54 positive cells appeared, there was a
considerable cell morphology difference with a significant
decrease in liver-specific gene expression .
Johannesson et al. found that Activin A-treated hESC
tend towards the hepatic lineage instead of towards
pancreatic progenitor cells if they are not treated with
FGF4 and retinoic acid (RA) . On the other hand,
Ameri et al. reported a dose-related response to FGF2
regarding cell fate selection. They demonstrated low levels
of FGF2 enhanced hepatic induction while intermediate
doses of FGF2 resulted in pancreatic fate selection . In
summary, many factors have been shown to affect hepatic
induction including the timing, concentration and specific
variety of growth factors, as well as the methods of
generating definitive endoderm.
In this step, differentiated cells present a polygonal
hepatocyte morphology with distinct round nuclei that have
cytoplasmic vacuoles [76, 81]. Hepatocyte maturation
strategies are designed to replicate the role of growth
factors in liver development (see above). In general,
sequential and different levels of HGF and/ or, OSM or
EGF with glucocorticoids and insulin in a hepatocyte-
optimized culture medium are used (See Table 1).
Differentiation toward Liver Precursor Cells
The isolation of a hepatic precursor cell line derived from
hESC has drawn the attention of some researchers because
the establishment of a method to drive hESC or iPS cells to
a stable bipotent liver stem cell line which has renewal
capability would bypass many expensive and difficult
differentiation steps and would enhance clinical applica-
tions that require a large quantity of cells. Haiyun Pei and
colleagues reported the isolation of liver progenitor-like
cells which were isolated from portal zone 1 that expressed
stromal progenitor markers, such as CD29, CD44, CD71,
CD90, and CD105, and were negative for CD34 and CD45
. Zhao et al. studied N-cadherin-expressing hepatic
endoderm that was derived from hESC by induction of
definitive endoderm and then treatment with BMP2 and
FGF4 for 5 days. These cells were maintained and passaged
on mouse embryonic stromal feeders cells for more than
100 days. They also reported that these cells can be
differentiated toward hepatocyte- and cholangiocyte-like
Differentiation of iPSCs Toward Hepatocytes
Induced pluripotent stem cells (iPSCs) are defined as
reprogrammed somatic cells that have properties of plurip-
otent stem cells. Since the first report of the generation of
iPSCs by Yamanaka and colleagues from mouse fibroblasts
in 2006 , many studies have reported iPSC formation
from species including mouse, rat, monkey and human
. Typically, iPSCs are generated by retroviral induction
of transcription factors, Oct3/4, Sox2, KLF4 and c-Myc, in
fibroblasts . Lentivirus and adenovirus induction,
induction with other gene combinations and virus free
approaches such as using plasmids, small molecules and
recombinant proteins have also been reported [85, 86]. In
addition, it has been shown that iPSCs can be generated
from a variety of cell types such as pancreatic cells,
meningiocytes, keratinocytes, hematopoietic cells differen-
tiated from ESCs, and primary human hepatocytes [85, 87,
88]. iPSC lines generated from patients suffering specific
diseases provide a unique source for study and disease
modeling. For example, iPSCs generation has been reported
from individuals with juvenile diabetes mellitus, Down
syndrome, muscular dystrophy, neurodegenerative diseases,
as well as ischemic heart failure, Parkinson’s disease,
Alzheimer’s disease, diabetes mellitus, sickle cell anemia
and Huntington’s disease, and the list is growing daily [87,
89]. In addition, iPSCs provide a potentially unlimited
source for autologous cell therapy for regenerative medi-
cine. It has been shown that human iPSCs can be
differentiated to many tissues such as hematopoietic
precursors and functional osteoclasts , pancreatic
insulin-producing cells , cardiomyocytes , photo-
receptors , as well as neural conversion . Recently,
mice have been cloned from iPSCs, which is another proof
of the pluripotency of these cells [95, 96].
iPSCs is a favorable choice for hepatocyte generation
because of their pluripotency and potential source for
autologous hepatocyte transplantation. Differentiation of
iPSCs toward a hepatic lineage has been shown in mice
[97, 98] and in humans [99–101] using similar protocols as
for hESC cells (Table 2). Si-Tayeb et al. demonstrated that
iPSC-derived mouse embryos similar to control fetal livers
consist of hepatocytes, endothelial cells, and sinusoidal
cells that express specific genes and markers at the same
level as in the control livers. In addition, they showed that
human iPSCs generated from foreskin fibroblasts by
lentiviral transduction of OCT3/4, SOX2, NANOG and
LN28 are capable of differentiating toward functional
hepatocytes by a four-step differentiation protocol and low
752 Stem Cell Rev and Rep (2011) 7:748–759
Table 1 Step-wise differentiation of human embryonic stem cell through definitive endoderm with Activin A
Activin A 3 days
HGF 5 day
OSM, Dex 5 days
Evaluation of entry of HIV-HCV
Pseudotype viruses into hESC-
derived hepatic cells. In vivo
study in SCID mice
Jun Cai et al.
