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Introduction
Embryonic stem (ES) cells are continuously growing cell
lines initially isolated from the inner cell mass of
blastocysts[1]. Under certain experimental conditions, ES cells
spontaneously differentiate into cell types from all 3 germ layers
in vitro. Due to the unlimited proliferating ability and
pluripotent differentiation potential, ES cells may provide an
alternative cell source for cell replacement therapy in the
treatment of various diseases. To date, attempts have been made
to direct the differentiation of ES cells into a variety of cell
lineages in vitro. When cultured in
vitro under certain conditions, ES cells can differentiate into
cardiac[2_4], neuronal[5_7],
hematopoietic[8,9], vascular smooth
muscle[10], adipogenic[11], or chondrogenic cell
types[12].
Thus far, studies on ES cell differentiation into
hepatocytes have mainly focused on genetic and protein
analyses of specific markers implicated in the development of the
liver[13]. However, little attention has been paid to how to
determine and improve the hepatic differentiation ratio. An
efficient hepatic differentiation system is very important for
subsequent research on hepatocyte transplantation and liver
engineering. According to the principle that cells in culture
survive when they are adapted to the existing
microenviron-ment, we developed an efficient culture system to enrich
hepatocyte-like cells from differentiated ES cells. In such a
system, only hepatocyte-like cells survive, while other cells
do not. This particular culture system contains factors that
selectively activate the proliferation of hepatocyte-like cells,
but inhibit the growth of other cells. Studies on related
mechanisms of liver regeneration and hepatic stem cells
corresponding to liver injury have suggested that the pathological
serum obtained from patients or animals with severe liver
injury may provide such
conditions[14_17]. Therefore, in the present study, we utilized a culture system containing
cholestatic serum to enrich hepatocyte-like cells from mouse
ES-derived differentiated cells in vitro. Our results showed
that cholestatic serum is efficient in lineage differentiation
and proliferation of hepatocyte-like cells from mouse ES cells,
and the resulting hepatocyte-like cells are capable of
accumulating glycogen, an important metabolic function of
hepatocytes.
Materials and methods
Preparation of the conditional selective
medium Cholestatic serum was prepared according to our previously
reported method[18]. Sprague-Dawley rats weighing 200_250 g (Laboratory Animal Research Center of Sun Yat-sen
University, Guangzhou, China) underwent ligation and
transection of the common bile duct under general
anesthesia with ether to induce cholestasis. Ten days after the
operation, the rats were sacrificed and whole blood was
collected. Serum was isolated from the whole blood and
then subjected to liver function testing, inactivated, and
sterilized for use in culture. Different doses of cholestatic serum
were added into the differentiating medium to achieve the
following final concentrations: 20, 50, and 100 mL/L.
Differentiating medium containing cholestatic serum served as the
conditional selective medium in the course of cell
differen-tiation. All animal experimental procedures were approved
by the Sun Yat-sen University Institutional Animal Care and
Use Committee.
Cell culture Undifferentiated E14 mouse ES cells (ATCC,
Manassas, VA, USA) were maintained on gelatin-coated
dishes in Dulbecco's modified Eagle's medium
(DMEM;GIBCO, Grand Island, NY, USA), supplemented with 15%
fetal bovine serum (Hyclone, Rockville, MD, USA), 1000
U/mL recombinant mouse leukemia inhibitory factor
(LIF;Chemicon, Temecula, CA, USA), 1% non-essential amino
acids, 1 mmol/L glutamine, 0.1 mmol/L β-mercaptoethanol
(Sigma, St Louis, MO, USA), and 10 ng/mL recombinant
human fibroblast growth factor-4 (FGF-4; R&D,
Minnea-polis, MN, USA). The above culture medium, excluding LIF,
was defined as the differentiating medium.
