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Regular Articles |
§¶
From the Marion Bessin Liver Research Center,*
the Division of Gastroenterology, Hepatology and
Nutrition,
and the Departments of
Medicine,
Cell
Biology,§
and
Pathology,¶
Albert Einstein College of Medicine,
Bronx, New York; the Department of
Gastroenterology,||
Tel Aviv
Sourasky Medical Center, Tel Aviv, Israel; and the Istituto di
Patologia Sperimentale,**
Ospedale Oncologico "A
Businco", University of Cagliari, Cagliari, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein (AFP) and albumin, but not CK-19; cells
expressing CK-19, but not AFP or albumin, and cells
expressing AFP, albumin, and cytokeratins-19 (CK-19).
Proliferation, differentiation, and expansion of
transplanted FLEC differed significantly in the two models. In normal
liver, 1 to 2 weeks after transplantation, mainly cells
with a single phenotype, hepatocytic (expressing AFP and
albumin) or bile ductular (expressing only CK-19), had
proliferated. In Rs-treated rats, in which the proliferative
capacity of endogenous hepatocytes is impaired, transplanted
cells showed mainly a dual phenotype (expressing both AFP/albumin and
CK-19). One month after transplantation, DPPIV+
FLEC engrafted into the parenchyma exhibited an hepatocytic phenotype
and generated new hepatic cord structures. FLEC, localized in
the vicinity of bile ducts, exhibited a biliary epithelial
phenotype and formed new bile duct structures or were incorporated into
pre-existing bile ducts. In the absence of a proliferative
stimulus, ED 14 FLEC did not proliferate or differentiate. Our
results demonstrate that 14-day fetal liver contains lineage committed
(unipotential) and uncommitted (bipotential) progenitor cells
exerting different repopulating capacities, which are affected
by the proliferative status of the recipient liver and the host site
within the liver where the transplanted cells become engrafted. These
findings have important implications in future studies directed toward
liver repopulation and ex vivo gene
therapy.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein (AFP) and then
albumin.6,7
Following the expression of these and other
hepatic markers, including also cytokeratins (CKs), most authors
conclude that hepatoblasts are bipotential cells, capable of
differentiating along the hepatocytic or bile duct epithelial cell
lineage.7-11
At ED 15–16, the rat liver already contains
committed immature hepatocytes and bile duct epithelial
cells.7,8,12,13
In both rats and humans, embryonic
hepatoblasts in large vascular spaces also form primitive ductal
structures, which ultimately give rise to the intrahepatic bile
ducts.14-16
A number of transcription, signaling, and growth factors have been
identified that play an essential role in gut endoderm differentiation
and fetal liver development. These include factors that bind to the
GATA DNA sequence motif (GATA), signal transducers and activators of
transcription (STATs), hepatocyte nuclear factors (HNF)-3- and -ß,
HNF-4, HNF-6, and certain fibroblast growth factors
(FGFs).17-25
However, the mechanisms by which primitive
pluripotential endodermal cells undergo hepatic specification and how
bipotential hepatoblasts differentiate further into hepatocytes and
bile duct epithelium remain largely unknown.
Studies in the adult liver have also provided strong evidence for the existence of putative liver stem cells, ie, undifferentiated liver epithelial cells that can be activated to proliferate and differentiate into hepatocytes or bile duct epithelial cells.26-28 These cells are thought to reside within or adjacent to the canals of Hering. Unlike stem cells in other tissues, such as bone marrow, skin, and intestine, which undergo continuous renewal, liver stem-like cells are facultative; they comprise a quiescent compartment of dormant cells that is activated only if the regenerative capacity of hepatocytes is impaired. Attempts have been made to identify their counterpart in fetal liver,7,28-31 and it has been suggested that the dormant stem-like cells originate most probably from bipotential fetal liver epithelial progenitor cells.28,32,33
To explore the ability of fetal liver epithelial progenitor cells (FLEC) to proliferate and differentiate into hepatocytes (Hc) and bile duct epithelial cells (BDEC) and become incorporated into structural components of the liver lobule, we have used a cell transplantation approach to monitor the fate of these cells under different experimental conditions. Cells were transplanted into the liver of a syngeneic mutant Fischer 344 (F344) rat strain, deficient in the exopeptidase dipeptidyl-peptidase IV (DPPIV).34 Because this enzyme is expressed in both Hc and BDEC, the genetically DPPIV-deficient F344 rat is an excellent model to follow the proliferation, lobular distribution, and morphological appearance of transplanted wild-type (DPPIV+) hepatic cells.34-38
