Current Stem Cell Research & Therapy, 2010, 5, 00-00
1574-888X/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.
Hepatic Endoderm Differentiation From Human Embryonic Stem Cells
Zara Hannoun*, Céline Filippi*, Gareth Sullivan, David C Hay and John P Iredale‡
MRC-Centre for Regenerative Medicine, University of Edinburgh, Chancellor’s Building, 49 Little France crescent, Edinburgh EH16
Abstract: Primary human hepatocytes are a scarce resource with variable function which diminishes with time in culture. As a conse-
quence their use in tissue modeling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of hu-
man tissue due to their properties of self-renewal and their ability to give rise to all three germ layers. hESCs have the potential to pro-
vide an unlimited supply of hepatic endoderm (HE) which could offer efficient tools for drug discovery, disease modeling and therapeutic
applications. There has been a major focus on developing protocols to derive functional HE from hESCs. This review focuses on human
liver biology and the translation of observations of in vivo systems into developing differentiation protocols to yield hepatic endoderm. It
also details the potential role of oxygen tension as a new regulatory mechanism in HE differentiation and points out the importance of the
mitochondrial function analysis in defining successful HE generation.
Keywords: Human embryonic stem cells, liver, endoderm, mitochondria, hypoxia.
The liver’s main functions include the elimination of toxins and
waste products, maintaining metabolic homeostasis and the produc-
tion of serum proteins required for blood clotting and immune re-
sponses . The structure of the liver facilitates the efficient deliv-
ery of these functions . Hepatocytes contribute to approximately
80% of hepatic tissue, and as such are responsible for the majority
of liver functions. The hepatic vascular system has evolved to allow
75% of all blood entering the liver to come via the portal vein. This
facilitates the efficient removal of xenobiotics or toxic insult from
the blood by hepatocytes before it returns to the heart via the vena
cava. The fenestrated endothelium of the liver sinusoids facilitates
the exchange of small and macro-molecules with hepatocytes. In
between the hepatocytes are a series of canaliculi and ducts ‘the
biliary system’ that provide a major excretory route for detoxified
substances and the by-products of metabolism, .
A detailed understanding of liver development during human
embryogenesis has contributed to identifying conditions for hepatic
differentiation in vitro. Definitive endoderm originates from the
primitive streak at the 8-12 somite stage in humans. This is fol-
lowed by the invagination of the endoderm forming the foregut.
The precise location of the ventral foregut allows it to receive sig-
nals from the developing heart to induce its development towards
hepatic fate. In response to the signals the primary liver bud forms,
growing outward from the ventral foregut at the 13-20 somite stage
and nascent hepatoblasts then invade the septum transversum mes-
enchyme. Angiogenesis and vasculogenesis, mediated by oxygen-
sensing factors, occur simultaneously during liver bud formation,
leading to the vascular anatomy necessary for hepatic function. The
liver fully matures after birth when hepatocyte proliferation dimin-
ishes and mature function develops [3-5].
IMPORTANT SIGNALING PATHWAYS IMPLICATED IN
The initial induction of hepatic development occurs in response
to fibroblast growth factors (FGF) 1 and 2 produced by the card-
*Address correspondence to these authors at the Centre for Inflammation
Research, University of Edinburgh, QMRI, 47 Little France crescent,
Edinburgh EH16 4SB, UK; E-mail: John.Iredale@ed.ac.uk
*Authors contributed equally to this work
iogenic mesoderm. In addition to FGF Signaling, hepatic develop-
ment also requires another Signaling molecule; secreted by the
septum transversum mesenchyme, bone morphogenetic protein
(BMP). Both FGFs and BMP Signaling are required to induce the
formation of the hepatic endoderm .These Signaling molecules
promote the expression of the homeobox transcription factor Hex,
essential for hepatoblast expansion . BMP also regulates the
expression of the GATA factors however the exact mechanism
remains unknown .
The fetal liver functions as a major haematopoietic organ which
develops the full complement of mature metabolic functions after
birth. As such liver development can be regarded both as a simple
maturation process and a functional switch occurring at or around
parturition . The transcription factor CAAT/enhancer binding
protein (C/EBPa) has been demonstrated to be associated with this
functional switch. In addition there are various soluble factors asso-
ciated with liver maturation, for example oncostatin M (OSM),
glucocorticoid and hepatocyte growth factor (HGF). These factors
promote mature hepatic gene expression, morphological changes
associated with adult hepatocytes and induce cell function; such as
detoxification, serum protein and bile synthesis, glycogen storage
and lipid metabolism.
The elucidation of the Signaling pathways involved in liver de-
velopment is already providing strategies for efficiently differentiat-
ing hESC to HE.
KEY FACTORS IN HEPATIC ENDODERMAL DIFFEREN-
In vivo and in vitro formation of hepatic endoderm is a complex
process regulated by growth factors, cytokines, transcription factors
and the cellular adhesions. Below we consider in greater details the
specific role of each of these regulators in the differentiation proc-
ess and how they might potentially contribute to in vitro hepatic
differentiation culture conditions.
