1 Stem Cell Regulation, Kanagawa Academy of Science and Technology (KAST),
Teikyo University Biotechnology Research Center, 907 Nogawa, Kawasaki,
Kanagawa 216-0001, Japan
2 Kirin Pharmaceutical Research Lab, Takasaki, Gunma 370-1295, Japan
3 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
4 First Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo
663-8501, Japan
* Author for correspondence (e-mail: miyajima{at}ims.u-tokyo.ac.jp)
Accepted 23 January 2003
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Summary |
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Key words: Signal sequence trap, Hepatocyte, Stem cell, Fetal liver, Epithelial cell
![]() |
Introduction |
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Hepatic progenitors are also known to be present in the adult liver
(Fausto, 1994;
Sell, 1994
). Oval cells with
the ability to differentiate into both hepatocytes and BECs appear in the
periportal region following hepatic injury when hepatocyte proliferation is
inhibited by a carcinogen, 2-acetylaminofluorene
(Sell et al., 1981
), or
D-galactosamine (Lemire et al.,
1991
). Rat oval cells have been shown histochemically to express
immature hematopoietic cell markers, c-Kit, CD34 and Thy1
(Fujio et al., 1994
;
Omori et al., 1997
;
Petersen et al., 1998
). In
addition, small hepatocytes in adult rat liver are suggested to be hepatic
progenitor cells (Mitaka et al.,
1999
; Tateno et al.,
2000
). However, it still remains unclear what roles oval cells and
small hepatocytes play in normal liver development, homeostasis and
regeneration. Besides adult liver, hepatic progenitor cells were shown to
exist in pancreas (Rao et al.,
1989
; Reddy et al.,
1991
; Zulewski et al.,
2001
; Tosh et al.,
2002
;). Surprisingly, recent results showed that bone marrow cells
could differentiate into hepatocytes
(Lagasse et al., 2000
;
Oh et al., 2000
;
Petersen et al., 1999
;
Wang et al., 2002
).
Furthermore, hepatocytes were induced from ES cells as shown by expression of
-fetoprotein and albumin, and they were engrafted into recipient liver
as hepatocytes (Chinzei et al.,
2002
). However, the mechanisms of the transdifferentiation from
bone marrow cells and the induction of hepatocytes from ES cells have not been
investigated. In order to understand the molecular mechanism underlying these
phenomena as well as the in vivo liver organogenesis, it is necessary to
isolate hepatoblasts and to investigate their differentiation and
proliferation.
The methodology using mAbs against cell surface antigens and a cell sorter
has been extensively used to isolate HSCs and can be applicable for the
isolation of the hepatic progenitor cells. In addition to the report that oval
cells were isolated as Thy1+ cells from regenerating adult liver
(Petersen et al., 1998),
recently, attempts have been made to purify progenitors from fetal liver based
on the expression of cell surface antigens by FACS. Suzuki et al. showed that
the
CD45-TER119-c-Kit-CD29+CD49f+
and
CD45-TER119-c-Kit-c-Met+CD49f+/lo
fraction of E13.5 mouse liver contained hepatic progenitor cells
(Suzuki et al., 2000
;
Suzuki et al., 2002
). Kubota
et al. showed that the RT1A1-OX18loICAM-1+
fraction of E13 rat fetal liver contained hepatoblasts
(Kubota and Reid, 2000
). While
these studies demonstrate the power of cell sorters to enrich hepatoblasts,
there are not enough surface antigens to identify hepatoblasts. In this study,
we used the signal sequence trap method to identify proteins with a signal
sequence in E14.5 mouse fetal liver cells and found that Dlk was abundantly
expressed in fetal liver.
