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Introduction
Although liver transplantation has become a standard
therapy for acute liver failure and end-stage liver disease, its
application has been limited due to the shortage of donor
organs. There has been extensive research in the field of
hepatocyte transplantation and bioartificial livers for
bridging and as an alternative to liver
transplantation[1,2]. A necessity for the development of bioartificial livers and
hepatocyte transplantation is the availability of sufficient
high-quality hepatocytes.
Embryonic stem (ES) cells are pluripotent and can
proliferate infinitely in an undifferentiated state in vitro. Therefore,
ES cell-derived hepatocytes could serve as potential cell
sources for bioartificial livers and hepatocyte transplantation.
Since the first report on the differentiation of mouse ES cells
into endodermal cells[3], many studies have demonstrated
that ES cells can differentiate into hepatocytes
in vitro and in vivo[4,5]. These differentiation protocols included the
addition of growth factors, sodium butyrate, collagen scaffold,
serum limitation, genetic manipulation, and
coculture[6_11].
Although many methods have been applied in the
differentiation of ES cells into hepatocytes, the research of
scalable and controlled culture systems is needed before the
clinical application of ES cells. The immobilization of cells
within alginate microbeads allows high-density cell culture
and free exchange of nutrients, oxygen, and bioactive
pro-ducts, whereas the cells are protected from sheer
strength[12]. Several investigators have described the induction of adult
stem cell differentiation and the mass production of
embryoid bodies following alginate
encapsulation[13_15]. Alginate encapsulation has also been reported to increase the
function of mature hepatocytes and maintain mature hepatocyte
function within bioartificial
livers[16]. However, studies describing the alginate encapsulation of embryoid-body cells
derived from ES cells to generate differentiated hepatocytes
in alginate microbeads have been limited.
In the present study, embryoid-body cells derived from
ES cells were cultured in alginate microbeads with exogenous
growth factors to promote hepatic histogenesis and
characterized cells by evaluating the expression of
hepatocyte-specific markers and function.
Materials and methods
Cell culture and formation of embryoid
bodies The mouse ES cells line CCE was grown on mouse embryo
fibroblasts treated with mitomycin C (Sigma, St Louis, MO, USA)
in Dulbecco's modified Eagle's medium with high glucose
(H-DMEM, GIBCO, Grand Island, NY, USA), supplemented
with 15% fetal bovine serum, 100 mmol/L non-essential amino
acids, 100 mmol/L 2-mercaptoethanol (GIBCO, USA), 2 mol/L
L-glutamine (GIBCO, USA), 100 U/mL penicillin, and 100
U/mL streptomycin (all from GIBCO, USA). In order to
maintain the ES cells in an undifferentiated state, we added 1000
U/mL mouse leukemia inhibitory factor (mL IF) (ESGRO,
Chemicon, Temecula, CA, USA) to the culture medium. The
media were changed every day. The cultures were split and
passaged every 3 d. To induce the formation of embryoid
bodies, undifferentiated cells were trypsinized and
dissociated to single-cell suspension and then plated at
6×104 cells/mL in petri dishes in culture medium without mLIF. The
media were changed every 48 h. All cell cultures were
incubated in a humidified 37 °C, 5%
CO2 environment. Only the ES cells between passages 10 and 20 were used for
differentiation.
Encapsulation of embryoid-body cells and the induction
of hepatic differentiation For the encapsulation in alginate
microbeads, 5 d-old embryoid bodies were trypsinized with
0.25% trypsin-EDTA (GIBCO, USA), and
5×106 cells were mixed in 1 mL sterile filtered 2% alginate (Sigma, USA)
dissolved in Ca2+ free DMEM (GIBCO, USA). The cell
suspension was extruded through a droplet generator NISCO
encapsulator (NISCO, Zurich, Switzerland). The beads
formed were allowed to gel in a hardening bath (100 mmol/L
CaCl2 [Sigma, USA], 10 mmol/L morpholinoethanesulphonic
acid [MOPS; Sigma, USA], pH 7.4). After 10 min of hardening,
the beads were washed 3 times with buffered saline (0.85%
NaCl [Sigma, USA], 10 mmol/L
4-(2-hydroxyethyl)-1-pipera-zineethanesulfonic acid (HEPES) [Sigma, USA], pH 7.4). The
beads were ultimately resuspended into H-DMEM medium
containing 20% fetal bovine serum (without mLIF). The media
were changed at every 3 days. The first day for
embryoid-body cells embedded in alginate microbeads was designated
as d 0. According to the method previously described with
modification[8], several growth factors were added into the
culture medium at varying days to induce the differentiation
of hepatocytes, including acidic fibroblast growth factors
(aFGF; R&D Systems, Minneapolis, MN, USA; 100 ng/mL)
added between d 0 and 7, hepatocyte growth factor (HGF;
R&D Systems, USA; 20 ng/mL) was then added between d 7
and 14, and oncostatin M (OSM; R&D Systems, USA; 10
ng/mL), dexamethasone (Sigma, USA; 10-7 mol/L), 5 mg/mL
insulin, 5 mg/mL transferrin, and 5 µg/mL selenium (ITS,
Sigma, USA) were added between d 10 and 14. The cells
were cultured until d 14. As a control treatment for
spontaneous differentiation, embryoid-body cells in alginate
microbeads were cultured in vitro for 2 weeks in the absence
of growth factors.
