Highly Efficient Generation of Human Hepatocyte–Like
Cells from Induced Pluripotent Stem Cells
Karim Si-Tayeb,1* Fallon K. Noto,1* Masato Nagaoka,1Jixuan Li,1Michele A. Battle,1Christine Duris,2Paula E. North,2
Stephen Dalton,3and Stephen A. Duncan1
There exists a worldwide shortage of donor livers available for orthotropic liver transplan-
tation and hepatocyte transplantation therapies. In addition to their therapeutic potential,
primary human hepatocytes facilitate the study of molecular and genetic aspects of human
hepatic disease and development and provide a platform for drug toxicity screens and
identification of novel pharmaceuticals with potential to treat a wide array of metabolic
As an alternative to using donor livers as a source of primary hepatocytes, we explored the
possibility of generating patient-specific human hepatocytes from induced pluripotent stem
(iPS) cells. Conclusion: We demonstrate that mouse iPS cells retain full potential for fetal
liver development and describe a procedure that facilitates the efficient generation of highly
can integrate into the hepatic parenchyma in vivo. (HEPATOLOGY 2010;51:297-305.)
See Editorial on Page 20
along with either Klf41–4or Nanog and Lin285raises the
he ability to generate induced pluripotent stem
of the reprogramming factors Oct3/4 and Sox2
possibility of generating patient-specific cell types of all
lineages. Differentiated cell types produced from a pa-
tient’s iPS cells6have many potential therapeutic applica-
tions, including their use in tissue replacement and gene
therapy. Although the use of iPS-based cell therapies is a
realistic long-term goal, if protocols that facilitated effi-
cient differentiation into specific cell lineages could be
developed, iPS-derived cells could be used immediately
for the analysis of disease mechanisms and the identifica-
tion and study of pharmaceuticals.
The generation of hepatocytes from iPS cells is a par-
ticularly appealing goal because this parenchymal cell of
the liver is associated with several congenital diseases,7is
the site of xenobiotic control, and is the target of many
pathogens that cause severe liver dysfunction, including
hepatitis B and C viruses. Moreover, unlike most other
organs, the introduction of exogenous hepatocytes into
the liver parenchyma is a relatively simple undertaking,
suggesting that the liver is highly amenable to tissue ther-
apy using iPS cell–derived hepatocytes.8–10We therefore
to adopt a hepatic cell fate in embryos and to establish a
protocol using defined culture conditions for the genera-
tion of human hepatocyte–like cells from iPS cells.
Materials and Methods
Human ES and iPS Cell Culture. Human H9
(WA09) ES cells and iPS cells were cultured using stan-
Abbreviations: AFP, alpha-fetoprotein; ALB, albumin; BMP4, Bone morpho-
genetic protein 4; EGFP, enhanced green fluorescent protein; ES cells, embryonic
stem cells; Fapb1, fatty acid binding protein 1; FGF2, fibroblast growth factor 2;
Fox, Forkhead box; GATA4, GATA binding protein 4; hiPS, human induced
pluripotent stem cells; HNF, hepatocyte nuclear factor; huES cells, human embry-
onic stem cells; iPS cells, induced pluripotent stem cells; mRNA, messenger RNA;
Rbp4; retinol binding protein 4; Sox17, Sex determining region Y box 17.
From the1Department of Cell Biology, Neurobiology and Anatomy; and the
2Department of Pathology, Division of Pediatric Pathology, Medical College of
Health Sciences, University of Georgia, Athens, GA.
*These authors contributed equally to the manuscript.
Received August 10, 2009; accepted September 17, 2009.
Address reprint requests to: Stephen A. Duncan, Department of Cell Biology,
Road, Milwaukee, WI 53226. E-mail: firstname.lastname@example.org; fax: 414-456-6517.
Copyright © 2009 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Supported by National Institutes of Health (National Institute of Diabetes and
Digestive and Kidney Diseases/National Heart, Lung and Blood Institute
[NHLBI]) grants to S.A.D. and S.D. (National Institute for Child Health and
Wolf Quadracci Memorial Fund, and the Dr. James Guhl Memorial Fund.