Dlk is a type I membrane protein that has six EGF-like repeats in its
extracellular domain and a short cytoplasmic domain
(Laborda et al., 1993;
Smas and Sul, 1993
). The
extracellular domain shows homology to Delta, one of the Drosophila
melanogaster Notch ligands, but lacks the DSL domain that is important
for binding to Notch. Dlk was found to be highly expressed in a small lung
carcinoma cell line (Laborda et al.,
1993
) and was also identified as preadipocyte factor-1 (Pref-1) in
3T3-L1 preadipocytes (Smas and Sul,
1993
). Because Dlk orthologues were identified in human, rat and
bovine as well as in mouse independently, many names were given to the same
molecule; pG2 (Helman et al.,
1990
), fetal antigen-1 (FA-1)
(Jensen et al., 1994
), Pref-1
(Fahrenkrug et al., 1999
;
Smas and Sul, 1993
), stromal
cell derived protein-1 (SCP-1) (GenBank/D16847), zona glomerulosa-specific
factor (ZOG) (Halder et al.,
1998
), and Dlk (Laborda et
al., 1993
). Here we show that Dlk is strongly expressed in the
fetal liver between E10.5 and E16.5 and that Dlk can be used as a marker to
enrich hepatoblasts.
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Materials and Methods |
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Cell preparation and culture
Fetal hepatic cells of E14.5 liver were prepared and cultured according to
the method of Kamiya et al. (Kamiya et
al., 1999). Hepatic cells were suspended in DMEM containing 10%
FBS (Gibco BRL), 2 mM L-glutamine (Gibco), 1x nonessential amino acid
solution (Gibco), 1x insuline/transferrin/selenium (ITS) (Gibco), 50
µg/ml of gentamycin, 10-7 M dexamethasone (Dex) (Sigma), and 10
ng/ml of mouse oncostatin M (OSM) and plated on gelatin-coated dishes.
Signal sequence trap
The signal sequence trap method using a retroviral vector, pMX-SST,
developed by Kojima and Kitamura (Kojima
and Kitamura, 1999) was used to identify cDNA clones encoding
secreted and membrane proteins. cDNA was synthesized from poly(A) RNA of E14.5
CD45-TER119- hepatic cells using the Timesaver cDNA
synthesis kit (Pharmacia, Peapack, NJ) with random hexamer primers. After the
addition of the BstXI adaptor (Invitrogen, Carlsbad, CA), cDNA was
inserted into the BstXI site of the pMX-SST vector. The cDNA library
used in this study contained 5.0x106 independent clones.
Whole mount in situ hybridization
A fragment of Dlk cDNA was amplified by the reverse-transcription
polymerase chain reaction (RT-PCR) with two primers: 5'-ATG CTT CCT GCC
TGT GC-3' and 5'-GCA CGG GCC ACT GGC-3'. The PCR fragment
was subcloned into the pCRII vector (Invitrogen). Sense and antisense
single-stranded RNA probes were prepared by in vitro transcription using the
dioxigenin (DIG) RNA labeling kit (Roche, Basel, Switzerland). E10.5 embryo
was fixed in 4% PFA and bleached in 6% hydrogen peroxide. After treatment with
proteinase K, the embryo was hybridized with 0.5 µg/ml sense or antisense
probes at 65°C overnight. After washing and blocking procedures, the
embryos were incubated with alkaline phosphatase (AP)-conjugated anti-DIG
antibodies (Roche) at 4°C for overnight. The signal was developed in
4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate
(NBT/BCIP) solution (Roche).
Northern blotting analysis
Total RNA was extracted from tissues or cultured cells with Trizol reagent
(Gibco). After electrophoresis, RNA transferred to a nylon membrane was
hybridized with DIG-labeled antisense probe at 48°C overnight and then
incubated with AP-conjugated anti-DIG antibodies (Roche) at room temperature.
The signal was developed with CDPstar (Roche).
Immunochemistry
Fetal and adult livers were embedded in OCT compound (Sakura Finechemical,
Tokyo, Japan). Frozen sections were prepared using a Microtome cryostat HM 500
(Microm, Walldorf, Germany) and mounted on glass slides coated with MAS
(Matsunami glass, Japan). They were then fixed in 4% PFA and incubated with
normal goat serum. Primary and secondary antibodies were diluted to 1 µg/ml
and 5 µg/ml, respectively, in 3% normal goat serum. The samples were added
with anti-Dlk mAb followed by biotinylated goat anti-hamster IgG (Vector,
Burlingame, CA). The samples were incubated in each antibody solution at
4°C in a moist chamber. Expression of Dlk was visualized with the
Vectastain ABC kit (Vector) and 3,3-diaminobenzidine tetrahydrochloride (DAB)
(Roche). To detect CK19, samples were incubated with 1000-fold dilution of
anti-CK19 serum followed by AP-conjugated goat anti-rabbit IgG (Vector). The
signal was developed with NBT/BCIP (Roche).
