Derivation and Characterization of Hepatic Progenitor Cells from Human Embryonic Stem Cells

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DOI: 10.1371/journal.pone.0006468 · Source: PubMed
Abstract
The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.
Derivation and Characterization of Hepatic Progenitor
Cells from Human Embryonic Stem Cells
Dongxin Zhao
1
, Song Chen
1
, Jun Cai
1
, Yushan Guo
1,2
, Zhihua Song
1,2
, Jie Che
1
, Chun Liu
1,2
,ChenWu
1,2
,
Mingxiao Ding
1
, Hongkui Deng
1,2
*
1 Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, China, 2 Laboratory of Chemical
Genomics, Shenzhen Graduate School of Peking University, The University Town, Shenzhen, China
Abstract
The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early
human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy.
Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells
were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a
newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells
yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of
more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker
KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which
expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and
KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential
hepatic progenitor cells from hES cells. These hES cellderived hepatic progenitor cells could be effectively used as an in
vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.
Citation: Zhao D, Chen S, Cai J, Guo Y, Song Z, et al. (2009) Derivation and Characterization of Hepatic Progenitor Cells from Human Embryonic Stem Cells. PLoS
ONE 4(7): e6468. doi:10.1371/journal.po ne.0006468
Editor: Catherine M. Verfaillie, KU Leuven, Belgium
Received April 5, 2009; Accepted July 3, 2009; Published July 31, 2009
Copyright: ß 2009 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by a Bill & Melinda Gates Foundation Grant (37871), a Ministry of Education grant (705001), National Basic Research Program
of China (973 Program 2009CB941200 and 2007CB947900), National Natural Science Foundation of China for Creative Research Groups (30421004), the Chinese
Science and Technology Key Project (2008zx10002-014 and 2008zx10002-011), and China Ministry of Education (111 project) to H Deng. The funders had norole
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hongkui_deng@pku.edu.cn
Introduction
Human embryonic stem (hES) cells have the ability to grow infinitely
while still maintaining the pluripotency required for differentiation into
almost any cell type [1]. Thus, hES cells constitute a potential cell
source for a variety of applications, such as studies of the fundamental
mechanisms of lineage commitment and cell-based therapy in a broad
spectrum of diseases. Among the different lineages that can be
generated from hES cells, hepatic cells are of particular interest because
the liver plays a major role in metabolism and has multiple functions,
including glycogen storage, decomposition of red blood cells, plasma
protein synthesis, and detoxification. A number of studies have
demonstrated the feasibility of differentiating human or mouse ES cells
into the hepatic lineage [2–6]. We have established a protocol for
efficient production of hepatocytes by mimicking natural embryonic
liver development in vivo [7]. During the differentiation process, we and
other groups have observed that hepatocytes and cholangiocytes are
generated concomitantly [3,7], which suggests a common ancestor;
that is, hepatic progenitor cells may exist. The existence of comparable
hepatic progenitor cells in the ES differentiation process, however, has
not been demonstrated. The properties and proliferation potential of
these cells have not yet been characterized, and the mechanism of
primary lineage transition has not been elucidated.
Hepatic progenitor cells serve as the major component of the
hepatic parenchyma in early stages of liver organogenesis [8]. Studies
of mouse and human embryonic development indicate that they are
common progenitors of mature hepatocytes and biliary epithelial
cells, the lineage commitments of which are determined around the
mid-gestation stage [9]. Much research has been carried out on the
development of in vitro culture systems for hepatic progenitor cells
isolated from both human and mouse fetal livers [10–15]. Human
hepatic progenitor cells exhibited phenotypic stability after extensive
expansion [13] and, when placed in appropriate conditions, could
differentiate into hepatocytes, which expressed ALB and stored
glycogen, and into bile duct cells, which expressed KRT19 [12,13].
Although the proliferation and bipotential capacity of hepatic
progenitor cells have been demonstrated, the origin and function of
hepatic progenitor cell populations are areas of ongoing debate [9].
The difficulty may be partly due to theshortageofmaterialfromearly
human embryos and undefined stages of development, given that
hepatic progenitor cells have been directly separated only from
human liver organs to date. Therefore, in vitro generation of hepatic
progenitor cells based on a hES cell differentiation system offers a
novel platform for further research on hepatic progenitor cells.
In this study, we first identified N-cadherin as a surface marker of
hepatic endoderm cells for purification from hES cellderivates, and
generated hepatic progenitor cells from purified hepatic endoderm cells
by co-culture with murine embryonic stromal feeders (STO) cells.
These hepatic progenitor cells could expand and be passaged for more
than 100 days. Interestingly, they co-expressed the early hepatic
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marker AFP and biliary lineage marker KRT7, suggesting that they
are a common ancestor of both hepatocytes and cholangiocytes.