I. The FGF/BMP Signaling Pathways – Hepatic Induction
FGF signaling plays a central role in liver development by de-
fining hepatic specification. It facilitates the stimulation of hepatic
gene expression and nascent hepatocyte stability via the RAS/MAP
kinase pathway (MAPK). FGF ligands can activate the PI3 Kinase
pathway, which is active in foregut endodermal cells. However,
studies have demonstrated that inhibition of the PI3K pathway has
no effect on hepatic induction as FGF ligands exert their effect via
the MAPK pathway . Both FGF 1 and 2 are expressed by the
2 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 Hannoun et al.
cardiac mesoderm at the point of induction of hepatic specification
; in concert with FGF receptors 1 and 4 in the ventral foregut
endoderm. Morphogenetic events lead to hepatic endoderm being
distanced from the cardiac mesoderm, and hence the concentration
of FGF is reduced preventing differentiation into a more anterior
fate; the lungs. Gradients of FGF concentration provoke varying
responses which are essential for coordinating developmental
BMP signaling partners FGF to sustain complete induction of
hepatic endoderm. Co-operation between FGF and BMP 2 and 4,
produced by the septum transversum mesenchyme, promotes both
competence and specification of the primitive endoderm down the
hepatic lineage by induction of the transcription factor GATA4
II. The Wnt/?-Catenin Signaling – Priming and Proliferating
Unlike the FGF/BMP signaling, the Wnt/?-Catenin pathway is
involved in both differentiation and proliferation of pre-hepatic
endodermal cells. This is dependent on the respective Wnt ligand
Frizzled receptor interaction . Wnt9a secretion by sinusoidal
and stellate cells has been demonstrated to stimulate hepatoblast
proliferation, essential in both liver development, hepatocyte re-
population during injury and linked to hepatocyte maturation .
Studies in chicken have reported Wnt9a promotes glycogen accu-
mulation and up-regulation of glycogen synthase . Chicken
models indicate that Wnt3a stimulates proliferation of the periphery
of the liver lobes and regulates liver morphology . Zebrafish
genetic screens isolated a Wnt2b homolog essential for liver speci-
fication and may be involved if hepatoblast proliferation . Hay
and colleagues  demonstrated that Wnt3a is expressed during
hepatoblast differentiation to hepatocytes. When applied to a cell
culture model, Wnt3a addition resulted in a homogenous population
of stem cell derived hepatic endoderm. ?-Catenin and its down-
stream effectors have also been shown to interact with FGF10 in
promoting hepatoblast proliferation .
III. HGF and OSM Signaling – Hepatocyte Maturation
A number of soluble molecules have been demonstrated to ini-
tiate hepatic maturity. These include Hepatocyte Growth Factor
(HGF) and Oncostatin M (OSM). HGF is secreted by the septum
transversum mesenchyme, hepatoblasts and endothelial cells and
binds to the c-Met receptor, activating both the SEK1/MKK4 and c-
Jun signaling cascades . This results in Glucose-6-phosphate,
Tyrosine amino transferase, Carbamoyl-phosphate synthase and
Albumin expression, all of which are associated with mature liver
. OSM is produced by hematopoietic cells present in mid-fetal
livers. OSM is an IL-6 related cytokine and exerts its effect by acti-
vating the STAT3 (signal transducer and activator of transcription)
and Ras Pathways via the OSM receptor . A number of hepatic
gene promoters are directly regulated by STAT3 via binding sites in
there 5’ regulatory regions . Like HGF, OSM induces the ex-
pression of various liver markers . At birth OSM is down regu-
lated whilst HGF is unregulated . The liver undergoes a func-
tional switch at birth which accelerates liver maturation and pre-
pares the organ to respond to environmental changes; these include
a significant increase in the levels of glycogen and changes in oxy-
gen tension . To recreate these environmental changes present
at parturition may prove to be important for successful differentia-
tion to mature HE, in vitro.
IV. The TGFß Pathway – Hepatocyte Specification
The Tumor growth factor ? (TGF) pathway has been implicated
in differentiation of hepatoblasts down the biliary lineages .
This is thought to be achieved by the formation of a TGF? signaling
gradient; the exact mechanism is still unknown. Studies have indi-
cated that transcription factors HNF6 and OC2 regulate TGF? an-
tagonists such as Follistatin and ?-2-Macroglobulin. HNF6 is also
responsible for the down regulation of tgf? receptor II gene. In
HNF6 knockout mice there is an increase in the activation of the
TGF? pathway, and the animals exhibit a of lack of segregation
between the biliary and hepatic cell lineages . Furthermore,
mice with Smad2 and 3 (modulators of TGF? ligands) knockouts
display liver hyperplasia; this can be rescued by addition of HGF
. One could speculate that HGF regulates hepatic cellular orga-
nization and further contributes to hepatic specification.
In terms of in vitro modeling, It is reasonable to speculate that
inhibitors of the TGF? pathway may commit hESCs towards the
hepatic lineage On the other hand, modeling the system in 3D and
the generation of a TGF? signaling gradient may lead to the speci-
fication of the two cell types, both the hepatic and biliary, thereby
potentially exhibiting hepatic architecture.
V. Other Pathways Implicated in Hepatic Differentiation
Glucocorticoids have an important role in maintaining the func-
tional integrity of various cell types . These effects are medi-
ated via receptor activation on ligand binding, resulting in nuclear
translocation. Several genes are regulated via this pathway using
both translational and post-translational mechanisms . Dex-
amethasone is a synthetically generated glucocorticoid and has been
implicated in the maintenance of a differentiated liver phenotype in
vitro. It induces the expression of late hepatic genes, enhances the
activity of the albumin promoter  and has been found to reduce
cellular apoptosis . Dexamethasone also enhances the expres-
sion of the constitutive androstane receptor (CAR), involved in
hepatocyte detoxification, .