To induce differentiation, the ES cells were incubated
using the suspension culture methods described
previously[19_21]. Briefly, the cells were suspended in the differentiating
medium, designated as d 0 of cell differentiation, and allowed
to develop into embryoid bodies (EB). After 4 d in the culture,
the resulting EB were plated onto 6-well plates coated with
0.1% gelatin, and cultured with the differentiating medium in
the presence of 3 mmol/L sodium butyrate. One week later,
to isolate and enrich the ES-derived hepatocyte-like cells
from mixed differentiated cells, the cells were further
cultured in the conditional selective medium supplemented with
3 mmol/L sodium butyrate and 10 ng/mL recombinant mouse
hepatocyte growth factor (HGF; R&D, USA). Under these
conditions, differentiated cells were cultured for another 10
d. The cells cultured in the differentiating medium without
sodium butyrate and cholestatic serum were used as controls.
Electron microscopy The culture plates were washed
with phosphate-buffered saline and fixed in cold 25 g/L
glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4)
for 48 h. After fixation, the cells were curetted and
centrifuged to form aggregates. After post-fixing in 10 g/L osmium
tetroxide in 0.1 mol/L sodium cacodylate (pH 7.4), the cells
were dehydrated in a graded series of alcohol and embedded
in low viscosity epoxy resin. Ultra-thin sections were stained
with uranyl acetate and lead citrate and viewed under an
electron microscope.
RT-PCR On d 0, 3, 6, 9, 12, 15, 18, and 21 of cell
differen-tiation, the cells were collected for an analysis of the mRNA
expression of the following liver-specific genes:
α-fetoprotein (AFP), albumin (ALB), transthyretin (TTR), α1-antitrypsin (AAT), glucose-6-phosphate (G6P), and tyrosine
aminotransferase (TAT). Total RNA was extracted from
1×106 differentiated cells by using Trizol reagent (Invitrogen,
Carlsbad, CA, USA ). cDNA was prepared from 2 µg of total
RNA by using a RevertAid first-strand cDNA synthesis kit
(Fermentas, Hanover, MD, USA). Samples of cDNA
corresponding to the input RNA were amplified in PCR reaction
buffers containing primers and LA Taq DNA polymerase
(Takara, Kyoto, Japan ). The primer sequences and the
expected sizes of the RT-PCR products are shown in Table 1.
The PCR reaction was performed with the following thermal
profiles: denaturation at 94 °C for 5 min, followed by 30 cycles
of 30 s at 94 °C, 30 s at 55 °C, and 45 s at 72 °C. Then the
program was finished by a 10 min extension at 72 °C. The
amplified products were subjected to electrophoresis in 20
g/L agarose gels and stained with ethidium bromide. All the
procedures were performed according to the manufacturers'
instructions.
Immunocytochemistry On d 18 of cell differentiation,
immunocytochemistry for AFP, ALB, and cytokeratin 18
(CK18) was performed using the methods described
previously[22,23]. The primary antibodies were obtained and
diluted as follows: goat monoclonal antimouse AFP antibody
(1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA),
sheep monoclonal antimouse ALB antibody (1:50; Biodesign,
Saco, ME, USA), goat monoclonal antimouse CK18 antibody
(1:200; Santa Cruz Biotechnology, USA), and relative
Cy3-conjugated secondary antibodies (Sigma, USA). Nuclear
DNA was dyed with Hoechst 33258 (Sigma, USA).
Hepatocytes from mouse livers were used as controls.
Glycogen detection (Periodic acid_Schiff
reaction) The cells cultured on plates were dried in air and fixed with 95%
alcohol for 10 min. After rinsing 3 times with distilled water,
the cells were oxidized in 1% periodic acid for 10 min, rinsed
in distilled water again, then exposed to the Schiff reagent
for 10 min at 37 °C. A third water wash to remove the reagent
was followed by an inspection of the cells with a light
microscope. Spontaneously-differentiated cells were used
as controls.
Determination of the hepatic differentiation
ratio On
d 18 of cell differentiation, immunocytochemistry for ALB
was performed as described above. One hundred cells per
well and ALB-positive cells within the sample were
enumerated; the percentage of ALB-positive cells was
determined. The values from each well were averaged to
obtain a mean±SD. After staining with periodic acid-Schiff
(PAS), the percentage of PAS-positive cells was also
evaluated using the same method. The cells cultured with sodium
butyrate, but without cholestatic serum, and
spontaneously-differentiated cells were used as controls.