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this
dard conditions5that are described in supporting infor-
Histological and Functional Assays. In most cases,
assays relied on well-established procedures, and details
are provided as supplemental material online. Antibodies
used are provided in Supporting Table S1.
Oligonucleotide Array Analyses. Each array analysis
was performed on three samples that were generated
through independent differentiation experiments. Spe-
cific experimental details are provided as supporting ma-
terial online. All original gene array files are available
through the Gene Expression Omibus (GEO) database
(http://www.ncbi.nlm.nih.gov/geo/) accession number
Fetal Mouse Livers Generated from iPS Cells Are
Indistinguishable from Wild-Type Livers. We first de-
termined whether iPS cells were competent to follow a he-
patic developmental program that produced all liver cell
lineages by examining embryos derived solely from mouse
iPS cells by tetraploid complementation. Mouse iPS cells
were generated from C57BL/6J-Tg(pPGKneobpA)3Ems/J
then produced from these iPS cells by tetraploid comple-
mentation using transgenic mice (Tg[CAG-EGFP]-
B5Nagy/J) that ubiquitously express enhanced green
fluorescent protein (EGFP) as donors of tetraploid em-
mouse iPS cells. (A) Brightfield (top)
and fluorescent (bottom) images of
CAG-eGFP?/?(eGFP), eGFP negative
13) E12.5 embryos. (B) Brightfield
and fluorescent images of CAG-
(CD1;right), and iPS cell–derived
E12.5 fetal livers (eGFP:iPS 13). (C)
Whole-mount images of E14.5 em-
bryos (top) and their livers (middle)
derived from wild-type (CD-1) and iPS
cells (iPS 13). Bottom panels show
through control (CD-1) and iPS cell–
derived (iPS 13) E14.5 livers. (D) Im-
munohistochemistry revealing marker
expression in hepatocytes (HNF4a),
endothelial cells (GATA4), sinusoidal
cells (Lyve-1), and macrophages/
and iPS cell–derived (iPS 13) E14.5
livers. Scale bars ? 100 ?m. (E)
chain reaction analyses of two control
(CD-1) and iPS-derived (iPS 13)
E14.5 livers showing steady levels of
characteristic liver mRNAs as well as
mRNA, which is only expressed in iPS
cell–derived livers. RNA polymerase II
mRNA (Pol2ra) was used as a loading
control, and reactions without reverse
transcriptase (?RT) or template (0)
confirmed the absence of contaminat-
Fetal livers derived from
298SI-TAYEB, NOTO, ET AL.HEPATOLOGY, January 2010
bryos. Fig. 1A shows that control CAG-EGFP embryos
ubiquitously express EGFP, whereas EGFP was not de-
tected in wild-type CD1 embryos. When embryos were
generated from mouse iPS cells, from which EGFP is
absent, all embryos (n ? 5), including their livers (Fig.
1B), were devoid of EGFP expression except in extra em-
bryonic tissues that were derived from the donor tet-
Gross examination of E14.5 iPS cell–derived embryos
and their livers (n ? 3) revealed that they appeared to be
identical to controls (Fig. 1C). We therefore determined
whether these livers contained the expected repertoire of
hepatic cells by identifying the expression of proteins that
like control CD1 fetal livers, iPS cell–derived livers con-
tained hepatocytes (hepatocyte nuclear factor [HNF]4a
positive), endothelial cells (GATA binding protein 4
[GATA4] positive), sinusoidal cells (lymphatic vessel en-
dothelial hyaluronan receptor 1 positive), and Kupffer
cells/macrophage (F4/80 positive). We also measured the
extent of hepatocyte differentiation using reverse tran-
scription polymerase chain reaction to detect messenger
RNAs (mRNAs) that are key markers of the hepatocyte
cell lineage. Fig. 1E shows that every hepatocyte marker
mRNA examined—alpha fetoprotein, albumin, aldolase b,
apolipoproteins A1, A2, and C2, liver fatty acid binding
protein (Fabp1), retinol binding protein (Rbp4), and tran-
sthyretin—was expressed at a level comparable to control
fetal livers. Moreover, expression of several mRNAs en-
FoxA1, FoxA2, Pxr (Nr1i2), and Hnf4a—was commen-
surate with control livers. From these cumulative results,
we conclude that mouse iPS cells are fully competent to
generate fetal livers in vivo.