When normal liver is subjected to partial hepatectomy (PH), liver regeneration occurs through proliferation of pre-existing mature hepatocytes.39-40 However, when rats are treated with retrorsine (Rs), this pyrrolizidine alkaloid is taken up by hepatocytes and metabolized to a bioactive form, which alkylates cellular DNA. This interferes with cell cycle progression and leads to inability of hepatocytes to proliferate.41-43 In the present study, we used both normal and Rs-treated DPPIV- rats to follow the proliferation, lineage progression, and differentiation of transplanted ED 14 FLEC cells. This was evaluated by their morphological appearance, histochemical expression of DPPIV, and expression of markers specific for Hc or BDEC, using dual label immunohistochemistry and in situ hybridization (ISH). Our results demonstrate that FLEC are a heterogeneous population of cells with a single or dual phenotype (unipotential or bipotential) and that their lineage commitment and proliferative activity varies depending on the engraftment site and functional status of the host liver.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs and diaminobenzidine (DAB) were purchased from Sigma Chemical (St. Louis, MO). The Vectastain Elite ABC kit was from Vector Laboratories (Burlingame, CA). Rabbit anti-rat red blood cells IgG was from Rockland (Gilbertsville, PA). Radioactive 35S-UTP (SJ603) and CK-19 antibody (RPN 1165) were obtained from Amersham Life Science Products (Arlington Heights, IL). CK-14 antibody (NCL-LL002) was from Novocastra Laboratories (United States distributor, Vector Laboratories). OV-6 monoclonal antibody was a generous gift from Dr. S. Sell (Albany Medical College, Albany, NY). Digoxigenin RNA labeling mix and anti-digoxigenin-POD, Fab fragments were from Boehringer Mannheim (Indianapolis, IN). Autoradiographic emulsion, type NBT2, was purchased from Eastman Kodak Company (New Haven, CT). Dr. N. Fausto (University of Washington, Seattle, WA) kindly provided plasmid BAF700, used for synthesis of the fetal form of AFP mRNA riboprobe.
Animals and Animal Treatment
Normal Fischer rats (F344) were purchased from Charles River Laboratories (Wilmington, MA). Mutant DPPIV-deficient (DPPIV-) F344 rats were obtained from the Special Animal Core Facility of the Liver Research Center, Albert Einstein College of Medicine. All studies with animals were conducted under protocols approved by the Animal Care Use Committee of the Albert Einstein College of Medicine and were in accordance with National Institutes of Health guidelines. Rs treatment of the animals was as described previously.38 In all experiments, cell transplantation recipients were female DPPIV- F344 rats. For experiments in which cell transplantation recipients were treated with retrorsine, rats weighing 90 to 100 g were given two intraperitoneal injections of Rs, 2 weeks apart, each of 30 mg/kg body weight. One month after the second injection, animals were subjected either to two-thirds PH and transplantation or to transplantation without PH. For stimulation of cell proliferation with triiodothyronine (T3), 1 day before cell transplantation and every week thereafter, animals received subcutaneous injections of T3 (Sigma) at a dose of 400 µg/100 g body wt, for a total of four T3 injections.
Isolation of FLEC
Fourteen-day FLEC from DPPIV+ animals were isolated by a modification of the procedure of Sigal et al.12 In brief, fetal livers were placed in ice-cold modified Hanks’ balanced salt solution (HBSS; Gibco BRL, Grand Island, NY) without Ca2+, containing 0.8 mmol/L MgCl2 and 20 mmol/L HEPES, pH 7.4, and then triturated gently several times in modified HBSS containing 1 mmol/L EGTA. After centrifugation for 5 minutes at 450 x g at 4°C, the pellet was suspended in modified HBSS containing 0.2% collagenase, 0.07% DNase, and 1 mmol/L CaCl2. Digestion was carried out for 15 minutes at 37°C, with gentle trituration every 5 minutes. The reaction was stopped by adding an equal volume of modified HBSS containing 1 mmol/L EGTA and fetal bovine serum at a final concentration of 10%. The cell suspension was filtered through a 45-µm nylon mesh, and cells were collected by centrifugation as above. The cell pellet was washed twice with modified HBSS/0.1% bovine serum albumin, centrifuged, and suspended at a concentration of 107 cells/ml. The cell suspension was subjected to two rounds of panning with rabbit IgG against rat red blood cells (Rockland), as described.12
Transplantation of FLEC
A total of 1.5 to 3.0 x 106 cells in a volume of 0.5 ml (of which approximately 15% or 2.25 to 4.50 x 105 cells were judged to be FLEC by detection of AFP mRNA using ISH of small aliquots fixed to cytospin slides) were infused into the liver through the portal vein immediately after two-thirds PH. Control animals received only cell transplantation. Four to five animals, including control animals, were used for each time point. The proliferation and differentiation of FLEC in the liver of the recipients was analyzed 1, 2, and 4 weeks after cell transplantation. The livers of T3-treated animals were analyzed 4 weeks after cell transplantation.