Results
Preparation of the conditional selective
medium Ten days after the ligation of the common bile duct, the level of
serum total bilirubin (STB) in the cholestatic rats was 104±46.2 µmol/L, obviously higher than that in the parallel normal
rats (data not shown). The STB concentrations of the
conditional selective medium containing 20, 50, and 100 mL/L of
cholestatic serum were 2.9, 5.6, and 10.2 µmol/L, respectively.
ES cell differentiation into hepatocyte-like
cells The morphological changes of the ES cells during the process of
cell differentiation were observed using light microscopy.
The mouse ES cells proliferated vigorously and EB were
formed in the suspension culture 4 d after the removal of LIF.
In the absence of sodium butyrate, the control ES cells
spontaneously differentiated into multiple lineages (Figure
1A,1B). One day after incubation in culture medium containing
sodium butyrate, cell differentiation occurred. By 3 d of
culture, cell death was observed in a significant number of
cells, while the remaining cells assumed homogenous
morphological changes. Most of these cells were epithelioid
and fibroblast-like cells. In the presence of conditional
selective medium, however, cell proliferation was
considerable and numerous epithelial cells resembling hepatocytes
were observed (Figure 1C, 1D). The cells were polygonal in
shape with large, round, and center-situated nuclei;
bi-nuclei were also present. Furthermore, electron micrographs
revealed that the differentiated cells were rich in
endoplasmic reticulum, ribosomes, ellipsoid mitochondria, and
glycogen (Figure 1E), which are typical ultrastructural features of
hepatocytes (Figure 1F). Furthermore, we also found that
conditional selective medium with 50 mL/L cholestatic
serum was optimal for hepatocyte-like cells to survive and
proliferate, while 100 mL/L cholestatic serum led to
significant cell death, and 20 mL/L cholestatic serum reduced the
number of hepatocyte-like cells as compared with 50 mL/L
cholestatic serum.
Detection of hepatic markers The results of the RT-PCR
analysis showed that undifferentiated ES cells did not
express any hepatic markers; however, in the presence of
sodium butyrate and conditional selective medium, hepatic
differentiation markers were detected. AFP and ALB were
expressed within d 6 and d 9. Other endodermal and hepatic
markers, TTR and AAT, were also expressed within d 6 and
d 12. Late fetal hepatic markers, G6P and TAT, were not
expressed until d 15 and d 18, respectively.
Moreover, the protein expressions of AFP, ALB, and CK18 were observed
in the cells treated with sodium butyrate and conditional
selective medium, but not in the ES cells. The experiments
were repeated 3 times and the representative data are shown
in Figure 2 and 3.
Functional test of differentiated cells Glycogen storage
is an important metabolic function of hepatocytes. In this
study, the capability of glycogen storage in the
differentiated cells was analyzed by the PAS reaction. Glycogen
storage was manifested as the accumulation of magenta staining
in the cytoplasm of hepatocyte-like differentiated cells. Very
few PAS-positive cells were detected amongst the
spontaneously-differentiated cells (Figure 4A).
Determination of hepatic differentiation
ratio The ratio of hepatic differentiation has rarely been reported in
previous studies involving ES cells[24]. In the current study, the
hepatic differentiation ratio was determined by evaluating
the percentages of ALB- and PAS-positive cells in
differentiated cells. On d 18 of cell differentiation in the culture
system containing sodium butyrate and cholestatic serum,
ALB and PAS staining was observed in 82% and 87% of the
cells, respectively. In contrast, in the same culture system
without cholestatic serum, ALB and PAS were expressed in
65% and 50% of the cultured cells, respectively. On average,
in the spontaneously-differentiated cells, 19% of the cells
expressed ALB and 5% of the cells were PAS positive (Figure 4B).
Discussion
In this study, we present a novel and highly efficient
method for generating functional hepatocytes from ES cells.