CAR is a nuclear hormone receptor responsible for regulating
the transcription of genes associated with drug metabolism; such as
the cytochrome p450 genes. It has been shown to dimerize with the
Retinoic acid X receptor (RXR?) and bind to the nuclear receptor
dimer complex which contains a phenobarbital response enhancer
module (PBREM) . The PBREM enhancer is found in a num-
ber of phenobarbital inducible genes, such as the Cyp 2B10 and
3A4 genes . Studies by Sidhu and co-workers have shown an
increase in both levels of CAR and RXR? in a dose dependent
manner on addition of dexamethasone. Interestingly, dexametha-
sone has been shown to exhibit a wide range of effects including
up-regulation of mature liver markers such as albumin, transferrin
and transthyretin. At low concentrations a significant increase in
phenobarbital induction response was observed for the cytochrome
P450s 2B1, 2B2 and 3A1, in rat hepatocytes. Induction and stabili-
zation of HNF3 and 4? and the maintenance of expression of vari-
ous liver enriched transcription factors including C/EBP?, -?, -?,
HNF1?, -1?, -3?, -4? and RXR?. Overall this demonstrates that the
glucocorticoid ‘dexamethasone’ is capable of enhancing hepatocyte
inducibility and maintain a mature hepatic phenotype. In fact sev-
eral groups have demonstrated the presence of dexamethasone in
differentiation culture media improves hepatocyte function and
I. Factors Governing Liver Specification
As previously mentioned three main transcription factors regu-
late hepatic specification during liver development . These are
Forkhead Box (FOX) A, GATA4 and CCAAT-enhancer binding
protein (C/EBP) ? transcription factors. These factors are involved
in the transcriptional activation of the albumin gene . FOXA
and GATA4 relax the chromatin around the promoter region of the
albumin gene allowing access of other factors and its activation
. This type of facilitative interaction is termed ‘competence’ as
it primes a cell of an unspecified fate down a particular lineage by
initiating its ability to respond to a specific set of signals.
The forkhead protein, FOXA1, also known as hepatocyte nu-
clear factors HNF3?, is a transcriptional activator of large number
Hepatic Endoderm Differentiation from hESC Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 3
of hepatocyte specific genes. In addition they have been implicated
in pancreatic development . FOXA1 binds to cis regulatory
elements within the promoters of albumin, AFP, transthyretin, tyro-
sine aminotransferase and PEPCK genes, triggering their expres-
sion . Further studies have isolated another factor, HNF1?,
demonstrated to be important for stimulating hepatic formation
. Interestingly, the phenotype of HNF1? null mouse models is a
lack of endoderm competence and thus unable to initiate hepatic
GATA4 is a member of the GATA zinc finger transcription fac-
tors which is involved in the regulation of the heart tube and foregut
formation during embryogenesis, more specifically linked to myo-
cardial differentiation . The GATA transcription factors recog-
nize a consensus sequence AGATAG located in the promoter re-
gions of a number of genes such as the insulin growth factor I gene
. It has been demonstrated that GATA4 is essential for hepatic
specification as deficient mouse ES cells failed to differentiate into
definitive endoderm . In conclusion, GATA4 is essential for the
differentiation of extra embryonic endoderm in developing em-
bryos, it also plays a vital role in pancreatic and hepatic formation
in conjunction with GATA 6, FOXA1and C/EBP.
C/EBP ? is a member of the bZIP transcription factors that rec-
ognize the, consensus CCAAT as either a homo- or heterodimer
with other members of the family . C/EBP ? is a regulator of
gluconeogenesis and is able to activate phosphoenolpyruvate car-
FOXA1, HNF1? and GATA factors specifically regulate he-
patic specification whilst C/EBP proteins modulate hepatocyte me-
tabolism. Modulating the expression of these factors in a physio-
logical manner may prove fruitful in generating homogenous he-
patic endoderm populations, which induce function in response to
II. Hepatocyte Specific Transcription Factors – The Extended
Analysis of the hepatic transcriptome at pre- and post-natal
stages has isolated a large number of liver enriched transcription
factors, the majority of which belong to the hepatocyte nuclear
factor family of transcription factors (HNF). These include HNF1?
and ? (previously mentioned), HNF3? and ?, HNF4? and HNF6.
The expression patterns of these factors have helped to distinguish
their various roles throughout liver development. HNF1? is exclu-
sively expressed in fetal hepatocytes suggesting its role in their
specification. HNF4? on the other hand is expressed in both fetal
and adult hepatocytes; as such its role may be implicated in both
differentiation and maintenance of the hepatic phenotype. HNF6 is
expressed in both fetal, adult hepatocytes and in biliary epithelial
cells, however, its expression is ablated on biliary maturation.
HNF6 is central to regulation of gluconeogenic, glycolytic, bile acid
synthesis pathways and is essential for hepatocyte proliferation.
HNF3? and ? is expressed in both fetal and adult biliary epithelial
cells (BEC) and fetal hepatocytes; but lost on maturation to adult
hepatocytes. As such each factor has a unique role in liver specifi-
HNF4?, HNF6, HNF1? and ? all work synergistically and co-
operatively to coordinate hepatocyte differentiation. HNF4 and 6
co-regulate glucose-6-phosphate expression whilst HNF1 and 4?
control glucose, lipid and amino acid metabolism. HNF1? is impor-
tant for regulating bile acid and fatty acid oxidation .
Although each of these factors is individually significant,
HNF4? appears to be central. Embryos deficient in HNF4 die dur-
ing gastrulation. This can be circumvented using conditional knock
outs; however this still results in the formation of embryonic livers
containing large red lesions, with discontinuous parenchyma. The
deletion of HNF4 has deleterious effects for hepatocytes differen-
tiation, including metabolic function and altered cell morphology
and adhesions. Therefore, HNF4 is essential for hepatocyte differ-
entiation, metabolism and morphology .
CELLULAR ADHESIONS AND INTERACTIONS IMPOR-
TANT IN DIFFERENTIATION
The Extra Cellular Matrix
Hepatocytes are polarized cells with the apical domain corre-
sponding to the bile canaliculi and the basal domain corresponding
to the sinusoids . The extracellular matrix (ECM) in contact
with the basal domain of hepatocytes contains laminin, collagen
types I - V, fibronectin and proteoglycans . The interactions
between hepatocytes and the ECM are essential to maintain hepatic
polarity and functionality . The exact effects of ECM on HE
will be discussed in section 7.