Activin A 100 ng/ml
Wnt3a 50 ng/ml,
RPMI 1640, B27
1% DMSO, KO DMEM,
20% SR, 4 days
HGF, OSM, L-15,
10% FBS, 7 days
Intra splenic injection of cells in
Hay D et al.
Activin A 0.5% FBS,
RPMI 3 days
FGF-4, HGF, 2% KOSR,
RPMI media on Collagen I days 5–8
MM media, BSA,
FGF-4, HGF, OSM,
Dex Days 8–10
HCM Media, FGF-4,
HGF, OSM, Dex day
Portal injection of definitive
endoderm cells in
Agarwal S et al.
2% KOSR instead
of FBS in
formation 2 days
Activin A bFGF
HGF DMSO 8 days
Dex 3 days
Splenic injection of ASGPR 1
positive cells to uPA-SCID
rat, NOD-SCID mice
Basma H et al.
Activin A FGF-2
BMP 2/4 FGF 1/2/4
EGF, Insulin, Transferri,
acid 18–45 days
Differentiation in three cell lines
Brolén G et al.
No Serum-Activin A
FGF4, HGF, Dex, BMP2/4,
Insulin, DMSO, IMDM
FGF4, HGF, Dex, DMSO,
OSM, HCM Until use
Drug metabolism evaluation
Duan Y et al.
Low serum, Activin A,
SB, in RPMI 1640
Activin A10 ng/ml
FGF2 12 ng/ml
100 ng/mL Activin A,
20 ng/mL FGF2, 10 ng/mL BMP4,10 μM LY294003
50 ng/mL FGF10, 10−7M
retinoic acid, 10 μM
SB431542 3+2 days
30 ng/mLFGF4, 50 ng/mL
HGF, 50 ng/mL EGF.
Touboul T et al.
Stem Cell Rev and Rep (2011) 7:748–759753
oxygen content. They demonstrated that these cells prolif-
erated in mouse fetal liver for 7 days after cell transplan-
tation in vivo as well . Song Z et al. reported the
differentiation of human iPSC toward functional hepato-
cytes with a multi-phasic protocol, with 60% producing
AFP and albumin, similar to hESC differentiated to
hepatocytes . Sullivan et al. generated three human
iPSC lines by retroviral induction of Oct4 Sox2, Klf4, and
c-MYC. They showed that all three cell lines can
differentiate to hepatic endoderm which they characterized
with albumin and E-cadherin production, AFP, hepatocyte
nuclear factor-4a and cytochrome P450 7A1 expression
Oxygen Pressure and Mitochondrial Function
in Hepatocyte Differentiation
Early embryonic development mostly occurs in low oxygen
concentration. In addition, hypoxic microenvironments in
adults provide adult stem cell niches that control cellular
differentiation [102, 103]. The effect of low oxygen on
embryonic stem cell culture is controversial . Low
oxygen seems to decrease the chance of spontaneous
differentiation in mESC culture conditions in some reports;
however other studies suggest that it reduces the expression
of pluripotency genes. Low oxygen concentration enhances
the production of some lineages such as neurons, cardio-
myocytes, hematopoietic progenitors, endothelial cells, and
chondrocytes (reviewed in references  and ).
Multiple O2-sensitive intracellular mechanisms such as
hypoxia-inducible transcription factors (HIFs), the environ-
mental sensing of mammalian target of rapamycin (mTOR)
and the endoplasmic reticulum (ER) stress response have
been described . It appears that hypoxia increases the
mitochondrial membrane potential and subsequently reac-
tive oxygen species which activate HIF-1 expression .
Si-Tayeb et al. used different oxygen pressures during
the differentiation of hESC and hiPSC to hepatocytes. They
maintained undifferentiated hESC and hiPSC at low
oxygen pressure then generated the definitive endoderm at
a normal oxygen pressure, and finally hepatic induction and
specification was performed in a hypoxic environment. A
high percentage of hepatic differentiation is reported.
However, the advantage of the different oxygen pressures
is not discussed in comparison to a control group .
Characterization and Functional Evaluation
of Differentiated Hepatocytes and Drug Metabolism
There is no consensus for the characterization of
hepatocyte-like cells derived from stem cells. Generally,
Table 2 iPS differentiation toward hepatocyte
Moue GFP expressing
50 ng Activin A, 50 ng Wnt, 6 days
10 ng/ml bFGF 1%
DMSO 3 days
10 ng/ml HGF 1%
DMSO days 9–18
10 ng/ml HGF, 10 ng/ml
OSM, Dex, days 18–25
Gai H et al.