Although ES cells can differentiate into hepatocyte-like cells
either spontaneously or induced by exogenous factors
in vitro[1,19_22], the resulting cells still contain multiple
heterogeneous lineages which are not suitable for therapeutic
transplantation[25,26]. To avoid the risk of teratoma formation after
cell transplantation, recent reports have highlighted the
importance of acquiring functional ES-derived hepatocytes with
uniform phenotype[24,27]. We found that a culture system
containing cholestatic serum was very helpful to selectively
enrich and isolate functional hepatocytes from the mixed
differentiated ES cells.
The first step in our protocol involved culturing the EB
with sodium butyrate, an inhibitor of histone deacetylase
that can induce ES cells to differentiate into
hepatocytes[13,28,29]. In comparison to the method described
previously[28], the present protocol did not contain DMSO. We demonstrated
that sodium butyrate alone could efficiently initiate hepatic
differentiation from ES cells. The expressions of AFP and
ALB mRNA were detected within d 6 and d 9 as compared
with d 9 and d 12 in spontaneously-differentiated ES cells.
Upon consecutive induction, late fetal hepatic markers, G6P
and TAT, were expressed within d 15 and d 18. Moreover,
65% and 50% of the differentiated cells treated with sodium
butyrate were ALB and PAS positive on d 18 of differentiation,
while only 19% and 5% were ALB and PAS positive in
spontaneously-differentiated ES cells. Therefore, our data
confirm that hepatocyte-like cells from
spontaneously-differentiated ES cells may arrest in an immature stage, while sodium
butyrate not only promotes hepatic differentiation from ES
cells, but also induces maturation of ES-derived
hepatocyte-like cells.
To enrich the hepatocyte-like cells and eliminate the
heterogeneous populations, a conditional selective medium
containing cholestatic serum was used 7 d after the
induction by sodium butyrate. It has been reported that cholestasis
after bile duct ligation might induce marked hepatic stem cell
proliferation[16], and sera from patients with liver failure could
stimulate mouse bone marrow cells to transdifferentiate into
hepatocytes[30]. In the present study, we found that
cholestatic serum could selectively enrich and isolate
ES-derived hepatocytes efficiently. After being treated with
cholestatic serum for several days, the differentiated ES cells
exhibited very uniform morphology resembling mouse
hepatocytes, with many hepatic characteristics in the gene
expression profile, phenotypic markers, and functional
features. In comparison to the treatment with sodium
butyrate alone (without cholestatic serum), the ALB- and
PAS-positive cells increased to 82% and 87%, respectively, and
no karyotypic and phenotypic changes were observed in
the ES-derived hepatocyte-like cells in the presence of
cholestatic serum. This indicated that cholestatic serum did
not have a negative effect on the procedure of hepatic
differentiation mediated by sodium butyrate, but the approximate
percentage between PAS- and ALB-positive cells suggested
that cholestatic serum could further promote the maturity of
ES-derived hepatocyte-like cells.
The efficacy of the conditional selective medium used in
this study may be ascribed to cholestatic serum containing
the metabolic products which accumulated following
common bile duct ligation and hepatic insufficiency (i.e. bilirubin,
bile acid, endotoxin, and ammonia). The current study
suggests that ES-derived hepatocyte-like cells are capable of
accumulating glycogen and express enzymes concerned in
metabolism such as G6P and TAT, and it has previously been
reported that ES-derived hepatocyte-like cells metabolize
ammonia and synthesize urea[13,22]. Thus, we assumed that
the functional hepatocyte-like cells metabolized toxic
products contained in cholestatic serum, survived, and
selectively proliferated in response to the signals characteristic
of cholestatic serum, while the non-hepatic cells could not
adapt to such a pathological environment and resulted in
apoptosis. Therefore, the conditional selective medium based
on cholestatic serum selectively enriched hepatocyte-like
cells from mixed differentiated ES cells in 2 ways: (1)
providing selective proliferative signals for hepatocyte-like cells;
and (2) eliminating non-hepatic populations.
In summary, we have demonstrated a novel method to
improve the hepatic differentiation ratio with a conditional
selective medium containing cholestatic serum. The present
results make an important contribution by providing a novel
source of donor hepatocytes for therapeutic transplantation.
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