Hepatic Directed Differentiation Protocols
Elucidating the key pathways involved in hepatic endoderm
formation in vivo has allowed us to apply these to the in vitro situa-
tion. Over the past decade a number of procedures have been pub-
lished demonstrating successful differentiation of hESCs to HE,
Fig. (1) . hESC derived HE is classified using a number of cri-
teria, specific gene expression, serum protein production, glycogen
storage, CYP450 and urease activity . Although this validation
is internationally used and accepted, a more systematic approach is
warranted. The resulting HE will provide a defined and reliable
model that can be utilised in disease modeling and drug discovery
and as culture conditions are defined. HE will prove to be indispen-
sable for cell based therapy and tissue regeneration, Fig. (3).
There are a large number of protocols for HE formation ranging
from embryoid body (EB) derivation in fetal bovine serum (FBS)
, to differentiating hESCs utilising 2-D systems using collagen,
gelatin or matrigel as the basement membrane. Agarwal and col-
leagues cultured hESCs on collagen in the presence of FBS, knock-
out serum replacement (KOSR) and bovine serum albumin (BSA)
supplemented with Activin A (AA), fibroblast growth factor (FGF),
hepatocyte growth factor (HGF), oncostatin M (OSM) and dex-
amethasone. The resulting HE expressed a number of hepatocyte
specific markers including albumin, alpha-fetoprotein (AFP),
CYP3A4, CYP7A1 and was also capable of glycogen storage and
albumin secretion . Schwartz and colleagues generated HE
solely in the presence of FGF and HGF on collagen which ex-
pressed GATA4 and HNF1? and 3?; capable of albumin and urea
production and exhibited Cytochrome P450 activity after treatment
with Phenobarbital . HE has also been formed on collagen in
the presence of FBS supplemented with insulin, dexamethasone,
transferrin and selenious acid, where expression of albumin,
transthyretin and albumin serum protein production was observed
. hESCs cultured on gelatin in the presence of HGF and nerve
growth factor (NGF) generated HE expressing various hepatocyte
specific genes, but showed no further functionality . Hay and
colleagues demonstrated treatment with either Activin A/Wnt or
sodium butyrate followed by treatment with dimethylsulfoxide
(DMSO) generated immature hepatocytes that could be matured
with HGF, insulin, OSM and hydrocortisone to give HE that ex-
pressed the majority of hepatocyte specific genes, were capable of
glycogen storage, produced significant levels of hepatic serum pro-
teins and had inducible Cyp P450 activity [16, 57-59].
There is debate whether or not transferring culturing conditions
into 3-D environments may enhance HE function; as this potentially
mimics in vivo development more accurately. It has been proposed
that culturing hepatocytes between double layers of ECM in 3-D
structures will establish polarity and enhance hepatic function and
viability mimicking the in vivo situation. One group has success-
fully differentiated hESCs into HE in a 3-D environment. Bahar-
vand et al., cultured hESCs in self renewing conditions and using
the hanging drop method formed EBs. These EBs were then seeded
4 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 Hannoun et al.
onto collagen coated 3-D scaffold in culture medium supplemented
with FGF, HGF, OSM, insulin, dexamethasone, transferrin and
selenium. The HE expressed a number of hepatic specific genes and
produced significant levels of both urea and albumin . Du and
colleagues successfully constructed an ECM free synthetic culture
by sandwiching a hepatocyte monolayer between two membrane-
like structures. The top support system consisted of a glycine - ar-
ginine - aspartic acid - serine (GRGDS) modified polyethylene
terephthalate (PET) membrane. The bottom substratum consisted of
a galactosylated PET membrane. This resulted in hepatic polarity
including biliary excretion and enhanced function when compared
to 2-D collagen coated cultures using HE derived from hESCs .
Ng and colleagues successfully combined methylated and galacto-
sylated collagen nanofibres that optimized the interactions required
for the maintenance of functional hepatocytes. This enhanced inter-
actions between the nanofibres and the asialoglycoprotein receptor
(ASGPR), hence promoting hepatic function [62, 63]. These results
signify the importance of ECM interactions for maintaining hepatic
functionality. Sufficient differentiation has been achieved on 2-D
culture systems. However one can speculate that HE differentiation
will never reach its full potential until culture systems incorporate
the correct signaling factors and ECM.
Basma and colleagues established an efficient protocol for the
purification of a hepatocyte population from a heterogeneous endo-
dermal population. The hESCs derived EBs were plated onto Ma-
trigel and treated with Activin A and FGF 2. The cells were then
placed into defined media supplemented with HGF followed by
dexamethasone. The resulting HE was further enriched by FACS
sorting for ASGPR positive cells, a specific feature of mature hepa-
tocytes. The pure population of HE expressed hepatic gene function
comparable to adult hepatocytes .
Cai and colleagues developed a physiological protocol that
mimicked the in vivo situation. This involves priming hESCs with
Activin A to direct them towards definitive endoderm, followed by
BMP and FGF generating hepatic endoderm. The resulting HE was
matured using HGF, OSM and dexamethasone. The HE expressed a
range of mature hepatic genes; however there was no expression of
AFP, indicative that the HE produced is more mature than those
derived using other protocols. The HE produced significant albumin
and interestingly was susceptible to infection by the hepatitis virus
As the field of regenerative medicine advances there will be a
requirement for more defined and reproducible culture systems.
This has been the goal of Baharvand and colleagues, where hESCs
Fig. (1). The schematic displays the various areas which affect human embryonic stem cell differentiation into hepatocyte like cells. 1. Growth factors
and cytokines 2. Transcription factors 3. Extracellular matrix and cell:cell interaction 4. Energy pathways. Successful coordination of these events leads to the
formation of a mature viable hepatocyte with Cyp inducibility, ureogenesis, secretion of specific hepatic proteins such as albumin, fibrinogen and fibronectin
and the expression of hepatocyte specific genes. Abbreviations – AFP, alpha feto protein; Alb, albumin; BMP, bone morphogenic protein; Cyp, cytochrome
p450; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HNF, hepatocyte nuclear factor; OSM, oncostatin M; TAT, tyrosine amino transferase;
TGF, tumor growth factor; TTR, transthyretin.