Establishing a Protocol for the Efficient Production
of Hepatocyte-like Cells from Human Pluripotent
Cells. The generation of clinically and scientifically use-
ful hepatocytes from iPS cells requires the availability of
completely defined culture conditions that support effi-
cient and reproducible differentiation of iPS cells into the
hepatocyte lineage. Existing published procedures that
have been applied to the differentiation of both human
and mouse ES cells generally include steps in which
poorly defined components are introduced into the cul-
if such cells are to be used therapeutically. We therefore
sought to optimize the differentiation procedure and
eliminate the use of serum, fibroblast feeder cells, embry-
oid bodies, and undefined culture medium components,
initially using human embryonic stem cells (huES) cells.
We based our protocol on an understanding of the mech-
anisms underlying mouse embryogenesis, the availability
of protocols published by others,12–14and the use of em-
pirically determined procedures that resulted in an in-
crease in the number of cells expressing a combination of
markers of definitive endoderm (forkhead box A2
[FOXA2], sex determining region Y box 17 [SOX17],
and GATA binding protein 4 [GATA4]), specified he-
patic cells (FOXA2 and HNF4a), hepatoblasts (FOXA2,
hepatocytes (FOXA2, HNF4a, and albumin [ALB]).
Fig. 2A illustrates the procedure that we have used.
Undifferentiated stem cells were maintained in mono-
layer culture on Matrigel in embryonic stem (ES) cell
culture media conditioned by mitotically inactivated pri-
mary mouse embryonic fibroblasts in 4% O2/5%CO2.
pluripotency markers, including Oct4 (Fig. 2B) and
stage-specific embryonic antigen 4 (not shown). To initi-
ate differentiation, monolayers of huES cells were cul-
tured in Roswell Park Memorial Institute (RPMI) media
containing B27 supplements and 100 ng/mL activin A,
of definitive endoderm.15,16After 5 days of culture in 5%
CO2with ambient oxygen, more than 90% of cells had
2B) and stage-specific embryonic antigen 4 (not shown).
Immunocytochemistry using antibodies to detect pro-
teins expressed in the definitive endoderm showed that
more than 80% of cells expressed FOXA2, GATA4, and
SOX17. Importantly, these cells did not express HNF4a,
which is highly expressed in extra embryonic endodermal
cells, thereby excluding the possibility that the endoderm
generated by activin A treatment was visceral (yolk sac)
endoderm. Culture dishes containing induced definitive
B27 media supplemented with 20 ng/mL bone morpho-
genetic protein 4 (BMP4) and 10 ng/mL fibroblast
growth factor 2 (FGF2) for 5 days. Both BMP4 and
FGF2 have been shown to have crucial roles during he-
patic specification in mouse embryos.17,18Fig. 2B shows
that culture in BMP4/FGF2-supplemented media re-
FOXA2 expression was maintained, and HNF4a expres-
sion was initiated. This pattern of expression closely re-
sembles that found during development of the mouse
liver. In particular, GATA4 expression is specifically
down-regulated in cells that are destined to follow a he-
patic fate but remains expressed in the gut endoderm,19,20
whereas HNF4a expression is restricted to the nascent
hepatic cells formed during hepatic specification stages of
development (10 somites).20,21The specification of he-
more than 80% of cells expressing HNF4a. Based on
HEPATOLOGY, Vol. 51, No. 1, 2010SI-TAYEB, NOTO, ET AL. 299
Hepatocyte differentiation from huES cells was monitored by immunocytochemistry at days 0, 5, 10, 15, and 20 using antibodies that recognized
OCT3/4, FOXA2, SOX17, GATA4, HNF4a, alphafetoprotein (AFP), and albumin. Results are representative of three independent differentiation
experiments. (C) Albumin secretion by huES cell–derived hepatocytes was identified after 3 days of culture in medium by enzyme-linked
immunosorbent assay. (D) Representative flow cytometry profile showing the average number of albumin-positive hepatocytes in three independent
differentiation experiments ? 80.9% ? 0.75. (E) HES cell–derived hepatocytes were shown to store glycogen by periodic acid-Schiff staining (a);
store lipids by Oil Red O staining (b); to display hepatocyte morphology including binucleated cells (black arrow) (c); efficiently uptake low-density
lipoprotein using fluoresceinated low-density lipoprotein (d); metabolize indocyanine green (e); and localize dichlorofluorescein diacetate to their
plasma membranes (white arrow) (f). (F) Heat map of gene array analyses demonstrating that a series of 40 mRNAs that are solely expressed in
livers23were increased (red ? high, blue ? low) after differentiation (Hep) compared with undifferentiated (ES) cells.