For double immunofluorescence staining, fetal hepatic cells mounted on glass slides were fixed in 4% PFA and incubated with anti-Dlk mAb and rabbit anti-albumin polyclonal antibody (Nordic, Sweden). Dlk was detected with biotinylated goat anti-hamster IgG (Vector) and FITC-conjugated streptavidin (PharMingen) and albumin was detected with rhodamine-conjugated goat anti-rabbit IgG (Chemicon, Temecula, CA). The samples were examined under a fluorescence microscope, Nikon Eclipse E800 (Nikon, Tokyo, Japan).
Dlk+ cells isolated by AutoMACS were mounted on glass slides and
fixed in 4% PFA. They were incubated with 1 µg/ml anti-albumin or 20
µg/ml rabbit anti-human -fetoprotein (AFP) antibodies (ICN
Biomedicals, Costa Mesa, CA). Both signals were detected by
rhodamin-conjugated anti-rabbit IgG (Chemicon).
Flow cytometric analysis of Dlk and other cell surface markers
E14.5 fetal hepatic cells were incubated with anti-Dlk mAb and rat mAbs
against CD45, TER119 and PECAM-1 (PharMingen, San Jose, CA). After washing
with PBS, cells were incubated with FITC-conjugated goat anti-hamster IgG
(Vector) and PE-conjugated goat anti-rat IgG (Cedarlane, Ontario, Canada). All
these antibodies were diluted 100-fold and used for staining. The samples were
then washed with PBS and mixed with 1 µg/ml propidium iodide (PI) before
flow cytometric analysis with a FACScallibur (Becton Dickinson, San Jose,
CA).
Isolation of Dlk+ cells from fetal liver
Dlk+ cells were isolated by an automatic magnetic cell sorter
(AutoMACS) (Miltenyi Biotec, Bergisch Gladbach, Germany). E14.5 hepatic cells
were incubated with anti-Dlk mAb, biotinylated goat anti-hamster IgG. After a
wash with PBS, cells were resuspended in AutoMACS running buffer
(1x108 cells/ml of PBS containing 0.5% BSA) and 100 µl/ml
of streptavidin-labeled microbeads (Miltenyi Biotec) were added. After a wash
with the running buffer, the cells were loaded onto a magnetic column and
Dlk+ cells were eluted from the column after the depletion of
Dlk- cells. Alternatively, E14.5 CD45-TER119- cells were
incubated with anti-Dlk mAb and FITC-conjugated goat anti-hamster IgG and
sorted into Dlk- and Dlk+ fractions by using a
FACSvantage (Becton Dickinson).
RT-PCR analysis
Total RNA (1 µg) was used to synthesize cDNA using the First-strand cDNA
synthesis kit (Amersham Pharmacia Biotech, Piscataway, NJ) and random hexamer
primers. The samples were denatured at 94°C for 2 minutes, followed by the
thermal cycles; denaturation at 94°C for 30 seconds, annealing at the
temperature set for each pair of primers for 30 seconds, extension at 72°C
for 2 minutes. The thermal cycle was repeated 20 times for AFP and albumin, 25
times for Dlk and GAPDH, and 30 times for other genes. The primers used for
RT-PCR are shown in Table 1.
The primers used for Dlk were the same as those used for in situ
hybridization.
|
Quantitative PCR analysis was also performed to measure the mRNA levels for AFP and albumin by using LightCycler (Roche).
Culture of Dlk+ cells
A low density culture was performed to examine the growth potential of
Dlk+ cells. E14.5 Dlk- and Dlk+ cells
isolated by AutoMACS were cultured in DMEM F-12 (Sigma) at a density of 1000
and 50 cells/cm2, respectively, on 6-well plates coated with type
IV collagen (Nitta Gelatin, Osaka, Japan). The medium was supplemented with
10% FBS, 1xITS, 10 mM nicotineamide (Wako, Tokyo, Japan), 0.1 µM Dex,
and 5 mM L-glutamine. Various combinations of 20 ng/ml epidermal growth factor
(EGF) (PeproTech, London, UK), hepatocyte growth factor (HGF) (R&D,
Minneapolis, MN), and OSM were added 18 hours after the initiation of the
culture. After 5 days of culture, cell nuclei were stained with hematoxylin
(Muto Pure Chemicals, Japan) and the cells in each colony were counted. Colony
numbers in three wells were determined in each set of culture and the
experiment was repeated four times. These data were analyzed statistically
using JMP program to obtain standard deviations and P-values.
