1 Department of Surgery, Institute of Clinical Medicine, University of Tsukuba,
Tsukuba, Ibaraki 305-8575, Japan
2 Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute
of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
3 Department of Regenerative Medicine, Faculty of Medicine, Yokohama City
University, Yokohama, Kanagawa 236-0004, Japan
* Author for correspondence (e-mail: rtanigu{at}med.yokohama-cu.ac.jp)
Accepted 26 February 2003
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SUMMARY |
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Key words: Stem cell, Liver, Hepatocyte, C/EBP, Mouse
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INTRODUCTION |
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In a previous study, using flow cytometry and single-cell-based assays, we
prospectively identified hepatic stem cells with multilineage differentiation
potential and self-renewing capability
(Suzuki et al., 2002). These
cells expressed the hepatocyte growth factor receptor Met and were
low-positive for CD49f (
6 integrin subunit), but did not express
Kit (stem cell factor receptor), CD45 (leukocyte common antigen) and TER119 (a
molecule resembling glycophorin, exclusively expressed on immature erythroid
cells). Sorted stem cells could be clonally propagated in culture for over 6
months, where they continuously produced hepatocytes and cholangiocytes as
descendants, while maintaining primitive stem cells that expressed neither
albumin nor cytokeratin 19 during the greater part of their expansion. Our
studies with highly enriched populations with stem cell activity showed that
HGF was a critical requirement for proliferation
(Suzuki et al., 2000
;
Suzuki et al., 2002
). It
remained unknown, however, whether the Met/HGF interaction had a role in stem
cell differentiation. Spagnoli et al.
(Spagnoli et al., 1998
)
established a bi-potential hepatic precursor cell line from transgenic animals
that constitutively expressed the activated form of Met. This and our previous
findings suggest that, while the Met/HGF interaction is crucially responsible
for maturation of differentiating hepatocytes in the postnatal liver
(Hu et al., 1993
;
Kamiya et al., 2001
) and
division of mature hepatocytes in liver regeneration
(Michalopoulos et al., 1984
;
Ishiki et al., 1992
), it is
also involved, through a separate mechanism, in stem cell growth and
differentiation.
In this study, we investigated the direct effects of GFs and extracellular matrix components (ECMs) on proliferation and bi-potential differentiation of prospectively isolated and clonally cultured hepatic stem cells. To analyze primitive stem cells and stem cell-derived differentiating hepatocytes, respectively, cell type was determined by the expression of ALB enhancer/promoter-EGFP. We demonstrated that HGF, but not FGFs, induced early transition from ALB-negative (ALB) stem cells to ALB-positive (ALB+) hepatic precursors through signaling via the CCAAT/enhancer-binding protein (C/EBP), a basic leucine zipper transcription factor. Subsequently, HGF was an effective mitogen for differentiating cells, while OSM inhibited their proliferation and induced their maturation, as assessed by expression of glucose-6-phosphatase (G6P) and tryptophan-2, 3-dioxygenase (TO). By contrast, these factors suppressed differentiation into cholangiocyte-lineage cells. Inactivation of C/EBPs, even in the presence of both HGF and OSM, strongly inhibited the differentiation of stem cells into hepatocyte-lineage cells and allowed cells to self-renew efficiently. Although several ECMs could also induce differentiation of stem cells by regulating C/EBPs, their effect was much weaker and may just work supportively for stem cell differentiation. Our present data show that gradual effects from HGF and OSM, mediated by the transcription factor C/EBP, lead stem cells to differentiate into hepatocytes rather than cholangiocytes through the efficient expansion of differentiating cells and permissive signals inducing the maturation of hepatocytes.
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MATERIALS AND METHODS |
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Isolation of total RNA
We prepared total RNA from test samples and, as RNA standards for real-time
PCR, from fetal, neonatal and adult liver, using an RNeasy Mini Kit (QIAGEN,
Tokyo, Japan) according to the manufacturer's instructions. Total RNA was
diluted and used for quantitative analyses.