Generation of hepatocytes from human ES cells. (A) Flow diagram outlining the procedure used to control hepatocyte differentiation. (B)
300SI-TAYEB, NOTO, ET AL. HEPATOLOGY, January 2010
findings by others,12,13we cultured the specified hepatic
cells in RPMI-B27 supplemented with 20 ng/mL hepa-
growth factor inclusion in the culture conditions resulted
in high levels of expression of alpha-fetoprotein, which
indicates that the specified cells have committed to a
hepatoblast fate (Fig. 2B). Co-staining with FoxA2 (not
shown) showed that more than 98% of FoxA2 expressing
cells co–expressed alpha-fetoprotein, implying that the
differentiation of endoderm into the hepatic lineage was
For the final stage of differentiation, cultures were
transferred to 5% CO2/ambient O2, and the media was
replaced with hepatocyte culture medium supplemented
Under these conditions, the cells were found to express
high levels of albumin that could be identified by immu-
enzyme-linked immunosorbent assay assay (Fig. 2C). On
cytometry analyses (Fig. 2D). At the completion of the
differentiation protocol, the cells were also found to dis-
cells, oil red O staining identified the presence of lipid
droplets, and incubation of the cells with fluoresceinated
low-density lipoprotein demonstrated the ability of the
nine green, which was metabolized overnight (Fig. 2E),
and analyses of the culture media revealed the ability of
cells to undertake urea metabolism (Supporting Fig. S2).
The morphology of the differentiated cells also shared
many characteristics with primary hepatocytes, including
a large cytoplasmic-to-nuclear ratio, numerous vacuoles
and vesicles, and prominent nucleoli. Several cells were
Fig. 3); moreover, the differentiated cells formed sheets
reminiscent of an epithelial layer and were capable of ac-
tively localizing dichlorofluorescein diacetate to their
plasma membranes (Fig. 2E panel f, arrow). We further
examined the extent of differentiation using gene array
analyses, which were performed on undifferentiated H9
ES cells and cells subjected to the complete 20-day differ-
nome-wide expression profiling studies by others23have
identified a cluster of 175 genes whose expression is re-
stricted to normal human liver compared with 35 other
tissues examined. A subset of 40 of these genes have suc-
cessfully been used to identify hepatic character in other
studies,23and so we believe that expression of these 40
genes provides an accurate transcriptional fingerprint of a
of genes is not expressed in undifferentiated huES cells
(Fig. 2F and Supporting Table S2); however, expression
of nearly the entire gene set is robustly increased after
completion of the differentiation protocol. Based on our
analyses shown in Fig. 2, we conclude that the we have in
erate hepatocyte-like cells from huES cells under well-
defined culture conditions.
Production of Hepatocyte-like Cells from Human
Induced Pluripotent Stem Cells. If hepatocytes could
be generated from human induced pluripotent stem cells
(hiPS) cells with efficiencies that resembled those
achieved using huES cells, the procedure would provide a
disease as well as potentially provide human hepatocytes
for toxicological studies and pharmaceutical screens.
However, the effect of somatic cell nuclear reprogram-
ming on hepatocyte differentiation from iPS cells is un-
known. We therefore generated human iPS cells (hiPS)
tion factor 1 (OCT3/4) SRY-box containing gene 2,
(SOX2), NANOG homeobox (NANOG), and Lin-28 ho-
molog (LN28) as described by Yu et al.5A detailed charac-
terization of these iPS cells is shown in Supporting Fig. S4.