Moreover, these progenitor cells could be expanded extensively while
still maintaining the bipotential of differentiation into hepatocyte-like
cells and cholangiocyte-like cells, as verified by both gene expression
and functional assays. Therefore, this work offers a new in vitro model
for studying liver development, as well as a new source for cell therapy
based on hepatic progenitors.
Results
Identification of N-cadherin as a novel surface marker of
hES cell–derived hepatic en doderm cells
We previously established a stepwise protocol to differentiate
hES cells into hepatocytes by mimicking embryonic development
[7]. We produced hepatic endoderm cells by using this protocol.
hES cells were first exposed to Activin A for three days to induce
definitive endoderm formation, and then were treated with BMP2
and FGF4 for another five days to induce hepatic endoderm cells.
During this process, reverse-transcription (RT)-PCR was per-
formed to assess the temporal gene expression of the hepatic
marker genes AFP, ALB, HNF4A and CEBPA. All of these genes
demonstrated similar expression patterns starting at around day 5
from the beginning of induction and reaching a maximal level at
day 8 (Figure 1A), indicating the generation of hepatic endoderm.
To better analyze the properties of the hepatic endoderm cell
population and eliminate possible interference from other cell lineages,
we searched for a surface marker to purify hepatic endoderm cells from
hES cell derivatives. We systematically tested a panel of putative
hepatic progenitor cell surface markers, including CD29, CD34,
CD49f, CD133, c-kit, c-met, Thy-1, N-cadherin, E-cadherin, EpCAM
and NCAM (Table S1). Immunofluorescence using antibodies specific
Figure 1. N-cadherin expression marks hepatic endoderm cells. (A) Temporal gene expression analysis of hepatic endoderm cells
differentiated from human ES cells (quantitative RT-PCR). Expression level of the differentiated cells was calculated relative to undifferentiated hES
cells. (B) Immunofluorescence showing that N-cadherin is co-expressed with the hepatic endoderm markers AFP, ALB, HNF4A, GATA4, and FOXA2 at
day 8. The upper left image was taken using a fluorescence microscope, while the others were taken with a laser scanning confocal microscope. Scale
bar = 50 mm. Cell nuclei are stained with DAPI (blue). (C) Flow cytometric isolation of N-cadherin-expressing hES cellderived hepatic endoderm cells
digested with trypsin and EDTA or with trypsin and Ca
2+
at day 8. (D) Quantitative RT-PCR results showing elevated expression of hepatic marker
genes in post-sorted N-cadherin
+
cells. Y-axis, relative expression to GAPDH, then normalized to the N-cadherin
2
cells.
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for N-cadherin revealed that nearly all cell labeling was restricted to
AFP-expressing cells in the mixture of hES cell derivatives; moreover,
no AFP-expressing cells lacked N-cadherin labeling. This result was
confirmed by repeat tests using both fluorescence microscopy and laser
scanning confocal microscopy (Figure 1B). Intracellular flow cytometry
staining produced similar results, with co-expression of N-cadherin and
AFP in a single cell (Figure S1). Further immunofluorescence analysis
with confocal microscopy revealed concomitant expression of N-
cadherin and the hepatic endoderm markers ALB, HNF4A, FOXA2
and GATA4 (Figure 1B).
To purify hepatic endoderm cells from hES cell derivatives, we set
out to isolate the N-cadherin
+
cell population and collected N-
cadherin
2
cells for comparison. N-cadherin is a calcium-dependent
cell-cell adhesion glycoprotein that is highly sensitive to trypsin
treatment but can be efficiently protected from protease digestion by
Ca
2+
[16]. When hepatic endoderm cells were digested with 0.25%
trypsin and 0.53 mM EDTA, most of the N-cadherin in the
extracellular domain was cleaved and the cells were no longer
recognized by the monoclonal antibody GC4 (Figure 1C, middle)[17].
In contrast, when hepatic endoderm cells were treated with 0.25%
trypsin and 2 mM Ca
2+
instead of EDTA to keep N-cadherin intact,
a substantial portion of the population displayed positive staining for
N-cadherin (60.9%69.1%) by day 8 of differentiation (Figure 1C,
right). Immunofluorescence of post-sorted cells revealed that the N-
cadherin
+
fraction consisted of .90% of the AFP-expressing cells,
whereas few N-cadherin
2
cells were AFP-positive (Figure S2).
Further analysis by quantitative RT-PCR showed that the isolated
N-cadherin
+
cells were enriched for expression of the hepatic-specific
genes AFP, ALB, HNF4A and FOXA2 (Figure 1D). Additionally, this
N-cadherin
+
cell population could further mature into ALB- and
AAT-expressing hepatocyte-like cells and KRT7-expressing cholan-
giocyte-like cells (Figure 2A) using a previously established protocol
[7], while the N-cadherin
2
cell population could not. Therefore, N-
cadherin could serve as a surface marker of hepatic endoderm cells
for purification from mixed hES cell derivatives.