Semi-quantitative RT-PCR analysis
Sorted 1x105 cells were used to prepare total RNA. After
various dilutions of template cDNA, we optimized their concentration for each
primer. In these concentrations, amplification by PCR did not reach plateau
but could be used semi-quantitative analysis. PCR was conducted using
hepatocyte-specific primers for albumin (ALB), -1-antitrypsin
(
AT), glucose-6-phosphatase (G6P), and tryptophan-2,3-dioxygenase (TO),
and for positive control hypoxanthine phosphoribosyltransferase (HPRT) as
described (Suzuki et al.,
2000
; Suzuki et al.,
2002
). PCR cycles were as follows: initial denaturation at
95°C for 4 minutes followed by 40 cycles of 94°C for 1 minute,
56°C for 1 minute, 72°C for 1 minute and final extension at 72°C
for 10 minutes. PCR products were separated in 2% agarose gel.
PCR primers and TaqMan fluorogenic probes
PCR primers and TaqMan fluorogenic probes for real-time quantitative PCR
were designed using the Primer Express software program (Version 1.0) (Applied
Biosystems, Tokyo, Japan). The sequences were as follows:
hepatocyte-differentiation primers for ALB (forward, 5'-TGT CCC CAA AGA
GTT TAA AGC TG-3'; reverse, 5'-TCT TAA TCT GCT TCT CCT TCT CTG
G-3'; and probe, 5'-ACC TTC ACC TTC CAC TCT GAT ATC TGC ACA
CT-3'), -fetoprotein (AFP) (forward, 5'-CCT GTC AAC TCT GGT
ATC AGC CA-3'; reverse, 5'-CTC AGA AAACTG GTG ATG CAT
AGC-3'; and probe, 5'-TGC TGC AAC TCT TCG TAT TCC AAC AGG
A-3'),
AT (forward, 5'-TCG GAG GCT GAC ATC CAC AA-3';
reverse, 5'-TCA ACT GCA GCT CAC TGT CTG G-3'; and probe,
5'-TTC CAA CAC CTC CTC CAA ACC CTC AA-3'), G6P (forward,
5'-GTT CAA CCT CGT CTT CAA GTG GAT-3'; reverse, 5'-TGC TGT
AGT AGT CGG T GT CCA GGA-3'; and probe, 5'-TTT GGA CAA CGC CCG TAT
TGG TGG-3') and TO (forward, 5'-CAA GGT GAT AGC TCG GAT
GCA-3'; reverse, 5'-TCC AGA ACC GAG AAC TGC TGT-3'; and
probe, 5'-TGT GGT GGT CAT CTT CAA GCT CCT GG-3');
cholangiocyte-differentiation primers for cytokeratin 19 (CK19) (forward,
5'-TGA AGA TCC GCG ACT GGT-3'; reverse, 5'-TAA AGT AGT GGT
TGT AAT CTC GGG A-3'; and probe, 5'-CCA GAA GCA GGG ACC CGG
ACC-3'), and
-glutamyltranspeptidase (GGT) (forward, 5'-TTT
GCC TAT GCC AAG AGG AC-3'; reverse, 5'-TTG CGG ATC ACC TGA GAC
A-3'; and probe, 5'-ATG CTC GGT GAC CCA AAG TTT GTC G-3');
or miscellaneous primers for hepatocyte nuclear factor 1 (HNF1) (forward,
5'-GCT AGG CTC CAA CCT TGT CAC G-3'; reverse, 5'-TTG TGC CGG
AAG GCT TCC T-3'; and probe, 5'-AGG TGC GTG TCT ACA ACT GGT TTG
CCA-3'), hepatocyte nuclear factor 4 (HNF4) (forward, 5'-TGG TGT
TTA AGG ACG TGC TGC-3'; reverse, 5'-ACG GCT CAT CTC CGC TAG
CT-3'; and probe, 5'-CAA TGA CTA CAT CGT CCC TCG GCA CTG
T-3'), hepatocyte nuclear factor 6 (HNF6) (forward, 5'-CCG GAG TTC
CAG CGC AT-3'; reverse, 5'-TCT TGC TCT TTC CGT TTG CA-3';
and probe, 5'-TCG GCG CTC CGC TTA GCA GC-3'), Met (forward,
5'-GAT CGT TCA ACC GGA TCA GAA-3'; reverse, 5'-GGA AGA GCC
CGG ATA ATA ACA A-3'; and probe, 5'-TGC AGG ATT GAT CAT TGG TGC
GGT C-3'), CCAAT/enhancer binding protein-alpha (C/EBP
) (forward,
5'-AGC AAC GAG TAC CGG GTA CG-3'; reverse, 5'-TTA TCT CGG
CTC TTG CGC A-3'; and probe, 5'-CGG GAA CGC AAC AAC ATC
GCG-3') and CCAAT/enhancer binding protein-ß (C/EBPß)
(forward, 5'-CGG ATC AAA CGT GGC TGA G-3'; reverse, 5'-CGC
AGG AAC ATC TTT AAG GTG A-3'; and probe, 5'-ACG TGT AAC TGT CTA
GCC GGG CCC TG-3'). All TaqMan probes used in this experiment carried a
5' FAM reporter dye (Applied Biosystems).