The expression of albumin and CK19 in colonies was analyzed by immunocytochemistry. The cells cultured on chamber slides (NUNC, Roskilde, Denmark) coated with type IV collagen were fixed in methanol at -20°C for 10 minutes. Alternatively, each large colony formed on 6-well plates was placed with a cloning ring and treated with trypsin. The cells removed from the dish were mounted on glass slides and fixed in methanol. After washing with PBS and blocking with 3% donkey serum (Chemicon) for 30 minutes, the cells were incubated with 2 µg/ml goat anti-mouse albumin antibody (Bethyl laboratories, Inc., Mongomery, TX) and rabbit anti-CK19 serum (1000-fold dilution) at 4°C overnight. After they were washed with PBS containing 0.05% Tween 20 (PBST), the samples were incubated with Cy3-conjugated donkey anti-goat IgG antibody (Rockland, Gilbertsville, PA) and FITC-conjugated donkey anti-rabbit IgG antibody (Rockland) for 2 hours at 4°C.
FACSvantage was used for the single cell sorting. Each Dlk+ cell sorted from E14.5 hepatic cells was individually plated in one well of a 96-well plate coated with type IV collagen. After 5 days of culture, cells were fixed in methanol and the expression of albumin and CK19 was examined as described above.
Transmission-electron microscopy
Ultrastructures of E14.5 Dlk+ cells were examined with a
transmission-electron microscope before and after the low density culture.
Purified Dlk+ cells from fetal livers were fixed in 2.5%
phosphate-buffered glutaraldehyde for 30 minutes at room temperature and in 1%
phosphate-buffered OsO4 for 15 minutes at room temperature.
Dlk+ cells cultured for 5 days on 6-well plates coated with type IV
collagen were detached from dishes by trypsin treatment and similarly fixed.
Then, both cells were dehydrated and embeded in epoxy resin. Thin sections for
electron microscopy were counterstained with uranil acetate and lead citrate,
and examined with a JEM-1220 electron microscope (JEOL, Tokyo, Japan).
Cell transplantation
Dlk+ cells were isolated from E14.5 fetal livers of GFP
transgenic mouse by using AutoMACS. Acute liver injury was induced in
recipient mice by intraperitoneal administration of anti-Fas antibody Jo2 (200
µg/kg; PharMinagen) before transplantation. After 24 hours of the injection
of anti-Fas antibody, 2x105 Dlk+ cells were
transplanted intrasplenically into the anesthesized recipient mice as
described in the previous work (Ponder et
al., 1991). After 8 or 36 weeks, the recipient mice were
sacrificed and frozen sections of their livers were prepared. Donor-derived
GFP+ cells were detected under a fluorescence microscope. The
sections including donor-derived GFP+ cells were used for
immunostaining with anti-albumin antibody.
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Results |
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|
Dlk is expressed in fetal liver and downregulated along with liver
development
Dlk/Pref-1 was previously shown to be expressed in E8.5 fetus and in liver,
pituitary, lung, vertebra and tongue at E13.5
(Smas and Sul, 1993). In order
to examine Dlk expression at the onset of liver organogenesis, whole-mount in
situ hybridization was performed using E10.5 embryo. In this experiment, Dlk
was detected in the liver bud as well as vertebra
(Fig. 1). The Dlk expression in
the E10.5 liver bud was also confirmed by RT-PCR
(Fig. 2A). To examine the
expression of Dlk during later liver development, Northern blot analysis was
performed using total RNA extracted from developing livers. Dlk mRNA was
strongly expressed in fetal liver between E12.5 and E16.5
(Fig. 2B). Its expression was
downregulated later in gestation and disappeared in the neonatal and adult
livers (Fig. 2B). The
expression of Dlk during liver development was also examined by using primary
cultures of fetal hepatocytes. We previously showed that E14.5 fetal
hepatocytes are induced to differentiate morphologically and to express
various metabolic enzymes such as TAT, G6Pase and CPS by OSM in the presence
of Dex in vitro (Kamiya et al.,
1999
). Northern blot analysis showed that Dlk expression gradually
disappeared without OSM, and was more rapidly disappeared in the presence of
OSM, a condition that induced expression of CPS and TAT
(Fig 2C). Expression of TAT and
CPS was induced after birth (Greengard,
1970
; Haber et al.,
1995
), while expression of Dlk was downregulated after E16.5
(Fig. 2B). It is thus likely
that E14.5 hepatocytes in the primary culture spontaneously differentiated to
a later fetal stage and OSM was required for further differentiation to the
postnatal stage in vitro.