Generation and expansion of hepatic progenitor cells
from N-cadherin
+
hES cell–derived hepatic endoderm
cells
In embryonic liver development, once hepatic specification is
initiated and the liver bud is generated, hepatic progenitor cells
Figure 2. Characterization of sorted N-cadherin
+
cells. (A) N-cadherin
+
hepatic endoderm cells can further differentiate into hepatocyte-like
cells and cholangiocyte-like cells. Immunostaining for ALB (left), AAT (middle), and KRT7 (right) in day 18 cultures generated from day 8 sorted cells.
(B–C) Day 8 N-cadherin
+
hepatic endoderm cells showed little proliferation potential, as demonstrated by Ki67 expression (B) and BrdU incorporation
(C). Note that most AFP
+
cells were negative for BrdU. Scale bar = 50 mm.
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greatly expand to generate the final volume of the liver [8]. When
hES cellderived hepatic endoderm cells were examined closely,
they exhibited little proliferation potential. Staining for Ki67 and
AFP at the end of the second stage revealed few AFP
+
hepatic
endoderm cells co-expressing Ki67 (Figure 2B). When BrdU was
added to the differentiated cells over the entire 5 days of the
hepatic endoderm generation stage, less than 5% of the AFP
+
hepatic endoderm cells demonstrated BrdU incorporation
(Figure 2C). Taken together, hES cellderived hepatic endoderm
cells differentiated rapidly without extensive expansion. This
observation suggested that the culture conditions used may not
have favoured the proliferation of hepatic progenitor cells and
prompted us to search for better culture condition.
Many culture conditions for generating hepatic progenitor cells
were tested by adding different growth factors, culturing on
different extracellular matrices, and co-culturing with several
different feeder cells, including mouse fibroblast cells, STO cells,
NIH-3T3 cells, human umbilical vein endothelia cells, and ECV
endothelial cells. We found that when hES cellderived hepatic
endoderm cells were plated on mitomycin-treated murine
embryonic stromal feeders (STO) and cultured in hepatic
progenitor expansion medium —a serum-free medium optimized
for the proliferation of progenitor cells from rat hepatocytes [18]—
parenchymal cell colony appeared (Figure 3A and B). As control,
no colonie could be generated when mitomycin-treated feeders
were cultured alone under the same conditions. The colonies had
compact, sharp boundary morphologies. In contrast to hepatic
endoderm cells, which were nonviable or lost hepatic character
after passage, the colonies continued to expand. Immunofluores-
cence staining using antibodies against human nuclear antigens
showed that these colonies were composed of human cells,
indicating that they were derived from the hES cells and not the
STO cells (Figure S3). We interpret these results to indicate that
the colonies corresponded to hES cell derived hepatic progenitor
cells. The majority of cells in these colonies expressed the
proliferation marker Ki67 (Figure 3C). Moreover, we monitored
colony growth by measuring the change in diameter over time, as
an increment in colony size could be used as an indirect indication
of cell proliferation. By 7 days after passaging on STO feeders, the
hepatic progenitor cells formed typical colonies that were
62.0615.4 mm in diameter; by 20 days, these colonies had
reached 225.4692.0 mm, indicating slow and stable cell growth
(Figure 3D). The cultures were routinely split at a ratio of 1:2 or
1:3 (see Materials and Methods) for more than twelve passages
with a population doubling time of ,5.4 day, and could be frozen
and thawed repeatedly (Figure 3E and data not shown).
To characterize the hES cellderived hepatic progenitor cells,
we assessed the marker gene expression using immunofluores-
cence. These hES cellderived hepatic progenitor cells expressed
the early hepatic lineage marker AFP, but demonstrated faint or
no expression of the mature hepatocyte marker ALB (Figure 4 A
and C). The colonies also expressed the bile duct lineage marker
KRT19 and KRT7 (Figure 4 A and B). Moreover, they were
positive for the putative hepatic progenitor cell markers EpCAM
and CD133 (Figure S4).
To compare the difference in hepatic progenitor generation
potential between cells from the N-cadherin
+
hepatic endoderm
population and the N-cadherin
2
cell population after hepatic fate
determination, we cultured the N-cadherin
2
cell population under
the same conditions and found that the number of colonies yielded
was at least 6-fold lower than that obtained from the N-cadherin
+
population (Figure 3F). In addition, these colonies lost rapidly
during passage, suggesting that they had little proliferative
capacity. This result further supported the use of N-cadherin as
a marker to purify hepatic endoderm for the generation of hepatic
progenitor cells.
hES cell–derived hepatic progenitor cells exhibit the
potential for differentiation into hepatocyte-like cells
When hES cellderived hepatic progenitor cells were maintained
in hepatic progenitor expansion medium, some cells at the periphery
of the colonies erupted. In contrast with the AFP
+
KRT7
+
progenitor
cells, these erupted cells differentiated into AFP
+
KRT7
2
cells,
indicating spontaneous hepatocyte differentiation potential
(Figure 5A). To further confirm this hepatocyte differentiation
potential of hES cellderived hepatic progenitor cells, we used HGF
and OSM to promote maturation into hepatocytes as reported
previously [7]. After 5 days of HGF treatment followed by another 5
days of OSM treatment in hepatocyte culture medium (HCM), we
evaluated the expression of hepatocyte markers in the induced
cultures by immunofluorescence staining. The induced clusters lost
their expression of the cholangiocyte marker KRT7 while maintained
the expression of AFP and began to express ALB (Figure 5B and C),
which was only faintly expressed in hepatic progenitor cells.