Real-time PCR conditions
The RT and the PCR were performed in one step by using TaqMan EZ RT-PCR
Core Reagents (Applied Biosystems). The reaction mixture (25 µl final
volume) includes 100 or 500 ng total RNA, 5xTaqMan EZ buffer (5 µl),
Mn(OAc)2 (3 mM), rTth DNA polymerase (0.1 U/µl), uracil
N-glycosylase (0.01 U/µl), dATP, dCTP, dGTP, dUTP (each 300 µM), and
forward and reverse primers (200 nM), and probe (100 nM). Reverse
transcription was performed at 60°C for 30 minutes. PCR was performed as
follows: initial denaturation at 95°C for 5 minutes followed by 60 cycles
of 95°C for 15 seconds and 60°C for 1 minutes. A template-free control
was included in each experiment. All template-free controls, standard RNA
dilutions and test samples were assayed in triplicate.
Analysis of real-time PCR data
The starting amount of mRNA in each test sample was calculated by preparing
a standard curve using known dilutions of RNA standards. For each dilution,
the ABI-PRISM 7700 software (Applied Biosystems) generated a real-time
amplification curve constructed by relating the fluorescence signal intensity
(DRn) to the cycle number. The Rn value corresponded to the variation
in the reporter fluorescence intensity before and after PCR, normalized to the
fluorescence of an internal passive reference present in the buffer solution.
The standard curve was then generated on the basis of the linear relationship
existing between the Ct value (cycle threshold; corresponding to the cycle
number at which a significant increase in the fluorescence signal was first
detected) and the logarithm of the starting quantity. Starting quantities of
mRNA in samples were quantified by plotting the Ct on this standard curve.
Gene transfer into hepatic stem cell cultures
Stable transfection of hepatic stem cell cultures was carried out by
lipofection. Briefly, 10 µg of a construct containing both the enhanced
green fluorescence protein (EGFP) driven by the ALB promoter (0.3 kb)
and enhancer, a region located 8.5-10.4 kb upstream of the ALB promoter
(Pinkert et al., 1987), and
the Zeocin resistance gene was used to transfect 1x106 cells
using Lipofectamine 2000 (Gibco BRL, Gaithersburg, MD). Stably transfected
cells were selected by growth on laminin-coated dishes (Becton Dickinson) in
our standard medium supplemented with 600 µg/ml Zeocin (Invitrogen,
Groningen, Netherlands) and isolated by using cloning rings (Iwaki Glass,
Tokyo, Japan). The frequency of EGFP-positive cells was assayed by
FACS-Vantage (Becton Dickinson). In this paper, representative data from a
transfected stem cell clone are shown because similar results were obtained
from others.