Teratoma formation neural
differentiation mesodermal differentiation
1% DMSO day 1–4 –
sodium butyrate day 5–11 Li W et al.
Activin A, RPMI
media B27 in 20%
O2 5 days
RPMI, B27, BMP4,
FGF-2 in 4%
O2 5 days
HGF in %
4 O2, 5 day
HCM + OSM 20% O2
Si-Tayeb K et
al. 2010 
Fibroblasts of normal
Activin A, Wnt 3a,
Activin A, RPMI,
B27 days 4–5
SR days 5–8
L15/10% FCS HGF OSM
Sullivan GJ et
al. 2010 
Testing expression of
OCT4, NANOG, SOX)2,
SSEA4, TRA1-60 a,
TRA1-81, by RT-PCR
Activin A, 3 days
OSM, Dex 5 days then
OSM, Dex, N2B27
Song Z et al.
754Stem Cell Rev and Rep (2011) 7:748–759
hepatocyte-like cells are recognized by their morphology,
liver-specific mRNAs, protein markers and their functional
abilities in each phase of differentiation . Each phase of
differentiation is delineated by specific markers. SOX17,
GSC and FOXA2 are well-known markers of definitive
endoderm [32, 73]. Primary hepatic differentiation is often
assessed by the expression of HNF3b, AFP, and trans-
thyretin (TTR). The intermediate phase of hepatogenesis is
recognized by HNF1α, HNF4α, Albumin, and CK18.
Finally, mature hepatocytes are defined by such markers
as tryptophan-oxygenase (TO), tyrosine amino-transferase
(TAT), C/EBPα , specific CYPs and asialoglycoprotein
receptor 1(AGPR1) . Several metabolic tests are used
for functional assays of differentiated hepatocytes. Glyco-
gen accumulation is examined by periodic acid shift
staining as one of the mature hepatocyte characteristics [4,
59, 67, 76]. Under electron microscopy the appearance of
polygonal-shaped cells containing multiple nuclei is sug-
gestive of hepatocytes. With transmission electron micros-
copy, glycogen granules within the cells, round nuclei with
evenly distributed chromatin and Golgi complexes, well-
developed bile canaliculi with apical microvilli and tight
junctions are considered to be characteristics of mature
hepatocytes in some studies [5, 67]. Hepatocyte-specific
functions such as urea synthesis and albumin production are
common functional evaluations in hepatocyte differentia-
tion [4, 32, 68]. Uptake of low-density lipoprotein (LDL)
has been utilized as well [67, 77]. Indocyanine Green is a
synthetic dye that is taken up by hepatocytes and secreted
into bile ducts without conjugation . Its uptake and
secretion is specific to hepatocytes and thus are used to
determine hepatocyte-specific function [4, 59, 67, 68].
Other hepatocyte-specific functions have been evaluated
infrequently, such as measurement of coagulation factor VII
activity  and entry of HIV-HCV pseudotype viruses into
hESC-derived hepatic cells .
Drug discovery is one of the important applications of
metabolism ability of hepatocyte like-cells derived from hESC
. For example, the pentoxyresorufin-O-dealkylase
(PROD) assay for the assessment of the cytochrome p450
system has been demonstrated in some studies, and Ek M
et al. demonstrated UDP-glucuronosyltransferases (UGTs),
drug transporters, transcription factors and other liver-specific
genes including several important CYPs at the mRNA and
protein levels in their hESC-differentiated hepatocytes .
In addition, CYP3A4 induction of hESC-derived hepatocytes
by rifampin and midazolam is described using LC-MS/MS in
comparison with Hep G2 by Agarwal and colleagues .
The conversion of testosterone to 6-beta-hydroxytestosterone,
a specific measure of CYP 3A4-mediated metabolism, and
inducible hepatic CYP 1A1/1A2-mediated Ethoxyresorufin-
O-deethylase (EROD) activities have been reported as well
. We first systematically evaluated the presence and
distribution of the biotransforming enzymes/proteins and
their regulators, the nuclear receptors, and determined the
real-time activities and functions of these biotransforming
enzymes/proteins in hESC-derived hepatocytes . Using
ultra performance liquid chromatography-tandem mass spec-
trometry technology our group identified seven metabolic
pathways of the drug bufuralol including four newly-reported
ones in hESC-derived hepatocytes, which were the same as
those in freshly isolated human primary hepatocytes. In
addition, the results of the metabolism of four drugs indicate
that our hESC-derived hepatocytes have the capacity to
metabolize these drugs at levels that are comparable to
primary human hepatocytes .