Furthermore, most of the ALB-expressing cells exhibited positive
AAT staining (Figure 5D). Approximately 20–30% cells differentiated
from the hES cellderived hepatic progenitor cells expressed ALB
and AAT, which was less efficient than those differentiated directly
from hES cells (approximately 50%) [7].
RT-PCR analyses revealed that the expression of genes charac-
teristic of hepatocytes, including ALB, PEPCK, AAT, TAT,andtwo
members of the cytochrome P450 superfamily, CYP3A7 and CYP2A6,
were increased in the differentiated hepatocyte-like cells comparing
with the hepatic progenitor cells; while the expression of ductal
marker KRT7 was decreased (Figure 5E). The gene expression
profiles of the hepatocyte-like cells differentiated from hepatic
progenitor cells, including AFP, KRT8, KRT18 and the functional
markers mentioned above, were similar with those obtained by our
published direct differentiation protocol from hES cells (Figure 5E).
Moreover, undifferentiated hES cells markers OCT3/4 and NANOG
were expressed neither in the expandable hepatic progenitor cells nor
in the hepatocyte-like cells (Figure 5E), which suggested N-CAD
derived population did not contaminate undifferentiated hES cells.
To test whether the induced cells possessed hepatocyte functions,
we performed a panel of assays on the hepatocyte-like cells
differentiated from hES cellderived hepatic progenitor cells.
Human albumin secretion of the hepatic progenitor cells was
48.865.5 ng/day/million cells by ELISA, but increased dramati-
cally to 439.5663.5 ng/day/million cells after hepatocyte induc-
tion, which was closed to that of the hepatocyte-like cells directly
differentiated from hES cells (Figure 5F). We also assayed for
glycogen storage of the differentiated cells by Periodic acid Schiff
staining on the hepatocyte-like cells. Most of the differentiated cells
within the clusters stained red, demonstrating a glycogen storage
function (Figure 5G). The uptake and release of indocyanine green
(ICG) were used to check the hepatocytes differentiated form
hepatic progenitor cells. The differentiated hepatocyte-like cells
could uptake ICG from medium and exclude the absorbed ICG six
hours later. In contrast, uninduced hepatic progenitor cells did not
take up any ICG (Figure 5H). We also confirmed that the
differentiated hepatocyte-like cells were capable of taking up Dil-
labeled acetylated low-density lipoprotein (Dil-Ac-LDL) (Figure 5I).
Furthermore, to analyze the detoxify ability of the differentiated
hepatocyte-like cells, we evaluated the cytochrome p450 activity by
a PROD assay. When the hepatocyte-like cells cultured in the
absence of phenobarbital sodium induction, only a few cells
exhibited weak PROD activity (Figure 5J); while after incubation
with inducer phenobarbital sodium, the PROD activity was
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increased, indicating that the induced hepatocyte-like cells possessed
inducible P450 activity. In the control experiment, few hepatic
progenitor cells had PROD activity even in the presence of
phenobarbital sodium induction (Figure 5J). Taken together, all
these results indicated that the hES cellderived hepatic cells had
the potential for differentiation into hepatocyte-like cells.
hES cell–derived hepatic progenitor cells exhibit the
potential for differentiation into cholangiocyte-like cells
To test the ductal differentiation potential of hES cellderived
hepatic progenitor cells, we cultured them in William’s E medium
for 7 days on plates coated with matrigel, which has been reported
to promote hepatic progenitor cells differentiation into cholangio-
cytes [19]. Immunofluorescence indicated that KRT19- and
KRT7-positive, AFP-negative cells appeared (Figure 6A and B),
suggesting that the hES cellderived hepatic progenitor cells had
differentiated into cholangiocyte-like cells. Furthermore, we
differentiated hES cellderived hepatic progenitor cells in a
three-dimensional (3D) culture system in which cells were grown
in a gel consisting of matrigel and collagen I. This system has been
widely used to investigate the mechanisms underlying polarization
and tubulogenesis of epithelial cells [20], and it can also be used to
Figure 3. Scheme, cell morphology change, and the proliferation capacity of hepatic progenitor cells derived from hES cells. (A)
Hepatic endoderm cells generated by two-step differentiation from hES cells were purified using N-cadherin and further cultured on STO feeders to
induce hepatic progenitor cell colony formation. (B) Cell morphology changes during the procedure. (C) Hepatic progenitor cells were highly
proliferative and expressed the proliferation marker Ki67. Scale bar = 50 mm. (D) Growth kinetics of the hepatic progenitor colonies. Data represent
the mean6s.d. for 8–10 clones selected at random from passage 7. (E) Cumulative growth curve for the hepatic progenitor cells. The number of initial
feeder cells was deducted to obtain the final count. (F) Hepatic progenitor colony-forming potential of the N-cadherin
+
and N-cadherin
2
populations.