Retrovirus production and FACS analysis of transduced cells
The retroviral vector pGCsam (MSCV) is described elsewhere
(Kaneko et al., 2001). A
dominant-negative form of C/EBP (A-C/EBP) followed by IRES EGFP or IRES nerve
growth factor receptor truncated in the cytoplasmic domain (tNGFR) were
subcloned into pGCsam (GCsam-A-C/EBP-IRES-EGFP, GCsam-A-C/EBP-IRES-NGFR,
respectively). To produce recombinant retrovirus, plasmid DNA was transfected
into 293gp cells (293 cells containing the gag and pol genes
but lacking an envelope gene) along with 10A1 env expression plasmid
(pCL-10A1) (Miller et al., 1996) by CaPO4 co-precipitation, and
supernatant from the transfected cells was collected to infect cells. To
detect the expression of tNGFR on the cell surface, cells were stained by
mouse anti-human NGFR (Chemicon, Temecula, CA) followed by phycoerythrin
(PE)-conjugated rabbit anti-mouse Igs (Dako, Carpinteria, CA) and analyzed by
FACS-Vantage (Becton Dickinson).
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RESULTS |
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To examine direct roles for GFs and ECMs in stem cell differentiation, stem
cell clones were placed in several culture conditions and the state of
differentiation was analyzed by using FACS and quantitative PCR
(Fig. 1). In our stem cell
cultures, however, in addition to self-renewing stem cells, differentiating
progeny such as hepatocytes and cholangiocytes were spontaneously produced
from stem cells. Owing to this heterogeneity, the target cells for the effects
of GFs and ECMs could not be determined. To elucidate which steps of stem cell
differentiation were affected by differentiation-inducible factors, we
separated the original cell population into ALB and
ALB+ cells using FACS, following gene transfer of the ALB
enhancer/promoter-EGFP construct into stem cell cultures
(Fig. 1,
Fig. 2A). After FACS-sorting,
semi-quantitative RT-PCR analysis was conducted to compare the expression of
hepatocyte markers in EGFP+ (ALB+) cells with that in
EGFP (ALB) cells. As expected, the
expression of hepatocyte markers in ALB+ cells was much higher than
in ALB cells (Fig.
2B). These data clearly show that stem cell-derived
differentiating heaptocyte-lineage cells can be visualized and specifically
separated from other lineage cells. We next examined the expression of the
liver-enriched transcription factors in ALB and
ALB+ cells. Interestingly, although there was little difference in
the expression of HNF1 and HMF4 between ALB and
ALB+ cells in the result of real-time quantitative PCR analysis,
the expression of HNF6, which is known to be a regulator of pancreatic
endocrine cell differentiation (Jacquemin
et al., 2000), was much higher in ALB+ cells
(Fig. 2C). These results
suggest that HNF6 could be involved in hepatocyte-differentiation from hepatic
stem cells in the liver development.
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Regulation of growth and differentiation of purified
ALB and ALB+ cells by GFs and ECMs
To examine the effects of GFs and ECMs on ALB and
ALB+ cells, sorted cells were independently cultured in six-well
plates (1x104 cells/well) using several conditions and
examined 10 days later. Although single cell cultures of sorted cells required
ECMs and HGF similar to primary cultures, this high number of purified cells
allowed slow growth even without GFs and ECMs. For ALB
sorted cells, HGF strongly induced their proliferation. FGFs had a smaller
positive effect, while OSM, by contrast, inhibited their proliferation
(Fig. 3A, upper left graph).
For ALB+ sorted cells, extensive proliferation was also found when
cultured with HGF, but not with OSM and FGFs
(Fig. 3A, lower left graph).
The laminin, type IV collagen, and type I collagen were more effective on
ALB+ cells than ALB cells
(Fig. 3A, right graph). FACS
analysis of cultured cells indicated that induction of ALB+ cells
from ALB sorted cells was stimulated by HGF and OSM, and, to
a lesser extent, by laminin, type IV collagen and type I collagen
(Fig. 3B, upper graph). By
contrast, HGF and OSM strongly inhibited the generation of
ALB cells from ALB+ sorted cells. Laminin, type
IV collagen and type I collagen-coated dishes had similar inhibitory effects
on the generation of ALB cells
(Fig. 3B, lower graph).
Interestingly, aFGF and bFGF, which induce hepatogenesis in ventral endoderm
at E8 (Jung et al., 1999), had
little effect on stem cell growth and differentiation in our culture
system.