RT-PCR To extract RNA from encapsulated
embryoid-body cells, the microbeads were washed with 0.1 mol/L
phosphate buffered saline (PBS), and 55 mmol/L sodium
citrate (Sigma, USA), containing 10 mmol/L MOPS (Sigma,
USA), and 27 mmol/L NaCl (Sigma, USA) was added for
15 min at 37 °C to induce depolymerization. The released
cells were then collected by centrifugation at
453×g (1500 r/min) for 5 min. Total RNA was extracted using Trizol
reagent (GIBCO, USA) according to the manufacturer's
instructions. The concentration of RNA extracted was
determined at wavelength of 260 nm using a biophotometer
(Eppendorf, Netheler-Hinz, Hamburg, Germany).
First-strand complementary DNA (cDNA) was synthesized by the
reverse transcription system (Promega, Madison, WI,
USA). Total RNA (1 µg) was reverse transcribed to
first-strand cDNA in a 20 µL mixture containing 25 mmol/L
MgCl2 (4 µL), reverse transcription 10×buffer (2 µL), 10
mmol/L dNTP mixture (2 µL), recombinant RNase
inhibitor (0.5 µL), avian myeloblastosis virus (AMV) reverse
transcriptase (15 U), and oligo (dT) 15 primers (0.5 µg). The
reactions were incubated at 42 °C for 60 min, and then the
samples were heated at 95 °C for 5 min. The PCR was
performed by using a housekeeping gene β-actin as an internal
standard. The total reaction volume was 50 µL for the PCR
reaction, which was performed in a DNA thermal cycler
(Supermix, GIBCO, USA). All primers were synthesized by
Shanghai Sangon Biological Engineering Technology and
Service (Shanghai, China). The primer sequences and PCR
reaction conditions are shown in Table 1. The PCR samples,
together with a 2000 bp DNA ladder, were analyzed by 2%
agarose gel and visualized by ethidium bromide staining.
Immunofluorescence staining Albumin (ALB) and
cytokeratin-18 (CK18) were used as markers of mature
hepatocytes. Cells, including directed differentiated and
spontaneous differentiated embryoid-body cells in alginate
microbeads, were depolymerized and coated on the glass
slides. The sections were fixed with acetone for 15 min at
4 °C and incubated in blocking buffer (0.1 mol/L PBS
containing 0.5% normal goat serum and 0.2% Triton X-100)
for 1 h and thereafter in primary antibodies overnight at
4 °C diluted in 0.1 mol/L PBS. The antibodies used in this
study were rabbit anti-ALB (1:100 Abcam, Cambridge, UK)
and mouse monoclonal anti-CK18 (1:100 Abcam, UK). The
negative controls were processed using identical
pro-cedures, except for incubation without the primary antibody.
The secondary antibody used was immunoglobulin G
fluorescein-isothiocyanate-conjugated goat antimouse (1:50,
Sigma, USA) and tetramethylrhodamine isothiocyanate
(TRITC)-conjugated goat antirabbit (1:50, Sigma, USA).
Incubation with the secondary antibody was performed at room
temperature for 30 min.