We next determined the ability of iPS.C2a cells to
form hepatocyte-like cells. Human iPS cells were sub-
jected to the same protocol used to induce formation of
hepatocytes from huES cells, and the same analyses were
performed. As was the case for huES cells, iPS cells re-
sponded to the inductive procedures by expressing all
markers of definitive endoderm in response to activin A,
hepatic specification in response to BMP4/FGF2, hepa-
toblast formation in response to hepatocyte growth fac-
tor, and hepatocyte-like differentiation in response to
oncostatin M (Fig. 3A). Quantification of albumin-posi-
tive cells revealed that the kinetics and efficiency of he-
patic differentiation was similar to that found for
differentiation of huES cells (Fig. 2A). Flow cytometry
revealed that at the completion of the differentiation pro-
tocol, more than 80% of cells expressed albumin (Fig.
3B), and the levels of human albumin in the media ap-
proached 1.5 ?g/mL after 3 days of culture (Fig. 3C). As
was the case with human ES cell–derived hepatocyte-like
cells, iPS cell–derived hepatocyte-like cells displayed sev-
eral hepatic functions, including accumulation of glyco-
gen, metabolism of indocyanine green, accumulation of
lipid, active uptake of low-density lipoprotein (Fig. 3D),
morphological characteristics associated with hepatocytes
HEPATOLOGY, Vol. 51, No. 1, 2010SI-TAYEB, NOTO, ET AL.301
SOX17, GATA4, HNF4a, alphafetoprotein (AFP), and albumin (ALB) by immunocytochemistry at 0, 5, 10, 15, and 20 days. Results are representative of three
independent differentiation experiments. (B) Representative flow cytometry profile showing the average number of albumin-positive hiPS-derived hepatocytes
in three independent differentiation experiments ? 81.0% ? 4.8. (C) Albumin secretion by hiPS cell–derived hepatocytes was identified in the medium after
3 days of culture using enzyme-linked immunosorbent assay. (D) Hepatocyte-like cells derived from hiPS cells were shown to store glycogen by periodic
acid-Schiff staining (a); store lipids by Oil Red O staining (b); uptake low-density lipoprotein using fluoresceinated low-density lipoprotein (c); and metabolize
indocyanine green (d); have similar morphology to hepatocytes with some cells being binucleated (black arrow) (e); and direct dichlorofluorescein diacetate
to plasma membranes (white arrow) (f). (E) Heat map of gene array analyses showing that expression of 40 liver-specific mRNAs23was increased (red) after
differentiation (Hep) compared with undifferentiated (hiPS) cells in which most of these mRNAs were expressed at relatively low levels (blue). (F) Bar graphs
showing the levels of mRNAs, determined by real-time quantitative reverse transcription polymerase chain reaction, encoding phase I and II enzymes in
hepatocyte-like cells derived from H9 huES cells (yellow) and C2 hiPS cells (orange) expressed as fold of levels found in cadaveric human liver samples.
Differentiation of hepatocytes from human iPS cells. (A) Hepatocyte differentiation from hiPS cells was followed by detecting OCT3/4, FOXA2,
302 SI-TAYEB, NOTO, ET AL. HEPATOLOGY, January 2010
(Fig. 3D and Supporting Fig. S3). In addition, oligonu-
cleotide array analyses revealed that iPS cell–derived hep-
atocyte-like cells expressed the same hepatocyte mRNA
fingerprint that was found for human ES cell–derived
hepatocyte-like cells (Fig. 3E and Supporting Table S2).
We also compared the expression of a series of genes en-
coding phase I and phase II enzymes, whose expression is
cadaveric liver samples and hepatocyte-like cells derived
from either huES cells or hiPS cells. In both cases, the
levels of such mRNAs showed similar trends in expres-
sion. Of note, however, the levels of expression of these
enzymes were lower in most cases when compared with
adult liver samples (Fig. 3F), suggesting that although
have differentiated to a state that supports many hepatic
ing phase I and phase 2 enzymes, they do not entirely
recapitulate mature liver function.