Hepatic Endoderm Differentiation from hESC Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 5
have been grown on matrigel under serum free conditions and
treated with a series of defined factors to produce HE that express
hepatic genes, produce serum protein, display, urease activity, gly-
cogen storage and uptake of low density of lipoproteins .
The above studies have indicated that a combination of FGF
and BMP factors could be used in vitro to mimic the initial stages
of liver development by priming hESCs down the hepatic endoder-
mal lineage . Wnt has also been linked to both the priming of
hESCs and enhancing their proliferation. As such, sequential addi-
tions of FGF/BMP followed by Wnt may prove to be good method
of mimicking the conditions in vitro resulting in more functional
The various differentiation protocols to produce HE have so far
focused on the effects of growth factors and transcriptional regula-
tors, extracellular matrices and have been studied in great detail, as
mentioned above. However, another component that has been over-
looked, whilst critical during liver development, is the influence of
oxygen on stem cell differentiation into hepatic endoderm. Oxygen
has been shown to influence many aspects of stem cell biology,
differentiation and embryo development. Since the 1970s we have
known that ex-vivo culture of embryos at 20% O2 leads to neural
fold defects, which does not occur in low O2 environments. In addi-
tion low O2 concentration improves neural crest stem cell growth,
increases the quantity of multipotent clones and generates various
differentiated cell types not observed at O2 concentrations lower
that 20% . Low O2 concentrations have also been shown to
maintain specific progenitor cells in an undifferentiated state [68-
73] through interactions with Oct4 , Nanog  and Notch [72,
74-77] Signaling pathways. The opposite was shown in chondro-
cyte and cardiomyocyte generation [78, 79]. To date, no investiga-
tion has been performed to study the effects of low oxygen tension
(hypoxia) on stem cells differentiation into hepatocytes. Interest-
ingly, studies have demonstrated the importance of the hypoxia
inducible factor (HIF) mRNA around 12-14 weeks of gestation
stage in the human fetal liver . Therefore one could speculate a
role for HIF and its downstream effectors in hepatoblast/liver de-
The next part of this review deals with the various pathways in-
volved in O2 sensing and their potential consequences in hepatic
endoderm differentiation as well as its link with mitochondrial biol-
THE ROLE OF OXYGEN IN hESC DIFFERENTIATION
i. Hypoxia Signaling
For several decades the classification of physiologically nor-
moxic or hypoxic conditions has been the matter of lengthy debate.
Physiological O2 concentration for embryonic or adult cells varies
widely, but the acknowledged physiological normoxic state now
falls between 2-13% O2. Indeed the HIF pathway has been shown to
become activated at O2 concentrations below 2% in cell culture and
the highest physiological O2 concentration, in arterial blood, is
13%.There are some exceptions to this rule such as the thymus,
kidney medulla and bone marrow, which can physiologically exist
at 1% O2, or lower, owing to their atypical vascular networks.
Since the derivation of hESC was first reported in 1998 , it
has been routine practice to culture these cells under atmospheric
oxygen (21% O2). The efficacy of this practice may prove question-
able as the pre-implantation human conceptus is subjected to O2
tensions well below this concentration in utero  and O2 concen-
tration may represent an important regulator of hepatic growth,
maturation and function. Culturing hESC under high O2 leads to
overt differentiation within ESC colonies. This can be alleviated by
maintaining hESC culture under physiologically low pO2 (2-5%),
supporting self renewal and pluripotency [68, 83]. The main path-
way leading the cellular adaptation to hypoxia is the Hypoxia in-
ducible factor (HIF) pathway. First described in 1992 , HIF1 is
a heterodimeric transcription factor consisting of HIF-1? and HIF-
1?, . Under physiological normoxia, HIF-1? becomes hydroxy-
lated at two proline residues [86, 87] and is targeted by the von
Hippel-Lindau (VHL) protein for ubiquitination and its subsequent
proteasome-mediated degradation [88-90]. Under hypoxia, the
HIF-1? protein is not hydroxylated and is stable. It then translo-
cates to the nucleus where it dimerizes with HIF-1? and initiates
gene transcription by binding to hypoxia-responsive elements
(HRE), Fig. (2A).
Although HIF-1? is mainly regulated by oxygen tension, other
factors also modulate its expression and consequent function: nitric
oxide (NO), for example, has been shown to regulate HIF-1? ac-
cumulation ; the cytokines interleukin-1? and tumour necrosis
factor ? stimulate DNA binding of HIF-1 ; the transcriptional
activity of HIF-1 can be enhanced by the activation of p44/42 MAP
kinase [93, 94] and by trophic stimuli such as serum, insulin, and
insulin-like growth factors (IGF-1, IGF-2) [95-97]. Over-expression
of the v-src oncogene  as well as inactivation of the tumour
suppressor genes p53  or loss of PTEN  induce HIF-1?
over-expression and enhance the transcriptional activity of down-
Genes induced by HIF-1 are involved in a wide range of cellu-
lar functions such as cell growth, survival, motility, angiogenesis,
energy metabolism and prevention of cellular differentiation [70,
73, 88, 101-107]. Conversely, the pro-differentiation gene PPAR? is
down-regulated as a result of HIF activation . HIF-2?, a ho-
molog of HIF-1? also interacts with HIF-1?. It shares a similar
mechanism of O2 regulation with HIF-1? but has a restricted tissue
distribution. Whilst HIF-1? is essential for early embryonic devel-
opment, HIF-2? seems to regulate the late fetal development of
certain tissue types due to its implication in VEGF expression. In-
terestingly, HIF-2? has been shown to regulate the expression of
Oct-4 a marker of ‘stemness’ in progenitor cells [68, 69].