|
|
Consistent with the results of in situ hybridization, immunohistochemical examination using anti-Dlk mAb on frozen sections indicated that Dlk was expressed in endodermal cells in the liver bud at the E10.5 stage, while it was not expressed in the foregut, from which the hepatic diverticulum was generated (Fig. 3A-D). Dlk was also detected in the E14.5 liver (Fig. 3E), but not in the adult liver (Fig. 3F). These results collectively indicate that Dlk is expressed in immature liver cells from the onset of the liver organogenesis.
|
Expression of Dlk in fetal hepatocytes
The fetal liver parenchyma consists of immature hepatocytes, BECs, and
their common progenitors, hepatoblasts. In order to know which endodermal
cells express Dlk, we performed double immunostaining analysis of Dlk with
either albumin or CK19. First, E14.5 fetal hepatic cells mounted on glass
slides were stained with anti-Dlk mAb and anti-albumin antibody. Since the
expression of albumin is induced at the beginning of liver organogenesis
(Jung et al., 1999), albumin
was expected to be detected not only in hepatocytes but also in hepatoblasts.
We found that large hepatic cells expressed both Dlk and albumin in fetal
liver between E12.5 and E18.5 (Fig.
3G-J and data not shown). We then stained fetal liver sections
with anti-Dlk mAb, and anti-CK19 antibody that stains BECs. Although the
formation of ductal plates has already started at the E14.5 or E15.5 stage
(Clotman et al., 2002
;
Coffinier et al., 2002
;
Shiojiri, 1997
;
Shiojiri et al., 2001
), the
cells comprising the ductal structure were not uniformly stained with
anti-CK19 antibody (data not shown). Therefore, in order to investigate
whether Dlk is expressed in BECs, we used E17.5 liver sections in which
CK19+ ductal plate structures became apparent. Double
immunostaining showed that CK19+ BECs were Dlk- at E17.5
(Fig. 3K-M). These results
suggest that Dlk is expressed in both immature hepatocytes and hepatoblasts,
and the expression is downregulated when hepatoblasts differentiate into
CK19+ BECs.
Isolation of Dlk+ cells using cell sorters
As Dlk is a membrane protein, it might be useful as a surface antigen to
separate immature hepatocytes from other types of fetal hepatic cells such as
hematopoietic and endothelial cells. Flow cytometric analysis using anti-Dlk
mAb showed that about 10% of total hepatic cells were Dlk+ and that
they were clearly separable from Dlk- cells, which mainly consisted
of CD45+, TER119+ and PECAM-1+ cells
(panspecific hematopoietic, erythroid and endothelial cells, respectively)
(Fig 4A). We then separated
Dlk- and Dlk+ cells from E14.5 fetal liver by AutoMACS.
Flow cytometric analysis showed that both Dlk- and Dlk+
fractions were over 95% pure (data not shown), and immunostaining revealed
that about 95% of cells in the Dlk+ fraction were AFP+
and albumin+ (Fig.
4B). Furthermore, we employed quantitative RT-PCR analysis to
examine the expression of AFP and albumin in
CD45-TER119-Dlk- and
CD45-TER119-Dlk+ cells, which were separated
by FACSvantage. Dlk+ cells expressed AFP and albumin about 60 and
40 times, respectively, higher than Dlk- cells
(Fig. 4C). These results
indicate that immature hepatocytes, possibly including hepatoblasts, are
enriched in the Dlk+ fraction.
|
To further characterize the E14.5 Dlk+ cells isolated by
AutoMACS, expression of several genes including hepatocyte and BEC markers was
examined by RT-PCR (Table 3).