Finally, we sought to determine whether the differenti-
ated hepatic-like cells generated from huES cells and hiPS
cells had the capacity to contribute to the liver parenchyma
in vivo (Fig. 4). To test this, cells were collected at the com-
pletion of the 20-day differentiation protocol, and approxi-
mately 3 ? 105cells were injected into the right lateral liver
lobe of newborn mice. Livers were harvested 7 days after
that specifically recognizes human but not mouse albumin
(Fig. 4A). In contrast to control mice, in which no human
either huES cell–derived or hiPS cell–derived hepatocyte-
that strongly expressed human albumin (Fig. 4A). Unin-
jected lobes had no human albumin-positive cells. At high
resolution, the human albumin-positive cells in injected
lobes could be seen to be integrated into the existing mouse
parenchyma. Because albumin is a secreted protein, it could
false-positive result. We therefore confirmed that the cells
detected as albumin positive were indeed of human origin
using polymerase chain reaction of genomic DNA isolated
microdissection (Fig. 4B). From these results, we conclude
that hiPS cells derived from human foreskin fibroblasts can
hepatic parenchyma in vivo.
anism for the treatment of both chronic and acute liver
failure. However, the need for orthotopic liver transplan-
tation far outweighs the availability of donor livers.10For
a subset of liver diseases, particularly those resulting from
enzymatic disorders, hepatocyte transplantation could be a
of cells expressing human albumin (brown) in human livers (upper right) and in mouse livers injected with huES cell–derived (lower left) and hiPS
cell–derived (lower right) hepatocytes but not in uninjected control mouse livers (top right). (B) PCR analyses using primers that specifically recognize
human Alu or mouse HPRT sequences on genomic DNA isolated from control mouse and human fibroblasts as well as cells collected by laser capture
from sections through mouse liver, human tonsil, and albumin-positive cells from mouse livers injected with huES cell–derived or hiPS cell–derived
hepatocyes. Amplifications performed without template ensured the absence of contaminating DNA.
Integration of huES and hiPS cell–derived hepatocytes into the mouse hepatic parenchyma. (A) Micrographs showing the identification
HEPATOLOGY, Vol. 51, No. 1, 2010 SI-TAYEB, NOTO, ET AL.303
of animal models have supported the safety and, in some
cases, efficacy of using hepatocyte transplantation therapeu-
tically.8Although primary human hepatocytes can be puri-
fied from donor livers, approximately 1 to 5 ? 109cells are
required per transplantation, which makes necessary access
to large numbers of donor livers or the need to expand pri-
tissue culture environment.24
The need to expand primary hepatocytes purified from
hepatocytes. Unlike many other stem cells, ES cells and iPS
appealofusingiPScellsisthattheycouldprovide a source
of autologous hepatocytes. Several studies have de-
scribed the differentiation of human embryonic stem
cells into cells that display hepatic characteristics7,12–
14,25–31; however, this is the first report demonstrating
that iPS cells can also be used to efficiently generate
hepatocyte-like cells. Using the described procedure,
the generation of hepatocyte-like cells from hiPS cells
appears to be as efficient as observed from huES cells,
although it was noted that subtle differences in the
timing of onset and level of expression of different
hepatic genes were found (Fig. 3). It is not clear at this
point whether such differences in gene expression sim-
ply reflect heterogeneity between different iPS lines, as
is seen for huES cells, or whether they are characteristic
of all hiPS cells in general. Work is underway to address
this. In addition, one hiPS cell line we had generated
(iPS C3a), although possessing most of the hallmarks
of pluripotency, immediately differentiated into a fi-
broblast-like morphology when plated on Matrigel and
hepatic lineages. Similarly, it has been noted by others
that some hiPS cell lines appear to be incompletely
reprogrammed, and still others maintain expression of
exogenous transgenes, which appear to interfere with
differentiation protocols.32With this in mind, we be-
lieve it is crucial that standards for the generation and
characterization of hiPS cells are adopted throughout
the community to ensure reproducibility of formation
of differentiated cells from hiPS cells from different
patients and tissue sources.
Although several groups have been able to produce
hepatocyte-like cells from huES cells, we believe that the
current protocol used to produce hepatocytes from either
huES or hiPS cells offers a number of advances. Differen-
tiation is extremely efficient and reproducible, with be-
tween 80% and 85% of cells expressing hepatic markers,
including albumin. In most other procedures, the differ-
entiation of cells relies on embryoid body formation, in-
cludes interactions with primary feeder cells, or requires
the inclusion of serum during the differentiation proce-
dure. Although using such approaches to produce hepa-
tocytes can be successful, the inherent variability
ibility. The approach we have described relies on well-
defined culture conditions. We believe that using such
conditions will facilitate accurate analyses of molecular
pathways that control human hepatocyte differentiation,
comparative studies between iPS cells derived from pa-
tients suffering from various congenital liver diseases, and
development of screens for novel pharmaceutical ap-
proaches to correct liver disease.