Besides HIF, mTOR, the mammalian target of rapamycin (also
known as FRAP, RAFT, RAPT or SEP) also regulates the temporal
and spatial growth of cells in response to hypoxia and other envi-
ronmental cues including growth factor Signaling, nutrient avail-
ability, the cell energy status, the cell redox status and DNA dam-
age, Fig. (2A) (for review see ). mTOR is a conserved Ser/Thr
kinase of the phosphoinositide 3-kinase related kinase (PIKK) fam-
ily. It can be part of two distinct multiprotein complexes, mTOR
complex 1 (mTORC1), sensitive to rapamycin, or complex 2
(mTORC2), not sensitive to rapamycin. When activated, mTORC1,
composed of the Regulatory Associated Protein of mTOR (Raptor)
and other associated proteins, phosphorylates the initiation factor
4E-binding protein-1 (4E-BP1) and the p70 ribosomal protein S6
kinase (p70S6K) resulting in decreased protein synthesis, growth and
proliferation, which helps to conserve ATP. By contrast, mTORC2,
which contains mTOR and the Rapamycin-Insensitive Companion
of mTOR (Rictor) phosphorylates and activates Akt/PKB, thus
regulating cell proliferation, survival and metabolism. mTORC2
also controls cell shape and motility, by regulating the actin skele-
ton. A number of studies have identified the mTOR pathway as a
positive regulator of HIF-1? , with evidence for regulation at the
level of transcription, translation and protein stability [110, 111].
Hypoxia rapidly and reversibly triggers the hypo-phosphorylation
of mTORC1 and its effectors: 4E-BP1, p70S6K1 and p70S6K2, the
ribosomal protein S6 (rpS6), and the eukaryotic initiation factor 4G
(eIF4G). The effects of hypoxia on mTORC1 predominate the oth-
erwise opposite effects of insulin, amino acids, phorbol esters, and
serum. Whilst hypoxia certainly affects the Akt/protein kinase B
(PKB) and AMP-activated protein kinase (AMPK) phosphoryla-
tion, ATP levels, ATP/ADP ratio variation, its effects on mTOR is
actually independent of these pathways .
6 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 Hannoun et al.
ii. Hypoxia and Embryo Development
Wild-type embryos initiated HIF-1? expression at day E8.5 to
9.5, whilst Hif-1? null embryos developed neural tube defects,
cardiovascular malfunction and exhibit cell death within the ce-
phalic mesenchyme , leading to developmental arrest and
lethality by day 11 . KO of Hif-2? leads to various phenotypes
depending on the mice strains (reviewed in ), including, yolk
sac and embryo vascular defects, retinopathy, anaemia, fatal respi-
ratory distress, hepatic steatosis and embryonic lethality due to
cardiopathy. Simon and colleagues have shown gestational lethality
of murine embryos lacking the HIF-1? protein, with defects in pla-
cental and yolk sac formation as well as a reduction in vasculature,
blood cell and heart formation . Hif-2? was shown to specifi-
cally increase the expression of VEGF and TGF? during fetal de-
velopment [115, 116].
iii. Hypoxia Effects are Mediated Through an Increase in ROS
Production by Mitochondria
In normal conditions, O2 is the final acceptor of electrons which
are channelled through the mitochondrial respiratory chain, partici-
pating in the synthesis of ATP via the oxidative phosphorylation,
Fig. (2). Mitochondrial production of reactive oxygen species and their effects in hypoxia signaling. (A) The reducing equivalents produced by the
intermediary metabolism (NADH and FADH2) are re-oxidized by the respiratory chain complex I and II, the electrons being driven along the chain up to the
complex IV where they are accepted by the O2 to form H2O. In this process, the protons from the NADH and FADH2 are expulsed into the intermembrane
space creating the protonmotive force, participating at the creation of the mitochondrial membrane potential (??m). The protons are then channeled back in-
side the mitochondria, mostly through the F0F1-ATP synthase which produces ATP from ADP. The protonmotive force assures the tight coupling between the
oxidation and phosphorylation, except in conditions of proton leak through the inner membrane. In physiological conditions, the oxidative phosphorylation is
controlled only by the availability of NADH or FADH2 and ADP, itself channeled inside the matrix by the ANT. The electrons can ‘escape’ the channeling
and form highly reactive O2
trons do not find their final acceptor at the complex IV. In the fetal liver, the respiration is mostly controlled by the ANT as its expression is only 50% of what
can be found in adult mitochondria. The regulation of ATP concentration by the Mg2+-ATP/Pi antiporter is not functional in the fetal liver as Mg2+ is seques-
tered high levels of mitochondrial-bound hexokinase (not represented) (B) Reactive oxygen species produced in the cell, by the mitochondria or other systems,
regulate both the HIF and mTOR pathways through the prolyl hydroxylase (PHD) inhibition and REDD1 activation, respectively. The inhibition of the PHD
prevents HIF-1? binding with VHL and its subsequent ubiquitination, hence stabilizing it and allowing its translocation to the nucleus, dimerizzation with
HIF-1? and binding to hypoxia responsive elements in target genes. REDD1 expression activates TSC1/2 potentially because of their competition for the same
14-3-3 repressor protein (not represented, for more details see ). TSC1/2 can then inhibit mTORC1 resulting in a decreased protein synthesis, mitochon-
drial biogenesis and cell proliferation. The effects of hypoxia on the mTOR pathway dominate any other signaling affecting Akt or AMPK function.