The experiments were performed three times, and a representative result is shown. N-CAD = N-cadherin.
doi:10.1371/journal.pone.0006468.g003
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investigate the epithelial polarity of differentiated cholangiocytes
[21]. When cultured in the 3D system for 7 days, differentiated
hepatic progenitor cells formed round cysts with a central luminal
space surrounded by a monolayer of cells (Figure 6C). We
examined the expression of lineage markers for liver epithelial
cells. KRT7 and KRT19, two conventional markers of cholan-
giocytes, were detected in the surrounding monolayer cells,
whereas the hepatic lineage marker AFP was undetectable
(Figure 6D and E). We then checked whether these cells acquired
apicobasal polarity as cholangiocytes and found that b-catenin was
localized to the basolateral cell surfaces and F-actin bundles were
enriched in the inner layer of the lumen, indicating the presence of
apicobasal polarity (Figure 6F-H). E-cadherin and intergrin a
6
were also localized to the basolateral region (Figure 6I-N). To
determine whether the differentiated cholangiocyte-like cells
possessed a secretory function, we analyzed the function of
MDR, which is an ATP-dependent transmembrane export pump
that may mediate the biliary secretion of cationic organic solutes
[22]. When the cysts were incubated in the presence of rhodamine
123, the fluorescence intensity was much greater inside the luminal
space than in the surrounding cells (Figure 6O). In addition,
rhodamine 123 was trapped inside cells and was not transported
into the central lumen in the presence of 10 M verapamil, an
MDR inhibitor (Figure 6P), indicating that the transport of
rhodamine 123 depended on functional MDR in the apical
domain. Taken together, these data demonstrated that the
differentiated cells derived from hepatic progenitor cells show
great similarity to cholangiocytes in vivo.
Discussion
In this study, we demonstrated for the first time that hES cells
could be differentiated into hepatic progenitor cells. These hES
cellderived hepatic progenitor cells could maintain their
proliferation capacity for more than 100 days of culture in vitro,
while maintaining their differentiation potential into both
hepatocyte-like and cholangiocyte-like cells. After they were
expanded, hepatic progenitor cell cultures could undergo
differentiation into hepatocyte-like cells that expressed ALB and
AAT and stored glycogen, or into cholangiocyte-like cells that
formed duct-like cyst structures, expressed KRT7 and KRT19
and acquired epithelial polarity.
We found that N-cadherin can be used as a surface marker for
the enrichment of hepatic endoderm cells differentiated from hES
cells. Previous studies tracking hepatic endoderm cells from mixed
hES cell derivates were limited to using an ES cell line with a
Figure 4. Unique features of hepatic progenitor cells. Hepatic progenitor cells co-expressed AFP and KRT7 (A), KRT19 (B), and stained faintly for
ALB (C). (D) Negative control. Scale bar = 50 mm.
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Figure 5. Differentiation of hepatic progenitor cells into hepatocyte-like cells. (A) AFP
+
KRT7
2
cells were spontaneously generated during
culturing of hepatic progenitor cells. Scale bar = 100 mm. (B–D) Hepatic progenitor cells could be induced into KRT7-negative (B), AFP (B and C), ALB
(C), and AAT-positive (D) hepatocyte-like cells. Scale bar = 50 mm. (E) RT-PCR analyses of the mRNA expression of marker genes revealed the similarity
of the hepatocyte-like cells differentiated from hepatic progenitor cells to those directly differentiated from hES cells. hES-HPC, hES cellderived
hepatic progenitor cells; HPC-H, hepatocyte-like cells differentiated from hES cellderived hepatic progenitor cells; hES-H, hepatocyte-like cells
directly differentiated from hES cells; HFL, human fetal liver cells. (F) Albumin secretion was determined in vitro by ELISA (n = 7). (G) The PAS assay
indicated the cytoplasmic glycogen storage (dark red) ability of these hepatocyte-like cells. (H) ICG taken analysis of the differentiated hepatocyte-like
cells (left) and undifferentiated hES cell-derived hepatic progenitor cells (right). Six hours later, ICG was excluded from the differentiated hepatocyte-
like cells which had taken ICG (middle). (I) Fluorescence microphotographs showed the uptake of Dil-Ac-LDL by hepatocyte-like cells. (J) PROD assay
of the differentiated cells with (left) or without (middle) PB induction and the undifferentiated hepatic progenitor cells with PB induction (right). PB,
phenobarbital sodium. Scale bar = 50 mm.