Although the efficiency of generating cells that exhibit
most hepatocyte characteristics is high, we noted that the
repertoire of mRNAs encoding phase I and phase II en-
zymes, which have important roles in controlling drug me-
tabolism and xenobiotic responses, is incomplete when
compared with cadaveric livers. Loss of CYP450 enzyme
expression is common when hepatocytes are grown under
trol of CYP450 expression and activity by several environ-
mental and physiological parameters that are lacking in the
to differentiate toward the hepatocyte lineage; however, we
toxicological and drug metabolic studies will require the es-
tablishment of culture conditions that more fully support
expression of a full panel of phase I and II enzymes. In this
regard, recent experiments using microengineering ap-
proaches have established conditions that allow extended
culture of primary hepatocytes that maintain phase I and II
enzymatic activities, and we have initiated studies to deter-
hiPS-derived hepatocyte-like cells.35
In summary, we have shown that mouse iPS cells can
be induced to efficiently generate intact fetal livers and
differentiated hepatocytes. We acknowledge that com-
pared with the in vivo environment of the liver, the con-
ditions in culture are relatively artificial, and this is likely
to impact the function of iPS-derived hepatocytes com-
pared with the native environment. Nevertheless, the
data provided above demonstrate the feasibility of gener-
ating cells with hepatic characteristics from skin cells
through an iPS cell intermediate and that such cells can
engraft into the mammalian liver parenchyma. Such
proof-of-concept opens up the possibility of producing
304SI-TAYEB, NOTO, ET AL. HEPATOLOGY, January 2010
patient-specific hepatocytes in a relatively simple and
fident that such cells could be immediately useful for the
study of hepatocellular disease and basic developmental
mechanisms and for drug screening.
for providing frozen liver samples.
The authors thank Charles Myers
Generation of human induced pluripotent stem cells from dermal fibro-
blasts. Proc Natl Acad Sci U S A 2008;105:2883-2888.
2. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, http://www.
with defined factors. Nature 2008;451:141-146.
3. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K,
RVAbstractPluset al. Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell 2007;131:861-872.
embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:
5. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL,
Tian S, et al. Induced pluripotent stem cell lines derived from human
somatic cells. Science 2007;318:1917-1920.
6. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, et al.
Disease-specific induced pluripotent stem cells. Cell 2008;134:877-886.
7. Burlina AB. Hepatocyte transplantation for inborn errors of metabolism.
J Inherit Metab Dis 2004;27:373-383.
8. Fisher RA, Strom SC. Human hepatocyte transplantation: worldwide re-
sults. Transplantation 2006;82:441-449.
9. Grompe M. Principles of therapeutic liver repopulation. J Inherit Metab
10. Gupta S, Chowdhury JR. Therapeutic potential of hepatocyte transplan-
tation. Semin Cell Dev Biol 2002;13:439-446.
11. Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation
of completely cell culture-derived mice from early passage embryonic stem
cells. Proc Natl Acad Sci U S A 1993;90:8424-8428.
12. Agarwal S, Holton KL, Lanza R. Efficient differentiation of functional
hepatocytes from human embryonic stem cells. Stem Cells 2008;26:1117-
ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus et al.
Directed differentiation of human embryonic stem cells into functional
hepatic cells. HEPATOLOGY 2007;45:1229-1239.
14. Shiraki N, Umeda K, Sakashita N, Takeya M, Kume K, Kume S. Differ-
entiation of mouse and human embryonic stem cells into hepatic lineages.
Genes Cells 2008;13:731-746.
15. D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE.
Efficient differentiation of human embryonic stem cells to definitive
endoderm. Nat Biotechnol 2005;23:1534-1541.