ALB and urea production Conditioned media from
embryoid-body cells in alginate microbeads (20 microbeads per
well in a 24-well dish) cultured both with and without
exogenous growth factors were collected on d 0, 4, 7, 10, and 14
and frozen at -20 °C until the assay. The conditioned media
were assayed for ALB production using a quantitative ELISA
kit (Bethel Laboratories, Montgomery, USA) according to
the manufacturer's recommendations. To analyze urea
production, the embryoid-body cells in alginate microbeads
(20 microbeads per well in a 24-well dish) were incubated with
1 mL medium containing 5 mmol/L NH4Cl (Sigma, USA) for 24
h in 5% CO2 at 37 °C on d 0, 4, 7, 10, and 14. Following this
incubation, the supernatant was collected and the urea
concentrations were measured through a colorimetric assay kit
(Randox Laboratories, Antrim, UK). The culture medium was
used as a negative control.
Results
Cell morphology and viability The cell morphology in
the alginate microbeads was observed by the use of a
contrast microscope (Olympus, Tokyo, Japan). Through our
encapsulation system, the beads were round in shape,
identical in size, with a mean diameter of 500_600 µm, and
contained 600 cells in each bead. The cells were discretely
scattered in the bead and there were no differences in shape and
density throughout the 2-week experiment. Living cells
appeared to be shiny in the alginate microbeads. Immediately
following depolymerization, cell viability was determined by
the trypan-blue exclusion test. In the alginate microbeads,
the viability remained high (>90%) throughout the 2-week
experiment. The duration of the in vitro culture had no
significant effects on the viability of the cells.
Gene characterization of the embryoid-body cells in the
alginate microbeads To determine the level of
differentiation that occurred in the encapsulated cells, RNA from the
capsules was extracted and gene characterization for
liver-specific genes, including α-fetoprotein (AFP), ALB, Cyp7a1,
CK18, transthyretin (TTR), and tyrosine aminotransferase
(TAT) was performed. AFP is a marker of endodermal
differentiation, as well as an early fetal hepatic marker, and
its expression decreases as the liver develops into an adult
phenotype. The expression of ALB, the abundant protein
synthesized by mature hepatocytes, starts in early fetal
hepatocytes and reaches the maximal level in adult hepatocytes.
Cyp7a1 is expressed in the liver, but not in the yolk sac
tissue, and thus it can be a good marker for
hepatocytes[17]. CK18 is a cytoskeletal protein and is expressed in mature
hepatocytes. TAT represents an excellent enzymatic marker
for perinatal or postnatal hepatocyte-specific differentiation.
Since hormone-regulated TAT activity is strictly limited to
the parenchymal cells of the adult liver, it has been used
extensively for monitoring cellular differentiation in
experimental models for liver development/maturation
in vitro. TTR represents endodermal differentiation and is first expressed
in early embryos and throughout liver
maturation[18].
In our results, embryoid-body cells in alginate microbeads
on d 0 expressed mRNA of TTR and AFP. In the
differentiating embryoid-body cells with growth factors, mRNA of AFP,
ALB, TAT, and TTR were expressed on d 7. Cyp7a1 and
CK18 were expressed until d 14, but in the embryoid-body
cells without growth factors in the alginate microbeads,
Cyp7a1, CK18, and TAT mRNA could not be detected on
d 14. Thus, growth factors, such as aFGF, HGF and OSM
seem to be preferable for inducing hepatic differentiation in
encapsulated embryoid-body cells.
As expected, the pluripotent marker Oct-4 was expressed
in undifferentiated ES cells. The gene was also expressed in
embryoid-body cells in alginate microbeads on d 0, but the
Oct-4 gene expression diminished completely on d 7.
Immunofluorescence staining To further confirm the
hepatic differentiation from embryoid-body cells in the
alginate microbeads, the expression of ALB and CK18 were
analyzed by immunohistochemistry. The directed differentiated
cells were stained positively for both CK18 and ALB (Figure
3) on d 14. Some of the positive cells were binuclear. From
Figure 3, we can see that ALB and CK18 were found in the
cytoplasm. The percentages of ALB- and CK18-positive cells
were approximately 49%±3.8% and 50%±3.3% in direct
differentiated embryoid-body cells, respectively.
ALB and urea production Spontaneous differentiation
groups did not secrete ALB, whereas after induction of
hepatic differentiation, ALB secretion increased significantly
on d 10 and reached maximal values on d 14 (Figure 4A).
In addition, urea production was performed to confirm
whether directed differentiated embryoid-body cells in
alginate microbeads maintain hepatic metabolic functions.