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The expression of C/EBPs changed dramatically during stem cell
differentiation
To reveal the transcriptional control of hepatocyte-differentiation from
stem cells, we first examined the effect of HGF on the expression of C/EBPs,
which are candidate key factors. C/EBP proteins comprise a family of
transcription factors that have a bZIP structure, consisting of a DNA binding
basic region and a leucine zipper dimerization domain (Lekstrom-Himes et al.,
1998). They directly control the expression of genes encoding
hepatocyte-specific proteins such as ALB and AT
(Costa et al., 1989
;
Maire et al., 1989
;
Trautwein et al., 1996
). The
expression of C/EBP
is particularly upregulated when hepatocytes shift
from proliferation to the differentiation state
(Rana et al., 1994
;
Runge et al., 1997
). In the
fetal liver of Cebpa/ mice, hepatocytes
exhibited biliary epithelial cell characteristics and many pseudoglandular
structures appeared, suggesting an involvement of C/EBP
in directing
differentiation of bipotent hepatic stem cells along the hepatocyte-lineage
(Tomizawa et al., 1998
). As
shown in Fig. 5A, the
expression of C/EBP
in an original stem cell population was stimulated,
in a dose-dependent manner, by HGF as well as laminin, type IV collagen and
type I collagen. The differentiation of hepatocytes, represented by the
expression of ALB,
AT and G6P, was also stimulated in stem cell
cultures by high concentrations of HGF in the presence of ECMs (data not
shown). By contrast, the expression of C/EBPß was decreased by these
culture conditions (Fig. 5A).
HGF and ECMs were found to have little affect on the expression of
C/EBP
and C/EBP
(data not shown).
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Lack of C/EBP proteins inhibits hepatocyte differentiation from stem
cells
Using a retroviral gene transfer system, we expressed C/EBP or
ß in stem cell cultures to examine their roles in differentiation.
Transduced cells, however, stopped proliferating and died within a few days
(data not shown). This suggested that too much C/EBP disrupted the homeostasis
of hepatic stem cells. Therefore, we transduced cells with the retroviral
vector GCsam-A-C/EBP-IRES-EGFP, which drives expression of both A-C/EBP and
EGFP (Iwama et al., 2002
),
which disrupts the function of C/EBP proteins. A-C/EBP, a dominant-negative
C/EBP that has the potential to antagonize all C/EBP members, is a 102 amino
acid protein consisting of an N-terminal 9 amino acid Flag epitope, a 13 amino
acid linker, a 31 amino acid designated acidic amphipathic helix, and a 49
amino acid leucine zipper domain of C/EBP
(Olive et al., 1996
). The
leucine zipper from A-C/EBP specifically interacts with endogenous C/EBP
leucine zippers, and the N-terminal acidic extension forms a coiled coil with
endogenous C/EBP basic regions. This heterodimeric coiled coil structure is
much more stable than C/EBP
bound to DNA, and thus, the
dominant-negative protein abolishes DNA binding of all endogenous C/EBP family
members.
After transduction, EGFP-positive cells (98.2±0.8%; n=3)
were sorted by FACS and then subjected to in vitro and in vivo assays. Stem
cells expressing A-C/EBP failed to express hepatocyte-differentiation markers
such as ALB, AFP, AT, G6P and TO, even when cultured with both HGF and
OSM, which normally induce hepatocyte differentiation
(Fig. 6A). By contrast, the
expression of CK19 and GGT, markers of cholangiocyte-differentiation, was
relatively enhanced in cells expressing A-C/EBP. The expression of the
transcription factors HNF1, HNF4 and Met was also activated by blocking C/EBP
function, but this change was not significant. Interestingly, the expression
of HNF6 was decreased in transduced cells, in a similar manner to other
hepatocyte-differentiation markers (Fig.
6B). Both ALB immunocytochemistry and PAS staining also revealed
that stem cells expressing A-C/EBP did not give rise to functionally mature
hepatocytes expressing ALB and containing abundant glycogen stores
(Fig. 6C). Instead of
differentiation along the hepatocyte-lineage, cultured cells expressing
A-C/EBP grew actively and formed a lot of large colonies including more than
100 cells in comparison with mock controls, suggesting activation or
maintenance of self-renewal status (Fig.