16. McLean AB, D’Amour KA, Jones KL, Krishnamoorthy M, Kulik MJ,
Reynolds DM, et al. Activin a efficiently specifies definitive endoderm
from human embryonic stem cells only when phosphatidylinositol 3-
kinase signaling is suppressed. Stem Cells 2007;25:29-38.
17. Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of mammalian liver
development from endoderm by fibroblast growth factors. Science 1999;
18. Rossi JM, Dunn NR, Hogan BL, Zaret KS. Distinct mesodermal signals,
including BMPs from the septum transversum mesenchyme, are required
in combination for hepatogenesis from the endoderm. Genes Dev 2001;
19. Watt AJ, Zhao R, Li J, Duncan SA. Development of the mammalian liver
and ventral pancreas is dependent on GATA4. BMC Dev Biol 2007;7:37.
20. Zhao R, Watt AJ, Li J, Luebke-Wheeler J, Morrisey EE, Duncan SA.
for early heart formation. Mol Cell Biol 2005;25:2622-2631.
21. Duncan SA, Manova K, Chen WS, Hoodless P, Weinstein DC, Bach-
varova RF, http://www.ncbi.nlm.nih.gov/sites/entrez?Db?pubmed&
DiscoveryPanel.Pubmed_RVAbstractPlusetal. Expression of transcription
the developing mouse embryo: HNF-4 is a marker for primary endoderm in
the implanting blastocyst. Proc Natl Acad Sci U S A 1994;91:7598-7602.
liver development requires a paracrine action of oncostatin M through the
gp130 signal transducer. EMBO J 1999;18:2127-2136.
23. Ge X, Yamamoto S, Tsutsumi S, Midorikawa Y, Ihara S, Wang SM, et al.
Interpreting expression profiles of cancers by genome-wide survey of
breadth of expression in normal tissues. Genomics 2005;86:127-141.
24. Strain AJ. Isolated hepatocytes: use in experimental and clinical hepatol-
ogy. Gut 1994;35:433-436.
25. Baharvand H, Hashemi SM, Kazemi Ashtiani S, Farrokhi A. Differentia-
systems in vitro. Int J Dev Biol 2006;50:645-652.
26. Baharvand H, Hashemi SM, Shahsavani M. Differentiation of human
embryonic stem cells into functional hepatocyte-like cells in a serum-free
adherent culture condition. Differentiation 2008;76:465-477.
27. Chiao E, Elazar M, Xing Y, Xiong A, Kmet M, Millan MT, et al. Isolation
and transcriptional profiling of purified hepatic cells derived from human
embryonic stem cells. Stem Cells 2008;26:2032-2041.
28. Cho CH, Parashurama N, Park EY, et al. Homogeneous differentiation of
hepatocyte-like cells from embryonic stem cells: applications for the treat-
ment of liver failure. FASEB J 2008;22:898-909.
29. Hay DC, Fletcher J, Payne C, et al. Highly efficient differentiation of
hESCs to functional hepatic endoderm requires ActivinA and Wnt3a sig-
naling. Proc Natl Acad Sci U S A 2008;105:12301–12306.
30. Hay DC, Zhao D, Fletcher J, Hewitt ZA, McLean D, Urruticoechea-
Uriguen A, et al. Efficient differentiation of hepatocytes from human em-
bryonic stem cells exhibiting markers recapitulating liver development in
vivo. Stem Cells 2008;26:894-902.
31. Muraca M, Gerunda G, Neri D, Vilei MT, Granato A, Feltracco P, et al.
1a. Lancet 2002;359:317-318.
32. Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, et al.
Dissecting direct reprogramming through integrative genomic analysis.
33. Waring JF, Ciurlionis R, Jolly RA, Heindel M, Gagne G, Fagerland JA, et
al. Isolated human hepatocytes in culture display markedly different gene
expression patterns depending on attachment status. Toxicol In Vitro
34. Madan A, Graham RA, Carroll KM, Mudra DR, Burton LA, Krueger LA,
RVAbstractPluset al. Effects of prototypical microsomal enzyme inducers on
cytochrome P450 expression in cultured human hepatocytes. Drug Metab
35. Khetani SR, Bhatia SN. Microscale culture of human liver cells for drug
development. Nat Biotechnol 2008;26:120-126.
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