-?, both in the matrix (not shown) and in the intermembrane space, at much greater fluxes when O2 becomes limiting and the elec-
Hepatic Endoderm Differentiation from hESC Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 7
Fig. (2B). Hypoxia decreases the availability of O2 thus leading to
an increase of the mitochondrial membrane potential. This drives
the escape of electrons at the 3rd complex of the chain and induces
the formation of reactive oxygen species (ROS) able to transfer into
the cytoplasm . These ROS have been shown to mediate the
activation of HIF-1? expression and its subsequent effects [118,
119]. Whilst not directly linking it to ROS production, Schieke et
al. demonstrated that mTOR activation in hESC was also a function
of the mitochondrial membrane potential and, subsequently, influ-
enced their differentiation capacity . In stem cells the overall
ROS synthesis is lower than in differentiated cells . This may
be linked to the reduced number of mitochondria within stem cells
as compared to their differentiated counterparts. However, the very
fact that mitochondria in stem cells are less active and hence do not
consume as much oxygen as in differentiated cells, results in the
cytosolic O2 concentration being higher and leading to elevated
production of ROS in microsomes .
iv. Oxygen Tension Regulates Stem/Progenitor Cell Differentia-
Oxygen tension has been shown to regulate cell differentiation,
although the effects vary depending on the precursor/target cells. A
low O2 concentration is necessary for neuronal differentiation using
central nervous system precursors [123-125]. It has also been
shown to enhance chondrogenesis from bone marrow derived mes-
enchymal stem cells  and cardiomyogenesis from hESCs and
mouse EBs [118, 126]. In contrast, low O2 concentration delays
myogenesis myoblasts  and decreases the capacity of preadi-
pocytes to mature into adipocytes [71, 108]. CNS precursors exhib-
ited enhanced proliferation and displayed decreased apoptosis rates
in low O2 environments in the presence of mitogens [123, 124].
Interestingly, hypoxia promoted the generation of multiple lineages
favoring a dramatic increase in oligodendrocyte formation and en-
hancing dopaminergic neurones differentiation.
As previously mentioned, no study has focused on the effects of
physiological O2 concentration on fetal liver differentiation or
hESC differentiation towards HE. It has been shown that Wnt acti-
vation leads to increased cell proliferation through the activation of
mTOR, via the inhibition of GSK3 and TSC2 . Hence, the
activation of mTOR during hypoxia, could complement or syner-
gise the action of Wnt in inducing hESC differentiation into hepatic
endoderm . In ESC and mouse embryos, Hif-2? ectopic expres-
sion induces an over-expression of Brachyury, a marker of meso-
derm, and ?-fetoprotein, a marker of definitive and extra-
embryonic endoderm. One could speculate that the level of O2 ex-
posure in the developing embryo may participate in the morpho-
genesis of endodermal tissues, in particular the hepatobiliary axis.
v. Oxygen Tension and Mitochondrial Function
A decrease in oxygen tension does not result in decreased mito-
chondrial oxidative phosphorylation – not until the oxygen level
falls to critically low levels. However, in differentiated cells, a
gradual decrease in oxygen concentration over a few hours does
regulate both mitochondrial respiration and cellular ATP concentra-
tion, well before the ‘hypoxia threshold’ values are reached .
Fig. (3). Differentiating human embryonic stem cells into viable mature hepatocyte like cells will provide the tools for a number of important applications.
These include drug discovery, toxicity analysis, cell therapy, the bio-artificial liver and disease modeling.
8 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 Hannoun et al.
One explanation may be a lower output of ROS that would reduce
mTOR activation and HIF translation/stabilization [91, 111, 118,
119, 130]. Because mTOR controls mitochondrial biogenesis, its
inactivation could then further reduce ROS production [131, 132].
vi. Hepatic Endoderm Differentiation and Mitochondria
The liver consumes one fifth of all inspired oxygen, with the
majority of oxygen being used during the oxidative phosphorylation
process within the cell. The proportion of oxygen consumption by
the liver reveals both the extent of its function within the body and
the importance of mitochondrial oxidative phosphorylation within
this organ. Besides its importance in oxidative phosphorylation, a
number of CYP450 activities reside in mitochondria. Consequently,
the study of mitochondrial function, mitochondrial biogenesis and
their regulations appears of the utmost importance for the develop-
ment of fully functional hepatocytes.
Mitochondria possess their own genome, a 16 kilobase double-
stranded circular DNA (mtDNA), found as 2 to 10 copies per mito-
chondria . mtDNA encodes 13 polypeptides, all involved in
the respiratory chain complexes, and 22 tRNAs. Hence, most of the
~1500 proteins found in mitochondria are encoded by the nuclear
genome, and require specific import into the outer-, inner mito-
chondrial membrane and the mitochondrial matrix, depending on
their final location . Mitochondrial biogenesis is coordinated
by nuclear and mitochondrial encoded transcription activators and
co-activators [135, 136], the central factor being PPAR? co-
activator-1 ? (PGC-1??) . Environmental signals induce the
expression of PGC-1? and PGC-1? a related co-activator (PRC)
, which in turn target specific transcription factors (NRF-1,
NRF-2, and ERR alpha) leading to altered expression of respiratory
genes (for an exhaustive list of the NRF-1 and -2 recognition sites
in nuclear genes required for the respiratory chain expression and
function, see ). Mitochondrial transcription itself is then di-
rected by a small number of nuclear-encoded factors (TFAM,
TFB1M, TFB2M, mTERF [140, 141]), whilst mitochondrial repli-
cation is controlled by the mitochondrial DNA Polymerase (Pol?).
Development of high integrity, functional mitochondrial may prove
essential to the development of functional HE (or indeed other line-
ages) from hESC.