6D). Sorted ALB+ and ALB cells were
also transduced with the retroviral vector GCsam-A-C/EBP-IRES-NGFR and
analyzed for differentiation potential into hepatocyte-lineage cells. In
ALB+ cultured cells that expressed A-C/EBP
(ALB+/NGFR+ cells) after FACS sorting,
ALB cells emerged efficiently. Transduction of
ALB cells, by contrast, strongly inhibited the generation of
ALB+ cells (Fig.
6E). Taken together, these data indicate that the expression of
C/EBPs, induced by HGF, OSM and ECMs, is a key event for primary
differentiation of stem cells into bipotent precursors and hepatocyte-lineage
cells, and that disruption of this transcriptional regulation leads to the
maintenance of stem cell status, including self-renewal activity.
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DISCUSSION |
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In ALB cells generated from ALB+ precursors in
clonal cultures, both CK19+ cholangiocyte-lineage cells and
ALB CK19 cells that possess a stem cell
phenotype were identified (data not shown). These results suggest that ALB
expression early during the differentiation of stem cells is flexible and
cells can flow between stem cells and ALB+ hepatic precursors
before they obtain gradual signals for differentiation first from HGF and
secondarily from OSM. In hepatogenesis from the endoderm layer starting at E8,
FGFs produced by cardiac mesoderm play a key role in the generation of
ALB+ cells (Jung et al.,
1999). Our present data, however, showed that FGFs have much
smaller effects on the transition from ALB stem cells to
ALB+ cells. The E13.5 fetal mouse livers that we used for isolating
stem cells had already been apart from cardiac mesoderm, indicating that FGFs
should have finished their role in early liver development by this stage. A
few cells receiving no signals from cardiac mesoderm or other lining
mesenchymal cells in early hepatogenesis may be maintained in an
undifferentiated state until the E13.5 mid-fetal stage. FGFs may directly
induce the differentiation of hepatocytes and/or ALB+ hepatic
precursors from foregut endoderm, but HGF stimulates the differentiation of
dormant stem cells preserved in developing livers. A number of factors are
likely to work mutually as inducers or repressors of liver organogenesis based
on subtle timing.
C/EBP proteins regulate liver-specific gene expression and cell
proliferation (Costa et al.,
1989; Maire et al.,
1989
; Rana et al.,
1994
; Trautwein et al.,
1996
; Soriano et al.,
1998
; Greenbaum et al.,
1998
). In particular, C/EBP
is highly expressed in
quiescent hepatocytes and positively regulates hepatocyte-specific gene
expression, such as ALB and
At
(Costa et al., 1989
;
Maire et al., 1989
;
Rana et al., 1994
;
Runge et al., 1997
;
Soriano et al., 1998
). In the
developing liver of C/EBP
knockout mice, a number of pseudoglandular
structures that co-express antigens specific for hepatocytes and
cholangiocytes were found in the liver parenchyma, but the formation of bile
ducts was not affected (Tomizawa et al.,
1998
). These data demonstrated that C/EBP
is an important
regulator of hepatocyte differentiation, but not of cholangiocytes in either
liver development or regeneration. In the results presented here, C/EBP
expression was highly induced during the progression of hepatocyte
differentiation from ALB stem cells to ALB+
hepatic precursors by HGF. We suggest that HGF directly regulates the
expression of C/EBP
, which plays a crucial role in the transition of
stem cells to ALB+ hepatic precursors. C/EBPb is also a liver
enriched transcription factor (Descombes et
al., 1990
), which, similar to C/EBP
, is involved in the
regulation of liver-specific genes such as ALB
(Trautwein et al., 1996
). In
the transition from proliferating to differentiated hepatocytes, the
C/EBP
:C/EBPß ratio was found to be increased
(Runge et al., 1997
). In the
present study, during the induction of hepatocyte-differentiation by HGF, the
C/EBP
:C/EBPß ratio was also highly increased. These results
demonstrate that the relative proportions of C/EBP
and C/EBPß may
be important for hepatocyte differentiation from stem cells in the developing
liver.