Dlk+ cells expressed strongly AFP and albumin, and significantly
CK8, CK18, Cx26, Cx43, GS and GGT. Although GGT is known to be expressed in
BECs of adult liver, it was previously reported that rat GGT was expressed
also in fetal hepatocytes (Shiojiri et
al., 1991; Holic et al.,
2000
). In contrast to early hepatocyte markers, expression of CPS,
TAT, G6Pase, TO and Cx32, which are known to be expressed in mature
hepatocytes, was undetected or barely detectable. Consistent with the result
that Dlk was downregulated in BECs (Fig.
3K-M), Dlk+ cells did not express CK19. In addition to
early hepatocyte marker genes, Dlk+ cells expressed liver enriched
transcription factors, HNF1ß, HNF3ß, HNF4 and HNF6, whereas none of
them was expressed in Dlk- cells. On the contrary, expression of
c-Kit was detected in Dlk- but not Dlk+ cells.
|
Highly proliferative potential of Dlk+ cells
To examine the proliferative potential of each sorted cell, we cultured
Dlk- and Dlk+ cells at a low density to evaluate clonal
growth. Dlk- and Dlk+ cells were isolated from E14.5
fetal liver using AutoMACS and were plated at a density of 1000 and 50
cells/cm2, respectively, on 6-well plates coated with type IV
collagen. After 5 days of culture, we counted the number of large colonies
containing over 100 cells, which were considered to be formed from highly
proliferative cells. First, we examined various combinations of cytokines,
EGF, HGF, and OSM, for growth in vitro
(Table 4). As Dlk+
cells efficiently proliferated with HGF or HGF plus EGF, HGF was the most
effective growth factor for Dlk+ cells among three cytokines,
consistent with the fact that Dlk+ cells expressed c-Met, the
receptor for HGF (data not shown). Since the number of large colonies formed
in the presence of HGF plus EGF slightly exceeded that with HGF in this low
density culture condition, we used the combination of HGF plus EGF in the
following experiments.
|
We then evaluated the growth potential of Dlk+ cells in a low density culture with EGF and HGF. The growth of a single Dlk+ cell was followed for 5 days. Dlk+ cells proliferated exponentially between culture day 2 and 4, and some colonies still actively proliferated beyond day 4 and reached 100 cells at the end of the culture. After 5 days of culture, 24±3% of input Dlk+ cells proliferated and formed colonies with various sizes (Fig. 5). Half of the colony-forming Dlk+ cells formed small colonies containing less than 40 cells, while 10% of the colony-forming Dlk+ cells were highly proliferative. Since the number of colonies formed from Dlk- cells was less than 5% of that from Dlk+ cells, colony-forming cells were enriched in the Dlk+ fraction.
|
Differentiation potential of Dlk+ cells
Dlk is expressed in the E10.5 liver bud in which hepatic parenchyma is
believed to consist of hepatoblasts. In order to know whether Dlk+
cells are bipotential at the mid-gestation, we tested expression of albumin
and CK19 in colonies formed from E14.5 Dlk+ cells. The results
showed that over 60% of colonies derived from E14.5 Dlk+ cells
contained both albumin+ and CK19+ cells
(Fig. 6A-C). As colonies with
various sizes contained both types of cells, there seemed no direct
correlation between differentiation potential and proliferation potential. In
this low density culture condition, the colonies contained cells which
expressed either albumin or CK19 and also those that expressed both. To
confirm the existence of albumin+CK19+ cells, a large
colony was picked from the culture dish and the cells mounted on glass slides
were subjected to immunostaining. We did find cells that expressed both
albumin and CK19 (Fig. 6D).