Embryo Development and Mitochondrial Function
It is known that mitochondria, because of their ability to gener-
ate ATP, have a central role in the normality of early mammalian
development . Metabolic, structural and numerical defects in
mitochondria have been associated with oocyte maturation and
premature arrest or abnormal development of the embryo [142-
144].Cell type- and location-specific domains of differential mito-
chondrial membrane potential exist in the peri-implantation blasto-
cyst. Indeed, cells from the trophectoderm and the inner cell mass
display distinct mitochondrial functions. The trophoblast contains
highly active mitochondria with high membrane potential whilst the
inner cell mass contains limited numbers of mitochondria with low
membrane potential . Houghton’s group demonstrated that
high nitric oxide production in the blastocyst mitochondria could
reduce the fluxes of the oxidative phosphorylation, through an inhi-
bition of the respiratory chain fourth complex, the cytochrome c
oxidase, which could potentially lead to this variation in the mem-
brane potential [146, 147].
Mitochondrial biogenesis and function are upregulated
throughout placentation (gestational days 11-13) in rat embryos
with an increase in the expression of nuclear proteins involved in
the co-ordination of mitochondria biogenesis (PGC-1?, NRF1 and
2, TFAM) as well as in the activity of the complexes of the oxida-
tive phosphorylation . Overall mitochondrial function in-
creases gradually in the final third trimester of rat and human fetus
development [136, 149-153]. A progressive improvement of the
mitochondrial function in the brain, heart, and livers [152, 154,
155], allowed the corresponding cells to rely on oxidative metabo-
lism rather than glycolytic metabolism for ATP generation. In the
liver, this gradual improvement is a consequence of progressive
augmentation of mitochondrial protein concentration and mito-
chondrial number within hepatocytes . Besides the slow matu-
ration of mitochondrial function happening during rat fetal devel-
opment, liver mitochondrial function increases dramatically right
after birth [151, 153, 155, 157, 158], resulting in a significant in-
crease in high-energy phosphate concentrations within the hepato-
cyte cytoplasm. The importance of the ATP and ADP transport in
fetal mitochondria function was highlighted by Hale and William-
son  who demonstrated that the adenine nucleotide transporter
(ANT), was controlling 98% of the cell respiration in the fetus,
whilst it had less or no control in the adult due to a large increase in
its expression in the adults as compared to the fetus. An increase of
the activity of the ANT occurs at birth probably due to hormonal
changes. In 1980, Sutton and Pollak [159, 160] demonstrated the
existence of another, Mg2+-dependent, ADP/ATP co-transportation
into the intermembrane space of the mitochondria and its atypical
regulation by the hexokinase, through a Mg2+ sequestration mecha-
In addition to the variations of ATP and ADP transport across
the mitochondrial membranes in the fetus versus the adult, Luis et
al. discovered that ?-F1-ATPase mRNA translation initiation was
regulated by a 3’ untranslated region binding protein (3’?-FBP)
which prevents the efficient translation of the ATPase transcripts
[161-164]. This results in the accumulation of a translationally-
repressed ribonucleoprotein particle. Interestingly, the formation of
the 3’ ?-FBPs complex with a 3’ ?-cis acting element on the AT-
Pase mRNA was later found to be regulated by the adenine nucleo-
tide concentration as well as the redox environment: indeed increas-
ing concentrations of ATP, ADP or AMP as well as oxidative con-
ditions, decrease the amount of complexes .
Very few studies have been undertaken to determine the effects
of endoderm development cues on mitochondrial functions in the
fetus. The only data present in the literature deal with the effects of
(i) activin and (ii) BMP Signaling alteration: (i) Animals hypomor-
phic for Activin A display a highly affected energy metabolism, as
measured by a large increase in mitochondrial oxidative phosphory-
lation uncoupling. They also showed a high increase in the expres-
sion of genes such as PGC1-? and NRF1 (see below for more de-
tails), which regulate mitochondrial biogenesis . Interestingly,
Meinhardt et al. demonstrated in germ cells that Activin produced
by stromal cells was maintaining condensed type of mitochondria
which exhibit a much lower oxidative phosphorylation efficiency
, as compared with its orthodox counterpart. Hence Activin
Signaling potentially participates in maintaining mitochondria in an
inactivated state. (ii) BMP Signaling, described previously in this
review, could also participate in mitochondrial biogenesis process.
Indeed it has been shown to induce the expression of the evolution-
arily conserved signaling intermediate in Toll pathways (ECSIT)
protein, that Vogel and colleagues later demonstrated to possess an
N-terminal mitochondrial targeting signal, where it interacts with
the assembly chaperone NADH dehydrogenase (ubiquinone)-
1alpha assembly factor 1 (NDUFAF1), hence regulating mitochon-
drial complex 1 assembly. Indeed, Ecsit knockdown using RNA
interference resulted in decreased NDUFAF1 and respiratory com-
plex I protein levels, accumulation of complex I sub-complexes,
and disrupted mitochondrial function .
Recent progress in the field of liver developmental biology has
provided efficient human models of stem cell-derived HE. Al-
though a number of successful techniques have been developed, the
repertoire of function in stem cell-derived HE is not yet as broad as
that of primary human hepatocytes. Given the improvements in cell
culture technology, hESC-derived HE could potentially be de-
ployed in high throughput screening, extra-corporeal liver device
Hepatic Endoderm Differentiation from hESC Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3 9
construction and ultimately in human cell therapy. Development of
high integrity, functional mitochondrial may prove essential to the
development of functional HLCs (or indeed other lineages) from
Zara Hannoun was funded by the Medical Research Council
and the University of Edinburgh. Céline Filippi was funded by a
grant awarded from the UK-Stem Cell Foundation and Scottish
Enterprise. David Hay was supported by a RCUK Fellowship.
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Received: 00 00, 2010 Revised: 00 00, 2010 Accepted: 00 00, 2010