Urea production in spontaneous differentiated groups did
not secrete urea. Directed differentiated embryoid-body cells
produced urea on d 7. On d 14, the amount of urea produced
by directed differentiated groups reached its maximum
(Figure 4B).
Discussion
Previous studies of the role of alginate encapsulation
in ES cell differentiation have provided conflicting
results[15,19_21]. Recently, Dean et al reported whether ES
cells or human ES cells, either in vivo or
in vitro, differentiate spontaneously in alginate
encapsulation[19]. Maguire et
al reported similar results[20]. As we know, the efficiency of
spontaneous ES cell differentiation into hepatocytes is very
low. It is a good way to direct differentiated ES cells into
hepatocytes by specific growth factor supplementation. Our
study showed that the alginate microbeads maintained
embryoid-body cell viability and gave rise to cells displaying
gene expression patterns, morphological features, and
metabolic activities characteristic of hepatocytes.
Previous studies have shown that adherent culture is
needed to further hepatocyte differentiation after the
formation of embryoid bodies, but adherent culture is limited by
the absence of large-scale processing considerations. It has
not been reported whether embryoid-body single cells keep
viability in alginate encapsulation. Our results show that
embryoid-body cells were cultured for 2 weeks with the
medium changed every 2_3 d. During the 2 week period, the
cell viability did not significantly decrease. The
encapsulated embryoid-body cells did not form large cellular
aggregates and remained in their current formation of single cells
and/or in small clumps.
After demonstrating that embryoid-body cells can
survive within the alginate microbeads, we evaluated the effect
of alginate microbeads on hepatocyte differentiation from
embryoid-body cells. It has been reported that ES cells
differentiate to form embryoid bodies in sodium alginate at low
concentrations, and sodium alginate at high concentrations
inhibits the differentiation of ES
cells[15]. Wang et
al[21] also reported that the expression of ES cells markers remains high
over 2 weeks of culture in alginate encapsulation in vitro.
Our result showed that embryoid-body cells in alginate
microbeads could not differentiate into functional
hepatocytes during spontaneous differentiation, although
hepatocyte genes, such as AFP and ALB were expressed. The
hepatocyte-related genes are expressed not only in the liver,
but also in the yolk sac of early
embryos[22], but Cyp7A1 could not be detected, which was thought to be a
liver-specific gene. This was similar to the results obtained by Asahina
et al, who found that embryoid bodies in suspension culture
could not differentiate into hepatocytes, and hepatocyte
genes could be detected only in adherent
culture[17].
Our next goal is to confirm whether embryoid-body cells
in alginate microbeads can differentiate into hepatocytes by
additional exogenous growth factors. Although the
mechanism of ES cell differentiation remains unknown, many
investigators have incorporated ES cell differentiation
strategies into the generation of a renewable hepatocyte cell source
using a variety of differentiation
techniques[8,9,23_28]. The most abundant factors that were used were aFGF, HGF, and
OSM, aFGF was added to imitate in vivo development, since
it is secreted by the mesoderm and is the first factor to
commit the foregut endoderm to form the liver primordium. HGF
supports fetal hepatocytes during mid-stage hepatogenesis.
OSM is produced by hematopoietic cells and induces the
maturation of fetal
hepatocytes[8,9,23,24]. Most of the
differentiation strategies required embryoid body intermediates. Our
results showed that embryoid-body cells in alginate
microbeads could differentiate into functional hepatocytes
by adding exogenous growth factors. This finding is
noteworthy because it indicates the absence of transport
limitations for exogenous growth factors in the alginate
micro-beads, and embryoid-body cells in alginate microbeads can
differentiate into hepatocytes by additional exogenous
growth factors.
The benefits of the alginate encapsulation system in
in vitro culture are numerous. Alginate microbeads can be
depolymerized through the use of divalent cation chelators,
facilitating rapid cell recovery for downstream analysis and
application. Alginate microbeads, through variations in the
encapsulation process (concentration, bead diameter, and
cell seeding density) can discretely control key culture
parameters. When alginate beads are applied for cell
transplantation in vivo, they can easily be coated in a polylysine
membrane for immune isolation.
In conclusion, our findings illustrate that the
differentiation of embryoid-body cells into hepatocytes in alginate
microbeads gives rise to functional hepatocytes containing
exogenous growth factors. ES cells may provide an
alternative source of hepatocytes for the development of the
bioartificial liver system and hepatocyte transplantation.
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