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reporter gene targeted to AFP, Foxa2 or another hepatic-specific
locus[2,3,5,23]. To our knowledge, the purification of hepatic
endoderm cells from derivates of an unmanipulated ES cell line
had not been reported. In the present study, we found that N-
cadherin expression specifically matched AFP expression after
the generation of hepatic endoderm cells (Figure 1B). These
results are consistent with previous reports describing t he N-
cadherin expression pattern during mouse and human liver
development [24–27]. In mouse e mbryos, N-cadherin is first
detected by immunohistochemistry in the liver at E10.5 and is
observed throughout the liver from E12.5 to adulthood. In the
human liver, N-cadherin is expressed during the fetal stage and
continues to be expressed in adult hepatocytes. In addition, N-
cadherin is uniquely expressed in hepa tic endo derm ce lls, but not
in undifferentiated hES cells in this differentiation system [28].
Therefore, compare d with other reported surfa ce markers of fetal
liver such as EpCAM and CD49f which are also expressed in
undifferentiated ES cells [29], N-cadherin can be used as a
specific surface marker for hepatic-committed cells to exclu de
undifferentiated ES cells under conditions that promote hepatic
lineage differentiation.
To date, the majority of studies have focused on the
differentiation of hES cells toward mature hepatocytes, and
there have been no previous reports investigating early hepatic
cell proliferation potential during the ES cell differentiation
process. In this study, we managed to couple the ability to
generate hES cellderived h epatic progenitor cells with the
capacity to expand this population in vitro. We gen erated hES
cellderived hepatic progenitor cells that co uld be p assaged more
than 12 times at a 1:2 or 1:3 split ratio and could be
cryopreserved and thawed repeatedly. The high purity, avail-
ability, and bipotential of hES cellderiv ed hepatic progenitor
cells will provide the basis for future th erapeutic efforts in
preclinicalanimalmodelsofdisease.
The hES cellderived hepatic progenitor cells obtained in the
present study appear to represent a population of cells similar to
those directly isolated from human fetal liver. Like the hepatic
stem cells and hepatoblast cells recently identified in fetal and
adult human livers by Reid’s group [12,30], our hES cellderived
hepatic progenitor cells could also be maintained on STO-feeder
cells in serum-free medium. Moreover, both of these cells
expressed AFP and CK19, as well as the surface makers EpCAM
and CD133, suggesting similar developmental origin.
Previous studies have reported a few biliary lineage cells in close
proximity to hepatocytes during the differentiation process [7];
however, the differentiation of cholangiocytes from hES cells has
been detected only on the basis of the expression of KRT7 and
KRT19. It is difficult to determine cholangiocyte differentiation
simply by analyzing gene expression, because only a few markers
are available and they are not closely related to cholangiocyte
function. In this study, we report a method to direct the
differentiation of hES cells into cholangiocyte-like cells through
the progenitor cell stage. Moreover, we employed multiple
standards including function assays to identify the biliary identity
of these differentiated cells. After induction, the differentiated
biliary cells (1) esxpressed KRT7 and KRT19, two conventional
markers of cholangiocytes, (2) formed a lumen with epithelial
polarity, demonstrating in vitro tubulogenesis, and (3) transported a
fluorescent dye, indicating functional MDR expression (Figure 6).
In conclusion, these multiple tests together showed that the
differentiated cells were similar to functional cholangiocytes.
In summary, this is the first report of the direct generation of
proliferative and bipotential hepatic progenitor cells differentiated
from hES cells. Because the generation process of hepatic
progenitor cells during in vitro differentiation mimics the
development of progenitor cells in vivo, these hES cellderived
hepatic progenitor cells could be used as an in vitro model for
Figure 6. Differentiation of hepatic progenitor cells into cholan-
giocyte-like cells. (A–B) Hepatic progenitor cells could be induced into
KRT7 (A)andKRT19-positive(B) cholangiocyte-like cells. (C) Morphology of
the hepatic progenitor cellderived ductal cysts formed in a 3D culture
system. (D–E) Co-immunofluorescence staining for AFP and cholangiocyte
markers. The cysts were positive for KRT19 (D, red) and KRT7 (E,red),but
were negative for AFP (E, green). (F–N) Localization of epithelial polarity and
ductal markers. b-ca tenin (F), E-cadherin (I), and Intergrin a
6
(L)localizedto
the basolateral membrane, while F-actin (G and J) localized to the apical
membrane. The ductal marker KRT19 localized to both the basolateral and
apical membranes (M). Merged images are shown in H, K,andN.(O)
Transport of rhodamine 123 into the central lumen of a cyst. (P) The MDR
inhibitor verapamil blocks rhodamine 123 transport. Scale bar = 50 mm.
doi:10.1371/journal.pone.0006468.g006
Hepatic Progenitors from hESCs
PLoS ONE | www.plosone.org 8 July 2009 | Volume 4 | Issue 7 | e6468
studying the early events of hepatic progenitor cell development.