These results strongly suggested that single Dlk+ cells were able
to differentiate into albumin+, CK19+, and
albumin+CK19+ cells. However, there still remained the
possibility that some colonies were derived from multiple cells even in the
low density culture. In order to exclude such possibility we performed single
cell sorting. By using FACSvantage each single Dlk+ cell was sorted
and plated in one well of a 96-well plate. After 5 days of culture, colonies
were formed in about 20% of wells inoculated with a single Dlk+
cell. About 10% of those colonies contained over 100 cells that consisted of
both albumin+ and CK19+ cells
(Fig. 6E), indicating that a
single Dlk+ cell was able to differentiate into both hepatocyte and
BEC lineages. Taken together, it is concluded that the majority of
colony-forming Dlk+ cells at E14.5 are hepatoblasts that display
bilineage gene expression and some of Dlk+ hepatoblasts are highly
proliferative.
|
We also analyzed ultrastructure of Dlk+ cells before and after 5 days of culture by electron microscope. The majority of E14.5 Dlk+ cells had a round-shaped nucleus, a small nucleolus, and elongated mitochondria (Fig. 7A). The former two characteristics suggest that E14.5 Dlk+ cells are rather primitive cells that are not actively synthesizing mRNAs. After 5 days of culture, most of the cultured cells exhibited a large number of microvilli on their cell surfaces and cleaved nucleus, larger nucleolus and many round-shaped mitochondria in the cytoplasm (Fig. 7B), suggesting that Dlk+ cells differentiated during the culture. However, because this culture condition was for proliferation rather than maturation, as described above, the Golgi apparatus, lysosomes and accumulation of glycogen were not apparent in the cytoplasm.
|
Transplantation of Dlk+ cells
In order to investigate differentiation potential of E14.5 Dlk+
cells in vivo, we transplanted Dlk+ cells into the spleen of
recipient mice that were pretreated with anti-Fas antibody to induce acute
liver damage. GFP+ cells were found in recipient liver at 8 weeks
after transplantation (data not shown) and GFP+ cells in the
recipient liver were more frequently found after 36 weeks
(Fig. 8A,B). These
GFP+ cells expressed albumin
(Fig. 8C), indicating that
Dlk+ cells can differentiate to hepatocytes in vivo under these
experimental conditions. By contrast, no GFP+ cells were found to
express CK19, suggesting that this protocol did not produce a condition to
replace recipient BECs.
|
![]() |
Discussion |
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We demonstrated that Dlk+ cells isolated from E14.5 liver were
able to proliferate to form colonies in 5 days of culture in the presence of
HGF and EGF. More than 60% of such colonies contained both albumin+
and CK19+ cells. Thus, E14.5 Dlk+ cells are mostly, if
not entirely, hepatoblasts. Furthermore, since 10% of colony-forming
Dlk+ cells formed large colonies that contained over 100 cells,
highly proliferative hepatoblasts are enriched in the Dlk+
population. Since the vast majority of Dlk+ cells expressed
albumin, and almost all the adherent cells were albumin+ after 1
day of culture, colonies were mostly formed from
Dlk+albumin+ cells. We also examined the ability of
Dlk+ cells at different stages of gestation to proliferate and to
differentiate. Dlk+ cells at E12.5 and E16.5 stages contained
highly proliferative and bipotential hepatoblasts, while E18.5 Dlk+
cells lost highly proliferative potential. As the expression of Dlk in each
cell was also downregulated at E18.5 (data not shown), the Dlk level may be
correlated with the growth potential of Dlk+ cells. We also
investigated the differentiation of Dlk+ cells in vivo by
transplantation of Dlk+ cells into the recipient mice treated with
anti-Fas antibody, Jo2, which induces apoptosis in hepatocytes. In this model
system, we detected donor-derived hepatocytes but not BECs. The inability to
demonstrate differentiation of hepatic progenitors to BECs seems to be a
common problem for in vivo transplantation assays because transplanted
progenitor cells differentiated into only hepatocytes in many cases using mice
as recipient animals (Suzuki et al.,
2000; Lagasse et al.,
2000
; Chinzei et al.,
2002
; Forbes et al.,
2002
; Malhi et al.,
2002
). One exception is that transplanted
CD45-TER119-c-Kit-c-Met+CD49f+/lo
cells formed bile-duct like structures; however, such structures were found
only in the spleen but not in liver
(Suzuki et al., 2002
). Thus,
it is possible that the protocol we used did not create a condition that
allowed the transplanted cell to replace the recipient BECs. However, the
possibility remains that Dlk+ cells at E14.5 have lost the ability
to become BECs in vivo.