Using knockout mice, several functions for C/EBPs in liver development have
been identified (Wang et al.,
1995; Soriano et al.,
1998
; Tomizawa et al.,
1998
). However, as redundancy exists among C/EBP family members,
their intrinsic roles in liver development remain to be clarified. For
example, liver cells in C/EBP
knockout mice could differentiate into
hepatocytes when they were cultured on Matrigel
(Soriano et al., 1998
),
suggesting that partial redundancy with other C/EBP proteins exists. To
eliminate these complicated interpretations, we used the dominant-negative
A-C/EBP to abolish endogenous DNA binding of all C/EBP family members. The
expression of A-C/EBP in ALB stem cells resulted in
semi-complete inhibition of the generation of hepatocyte-lineage cells, even
in cultures including both HGF and OSM. In addition, A-C/EBP expression in
ALB+ cells advanced the transition to ALB cells.
These findings, collectively, show that the C/EBP family, especially
C/EBP
and ß, which are directly regulated by HGF, are required for
the early steps in hepatic stem cell differentiation. In addition to
inhibiting differentiation, lack of all C/EBP functions in stem cells enhances
their self-renewal divisions in culture. In the developing liver, however, the
number of stem cells is very low and their proliferation is restricted, even
with the low expression of C/EBP proteins. Thus, other mechanisms may exist to
maintain their quiescent status in liver development.
A number of molecular events in the differentiation of hepatic stem cells,
such as the interaction of C/EBPs and other elements, are required for the
determination of hepatocyte or cholangiocyte lineage. C/EBP-mediated
growth arrest is known to require interaction with p21, a cyclin-dependent
kinase (CDK) inhibitor and CDK2 (Timchenko
et al., 1997
; Harris et al.,
2001
). This mechanism may be involved in OSM-mediated growth
arrest of hepatic precursors, in order to then induce differentiation into
mature hepatocytes. Because such growth inhibition is irrelevant to the
transcriptional activity of C/EBP
(Harris et al., 2001
), another
mechanism should exist to control transcription of genes important for liver
development. Actually, in the transcriptional control regions of ALB and
At, liver-enriched transcription factors such as HNF1
(Baumhueter et al., 1990
), HNF3
and HNF4 (Costa et al., 1989
),
and widely distributed proteins such as activator protein 1 (AP1) (Hu et al.,
1994) bind to regulate these genes along with C/EBP proteins. The currently
proposed cascade of sequential transcriptional control of hepatic stem cell
differentiation, however, is still unreliable. Our present results show that
HNF6 was expressed more highly in ALB+ cells than
ALB cells during the early differentiation of stem cells,
and HNF6 expression was also suppressed when stem cell differentiation was
inhibited by dominant-negative C/EBP proteins. The HNF6-binding sequence, in
fact, is present in the promoter regions of several hepatocyte-enriched genes,
such as
At, AFP, cytochrome P450, GLUT2 and TO (Samadani et al., 1996;
Tan et al., 2002
). Thus, HNF6
may be one possible candidate involved in the early transition of stem cells
to hepatic precursors, and its expression may be regulated by C/EBPs. In
Hnf6/ mice, abnormalities of the
intrahepatic and extrahepatic bile ducts and of the gallbladder were observed
(Clotman et al., 2002
). These
data suggest that HNF6 is essential for differentiation and maturation of
biliary lineage cells rather than hepatocytes. However, in the E13.5
developing liver of these mice a number of cytokeratin-positive biliary
lineage cells emerged compared to normal mice, suggesting that HNF6 regulates
not only morphogenesis of biliary tract but turning point of the
differentiation of primitive hepatic stem cells.
A precise description of stem cell differentiation would allow the control of hepatocyte differentiation and the induction of liver regeneration by manipulating the endogenous stem cell compartment. Our clonal culture assay with hepatic stem cells should reveal the mechanism that regulates their self-renewal potential and the signals that restrict their proliferation and differentiation in the developing liver. Exploring diverse gene programs activated in stem cells or differentiating cells should provide a molecular framework for future research into liver development. Key elements of stem cells, such as quiescent status and pluripotency, would be elucidated by comparing hepatic stem cells with other tissue-derived stem cells. The prospective isolation and characterization of stem cells is required for better understanding what exactly a stem cell is and what it does.
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ACKNOWLEDGMENTS |
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REFERENCES |
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