In Vivo Studies
Although transplantation is one of the potential goals of
hepatocyte generation from hESC, only a few studies have
evaluated these cells in vivo. Jun Cai et al. showed that
differentiated hESCs were able to engraft in the livers of
SCID mice injured by CCl4 and they expressed human
alpha-1 antitrypsin for at least 2 months . Our group
transduced hESC-differentiated hepatocytes using a lenti-
viral vector containing triple fusion protein and transplanted
transduced hESC-differentiated hepatocytes into the livers
of NOD-SCID mice . Then we investigated the
luciferase-induced bioluminescence in the animal livers by
a charge-coupled device camera. We showed that the
transplanted cells had engrafted into the liver and were
functional . Agarwal et al. transplanted definitive
endoderm cells derived from hESCs into the portal vein
of NOD-SCID mice that previously were treated with CCl4
and retrosine, and they detected that the cells expressed
human-specific mitochondrial antigen, as well as CD26 and
alpha-1-antitrypsin in the livers of the injected mice over
time . Hay et al. showed that hepatocyte-like cells could
engraft in the NOD-SCID mouse liver. They reported that
Wnt3a-treated cells expressed higher levels of human
albumin in the serum than did the control cells . Basma
et al. isolated the ASGPR positive cells from differentiated
hepatocyte-like cells by flow cytometery. Then, they trans-
planted these cells into the spleen of Alb-uPA SCID mice.
Very high levels of human albumin (1,000–2,000 ng/ml)
and alpha-1-antitrypsin were detected in the animals’ serum
75 days after transplantation. In histological evaluation,
small clusters of human CD18-expressing cells without any
tumor formation were observed. Then intrasplenic injection
of one million sorted cells was performed in an FK506-
immune suppressed Nagase analbuminemic rat or NOD-
SCID mice after 70% or 50% partial hepatectomy and
retrosine treatment. They reported 20,000 ng/ml human
Stem Cell Rev and Rep (2011) 7:748–759755
albumin in the transplanted animal’s serum with large
clusters of human engrafted cells in histological evaluation.
However, a well-differentiated adenocarcinoma was
detected as well . hiPSC-generated hepatocytes were
injected into the right lateral liver lobe of neonatal mice by
Si-Tayeb et al. After 7 days, human albumin-expressing,
integrated hepatocyte-like clusters of cells were observed in
the injected lobe .
The differentiation of hESC and other stem cells along a
hepatocyte lineage has undergone enormous changes over the
last decade. The progress has been substantial, with the
greatest paradigm shift being the emergence of hiPSC which
resolve many of the ethical issues surrounding hESC.
Although the progress towards the development of improved
protocols isimpressive, a numberofproblemsexist beforethe
toxicology or pharmacology studies. For example, xeno-free
and feeder free growth and differentiation conditions must be
established which are effective, reproducible, robust, and
relativelyinexpensive. This may include the developmentand
use of small molecules and synthetic biocompatible ECMs
which can substitute for the highly expensive growth factors
and xeno-derived ECMs.
New approaches may be developed in the near future that
may be even more effective than our present technology of
differentiating hepatocytes. For example, recent reports have
shown the transdifferentiation of fibroblasts to neuronal cells
and cardiomyocytes [108–111]. Thus one of the future
approaches may be the similar direct differentiation of
fibroblasts to hepatocytes or hepatic progenitor cells.
Despite continual improvements in in vitro function, the
engraftment and proliferation of hepatocyte-like cells from
hESC and hiPS has in general been disappointing in rodents.
One likely explanation is that the differentiated human cells
may not be compatible with the extracellular matrix and
growth factor niche of rodent livers. Thus it may be that
nonhuman primates will provide a better environment for
hepatocyte engraftment, differentiation, and proliferation.
Moreover, studies conducted in nonhuman primates will
obviously be better indicators of results in humans.
Tumorogenisity is still one of the main obstacles in the
clinical application of hiPSC and hESC, and a major
question is how safe must the cells be before they can be
used? How does one define lack of tumorogenisity? What
is an acceptable risk, and how does one determine that a
differentiated cell population is free from the presence of
any early progenitor cells? Another major issue is the loss
of proliferation of cells when they become significantly
differentiated. This leads to the question of whether a
sufficient number of mature hepatocyte-like cells that have
been derived from hESC or hiPSC can be obtained so that
cell transplantation can be clinically effective. Another
variant of this question is at what stage should cells that are
undergoing differentiation be transplanted? Should they be
transplanted at an early progenitor stage when they are
rapidly proliferating, yet not mature, or wait until they are
fully differentiated yet much less proliferative?
These are but a few of the many technical questions that
must be addressed in the coming years by investigators in
the field as our new-found success makes technical
questions take the forefront of research endeavors, and
basic research yields to translational approaches.
DK075415 (to M.A.Z.), California Institute of Regenerative Medicine
RC1-00359 (to M.A.Z.), Alpha-1 Foundation (to M.A.Z.), and a pilot
grant from the UC Davis Genome Center (to Y.D.).
This work was supported by NIH grant
The authors indicate no potential conflicts of interest.
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
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