Recent studies indicated that hepatoblasts could be enriched by using FACS:
mouse hepatoblasts were enriched in the
CD45-TER119-c-Kit-CD29+CD49f+
fraction (Suzuki et al., 2000)
and were further enriched in the
CD45-TER119-c-Kit-c-Met+CD49f+/lo
fraction (Suzuki et al.,
2002
), and rat hepatoblasts were present in
RT1A1-OX18loICAM-1+ cells
(Kubota and Reid, 2000
). In
the present study, we demonstrated that hepatoblasts are enriched in the
Dlk+ population of mouse fetal hepatic cells. Flow cytometric
analysis indicated that Dlk+ cells were
CD45-TER119- and cKit-. Although both
Dlk+ and
CD45-TER119-c-Kit-c-Met+CD49f+/lo
cells contained highly proliferative and bipotential cells, there is a
substantial difference in abundance between Dlk+ cells and
CD45-TER119-c-Kit-c-Met+CD49f+/lo
cells. Dlk+ cells constituted about 10% of E14.5 total fetal
hepatic cells, while
CD45-TER119-c-Kit-c-Met+CD49f+/lo
constituted only 0.3% of them. The potential for differentiation also
highlights a significant difference between Dlk+ cells and
CD45-TER119-c-Kit-CD29+CD49f+
or
CD45-TER119-c-Kit-c-Met+CD49f+/lo
cells. For example, a longer incubation time was required for
CD45-TER119-c-Kit-c-Met+CD49f+/lo
cells to differentiate into hepatocytes and BECs: 70% of large colonies
derived from Dlk+ cells contained albumin+ and
CK19+ cells only after 5 days of culture
(Fig. 6), while 80% of large
colonies derived from
CD45-TER119-c-Kit-c-Met+CD49f+/lo
cells contained both albumin+ and CK19+ cells only after
21 days of culture. Thus, it is likely that
CD45-TER119-c-Kit-c-Met+CD49f+/lo
cells represent more immature cells than Dlk+ cells. Rat
RT1A1-OX18loICAM-1+ hepatoblasts formed
colonies containing albumin+ and CK19+ cells after 5
days of culture. Although they were derived from different animals, mouse
Dlk+ cells and rat
RT1A1-OX18loICAM-1+ cells appear to exhibit
similar characteristics as a progenitor of hepatocytes and BECs.
The result that Dlk is expressed specifically in fetal liver suggests that
Dlk might be implicated in proliferation and/or differentiation of
hepatocytes. Consistent with this idea, Dlk, also known as Pref-1, was
previously shown to be involved in differentiation of pre-adipotcytes as
overexpression of Dlk resulted in inhibition of adipogenesis
(Smas et al., 1997;
Smas and Sul, 1993
). It was
also reported that Dlk expression increased when proliferation of pancreatic
ß cells reached a maximum (Carlsson et
al., 1997
). To test whether Dlk might have a role for hepatic
differentiation in a manner similar to adipocyte differentiation, we expressed
Dlk in the fetal hepatocyte primary culture by using a retrovirus vector.
However, expression of hepatic differentiation marker genes was not altered
(data not shown). Another possibility is that Dlk is involved in
hematopoiesis. Moore et al. reported that Dlk was expressed in fetal liver
stroma cells that were able to support hematopoiesis
(Moore et al., 1997
). In
addition, there are reports that Dlk modulated proliferation of thymocytes
(Kanata et al., 2000
), fetal
liver hematopoietic cells (Ohno et al.,
2001
) and pre-B cells (Bauer et
al., 1998
). Interestingly, fetal liver hematopoiesis is most
active in mid-gestation when Dlk is strongly expressed, which suggests that
Dlk is involved in hematopoiesis. However, recent studies on Dlk deficient
mouse show that the mutant mice are viable without apparent defects in liver
formation and hematopoiesis, while they show growth abnormality and altered
lipid metabolism (Moon et al.,
2002
). Thus, roles of Dlk in hematopoiesis as well as liver
development still remain elusive and await further investigation.
In this study, we successfully isolated Dlk+ cells and demonstrated that hepatoblasts are abundant in E14.5 Dlk+ cells. Using the present method, 95% pure Dlk+ cells can be easily isolated from total fetal hepatic cells using AutoMACS. In addition, since Dlk expression in fetal liver is similar between mouse and human, the present method may be applicable for the isolation of human hepatoblasts.
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References |
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