In addition, they displayed proliferation and bipotentiality, which
will facilitate studies of the molecular mechanisms of hepatic stem/
progenitor cell origin, self-renewal and differentiation in vitro.
Materials and Methods
Culture and differentiation of hES cells into hepatic
endoderm cells
The human ES cell line H1 was obtained from WiCell Research
Institute (Madison, WI) and maintained as described previously
[7]. For hepatic differentiation, hES cells were induced following
the first two steps of a previously established stepwise protocol [7].
Flow cytometry and cell sorting
Day 8 hepatic endoderm cells were dissociated by treatment
with 0.25% trypsin containing 2 mM Ca
2+
instead of EDTA. The
resulting cells were stained with anti-N-cadherin (Clone GC4,
Sigma-Aldrich, St Louis, MO) or mouse IgG (Sigma-Aldrich),
followed by phycoerythrin-conjugated anti-mouse IgG antibodies
(Jackson ImmunoResearch, West Grove, PA) in phosphate-
buffered saline containing 1 mg/ml albumin. Cells were sorted
with a MoFlo cell sorter (Dako Cytomation, Carpinteria, CA) and
the data were analyzed using Summit Software, version 4.0 (Dako
Cytomation).
Culture, expansion and differentiation of hepatic
progenitor cells
Hepatic endoderm cells were plated on a monolayer of
mitomycin-treated STO feeder cells in hepatic progenitor
expansion medium [18]. Colonies were observed within 7–10
days. The hepatic progenitor cells were passaged at a ratio of 1:2
or 1:3 every 8–12 days. For hepatocyte differentiation, hepatic
progenitor cells were cultured in HCM containing 20 ng/ml HGF
(Peprotech, Rocky Hill, NJ) for 5 days, and 10 ng/ml OSM (R&D
System, Minneapolis, MN) plus 0.1 mM dexamethasone (Sigma-
Aldrich) for the next 5 days. For cholangiocyte differentiation,
hepatic progenitor cells were plated in Matrigel-coated (Becton-
Dickinson, Bedford, MA, 1:30) cell culture plates for attachment,
and then incubated with William’s E medium (Sigma-Aldrich)
supplemented described previously [31]. For biliary differentiation
in a 3D system, 1610
5
hepatic progenitor cells were suspended in
a mixture of 240 ml of type-I collagen gel (R&D System), 400 mlof
Matrigel and 360 ml of William’s E medium with supplements.
After incubation at 37uC for 2 hours to solidify the gel, 800 mlof
William’s E medium with supplements was added on the top of the
solid gel and changed every 2 days.
Immunofluorescence
Cells or tissue sections were fixed and stained as described
previously [7]. Optimized concentrations of primary antibodies
are shown in Table S2. FITC or TRITC-conjugated secondary
antibodies were purchased from Jackson ImmunoResearch. F-
actin was detected with AlexaFluor 546-conjugated phalloidin
(Invitrogen, Carlsbad, CA) at a dilution of 1:200.
RT-PCR and qPCR analysis of gene expression
Total RNA was isolated from cells using TRIzol Reagent
(Invitrogen) and genomic DNA was removed using TURBO
DNA-free Kit (Ambion, Austin, TX) according to the manufac-
turer’s protocol. RT-PCR and qPCR were performed as
previously described [7]. Primer sequences and annealing
temperatures are shown in Table S3 and S4.
Assays of hepatocyte functi on
PAS stain for glycogen, uptake of LDL, ICG uptake and PROD
assay were performed as described previously [7]. For the
measurement of albumin secretion, the human albumin content
in the supernatant was determined by Human Albumin ELISA
Quantitation kit (Bethyl Laboratory, Montgomery, TX) under the
manufacturer’s instructions. The albumin secretion was normal-
ized to the cell count.
Assay for transp ort of fluorescent dye
Hepatic progenitor cells were cultured in a Chambered
Coverglass (Nalgene Nunc, Naperville, IL) for biliary differentia-
tion in a 3D system for 7 days. Further assays to assess the
transport of rhodamine 123 and R-(+)-verapamil effect were
performed as described proviously [21].
Supporting Information
Figure S1 Flow cytometry analysis of hepatic endoderm cells.
Day 8 cells were dissociated and stained with anti-N-cadherin and
anti-AFP antibody.
Found at: doi:10.1371/journal.pone.0006468.s001 (2.30 MB TIF)
Figure S2 Immunofluorescence staining of post-sorted N-
cadherin
+
and N-cadherin
2
cells. Left, AFP-expressing (green)
cells were enriched in N-cadherin
+
cells by cell sorting. Right, AFP-
expression is hardly detected in N-cadherin
2
cell population. Cell
nuclei are stained with DAPI (blue). Scale bar = 50 mm.
Found at: doi:10.1371/journal.pone.0006468.s002 (3.50 MB
DOC)
Figure S3 Immunofluorescence staining demonstrated the
human cell origin of colonies yielded on STO feeder cells. Upper
panel, colonies were stained using antibodies against AFP and
human nucleus (HuNu). Lower panel, STO feeder cells stained as
control. Cell nuclei are stained with DAPI (blue). Scale
bar = 100 mm.
Found at: doi:10.1371/journal.pone.0006468.s003 (3.54 MB TIF)
Figure S4 Flow cytometry analysis of putative hepatic progen-
itor marker expression in hES cell2derived hepatic progenitor
cells. A substantial portion of hepatic progenitor cells cultured on
the feeder cells showed the expression of EpCAM and CD133. As
control, STO feeder cells did not express either EpCAM or
CD133.
Found at: doi:10.1371/journal.pone.0006468.s004 (4.44 MB TIF)
Table S1 Relationship between expression of AFP and some
surface proteins in day 8 differentiation.
Found at: doi:10.1371/journal.pone.0006468.s005 (0.04 MB
DOC)
Table S2 Primary antibodies and dilution factors.
Found at: doi:10.1371/journal.pone.0006468.s006 (0.04 MB
DOC)
Table S3 Semiquantitative RT-PCR primers.
Found at: doi:10.1371/journal.pone.0006468.s007 (0.04 MB
DOC)
Table S4 Quantitative RT-PCR primers.
Found at: doi:10.1371/journal.pone.0006468.s008 (0.04 MB
DOC)
Acknowledgments
We thank Liying Du and Yizhe Zhang for technical support on
Fluorescence Activated Cell Sorting and real-time PCR respectively. We
also thank Chengyan Wang, Hongxia Lv, Wei Jiang, Wei Wei, Pengbo
Hepatic Progenitors from hESCs
PLoS ONE | www.plosone.org 9 July 2009 | Volume 4 | Issue 7 | e6468
Zhang, Yanxia Liu, Xiaolei Yin, Haisong Liu and other colleagues in our
laboratory for technical assistance and advice in carrying out these
experiments.
Author Contributions
Conceived and designed the experiments: DZ MD HD. Performed the
experiments: DZ SC JC YG ZS JC CL CW. Analyzed the data: DZ SC.
Wrote the paper: DZ SC HD.
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Hepatic Progenitors from hESCs
PLoS ONE | www.plosone.org 10 July 2009 | Volume 4 | Issue 7 | e6468
    • "Les cholangiocytes obtenus exprimaient la cytokératine (CK) 19, l'aquaporine (AQP1), l'intégrine α6, l'intégrine β4 et le récepteur de la sécrétine (SCTR) et internalisaient la rhodamine 123 fluorescente dans lalumì ere des cystes. Les mêmes caractéristiques ontétéontété mises enévidence enévidence sur des hépatoblastes différenciésdifférenciésà partir des hESC (Zhao et al., 2009) et après reprogrammation de fibroblastes murins en cellules souches hépatiques (Yu et al., 2013). Aucun de ces travaux n'a montré de caractérisation extensive des cellules incluant des marqueurs spécifiques, en particulier de récepteurs et de facteurs de transcription, ni de fonctionnalité. "
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    • "LPCs can also be isolated from normal as well as injured adult livers and maintained in culture for long term, although their role in vivo remains elusive (Miyajima et al., 2014). It has been reported that LPC-like cells were established from hESCs/hiPSCs (Takayama et al., 2013; Yanagida et al., 2013; Zhao et al., 2009), and these cells were shown to proliferate and differentiate into hepatocyte-like cells or cholangiocyte-like cells. These LPCs were either isolated by cell sorting using a combination of specific cell surface markers or generated by adenovirus-mediated gene transfer to promote hepatic lineage differentiation. "
    [Show abstract] [Hide abstract] ABSTRACT: To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.
    Full-text · Article · Sep 2015
    • "During the course of the differentiation process from PSCs, hepatoblasts with the potential to differentiate to both hepatocytes and cholangiocytes are expected to emerge. Accordingly, hepatoblast-like cells expressing both hepatocyte and cholangiocyte markers were shown to emerge from PSCs, and they were able to differentiate to hepatocytes and form cysts of cholangiocytes in vitro (Yanagida et al., 2013; Zhao et al., 2009). The liver progenitors were expanded on stromal cells in vitro, and transplantation into mouse liver injury models, such as fumarylacetoacetate-hydrolase (Fah) deficiency, allowed engraftment and subsequent differentiation to hepatocytes. "
    [Show abstract] [Hide abstract] ABSTRACT: The liver is a central organ for homeostasis with unique regenerative capacities. Mature hepatocytes possess a remarkable capacity to proliferate upon injury, challenging efforts to discern the role of adult liver stem cells in this process. In contrast, stem/progenitor cells in the developing liver have been extensively characterized, and these investigations have informed efforts to produce functional hepatocytes in vitro for cell therapy and drug screening. In this Review, we describe recent advances in the characterization of liver stem cells and discuss evidence supporting and refuting whether self-renewable and bipotential liver stem cells exist in development, homeostasis, regeneration, and disease.
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