Histochemical Detection of DPPIV, 
-Glutamyl
Transpeptidase (-GT), and Glucose-6 Phosphatase (G6-P)
To detect DPPIV+ transplanted cells in the
liver of DPPIV- F344 rats, histochemical
staining was carried out as described previously.37
-GT
was detected by the method of Rutenburg et al44
as
described previously45
and G-6P by the method of
Teusch,46
with modifications described
previously.47
Immunohistochemical Detection of CK-19, CK-14, and Oval Cell (OV)-6 Antigen
Immunohistochemical detection was performed after 10 minutes’ fixation in cold 4% paraformaldehyde (PFA) prepared in phosphate buffered saline (PBS). The slides were washed in PBS and then in PBS/0.1% Triton X-100. Endogenous peroxidase was blocked for 5 minutes with 5 mmol/L periodic acid, and the sections were washed for 30 minutes with 3 mmol/L sodium borohydride in PBS. Further blocking was performed according to instructions in the Vectastain ABC Elite kit (including biotin/avidin blocking). CK-19 antibody (RPN 1165) at a dilution of 1:10, CK-14 antibody (NCL-LL002) at a dilution of 1:20, and OV-6 antibody at a dilution of 1:100 were applied for 2 hours at room temperature. Biotinylated anti-mouse IgG (BA-2001) was used as a secondary antibody in combination with the Vectastain Elite ABC kit. Peroxidase activity was developed by diaminobenzidine (DAB) staining.
ISH and Dual Immunohistochemistry/ISH Labeling
ISH was conducted on frozen sections as described previously.45 ISH of cells collected on plus charged slides (Fisher Scientific, Springfield, NJ) by the cytospin method, including several additional steps before acetylation. In brief, after fixation, washing, and dehydration, the slides were rehydrated for 10 minutes in PBS/5 mmol/L MgCl2 and permeabilized for 20 minutes in 0.1% Triton X-100, prepared in the same buffer. The slides were then washed for 5 minutes with the same buffer, treated for 5 minutes at room temperature with 5 µg/ml of Proteinase K (in 0.1 mol/L Tris/HCl, pH 8, and 5 mmol/L EDTA), washed for 3 minutes with 0.2% glycine, fixed again for 5 minutes in PFA, washed with buffer, and acetylated.
Dual ISH labeling was performed with 35S-labeled rat albumin antisense riboprobe and digoxigenin-labeled AFP antisense riboprobe. For detection of digoxigenin-labeled RNA hybrids, anti-digoxigenin POD (Fab fragment) was applied and peroxidase activity revealed by DAB.45 The slides were dehydrated, dipped in autoradiographic emulsion (NBT2), and exposed for 1 week to detect autoradiographic grains representing albumin mRNA.
For combined (dual) immunohistochemistry and ISH labeling, frozen sections were first processed with CK-19 antibody, peroxidase activity developed with DAB, and ISH was then performed with 35S-labeled AFP or albumin antisense riboprobe as described previously.47 After washings and dehydration, the slides were exposed with autoradiographic emulsion for 1 to 3 days and stained with hematoxylin.
Screening for Y Chromosome Marker Sry in Female Rats Transplanted with FLEC
This procedure followed the protocol described by An et al.48 First, a Sry fragment was amplified from rat genomic DNA by polymerase chain reaction, using primers homologous to the mouse sry gene. The amplified product of 459 bp was cloned into pGEM T-Easy vector (Promega, Madison, WI). Screening for Y chromosome DNA in female livers transplanted with FLEC was carried out using rat Sry primers as follows: Primer 1, Rat Sry (5'-CATCGAAGGGTTAAAGTGCCA-3') and primer 2, Rat Sry-R (5'-ATAGTGTGTAGGTTGTTGTCC-3'). These primers amplify a stretch of 104 bp nested within the 459-bp fragment. Rat liver DNA from the recipient livers and control male and female rats was purified using the DNEasy kit (Qiagen Inc., Valencia, CA). Serial dilution of the DNA samples beginning with 50 ng of DNA per reaction were prepared in a 50-µl reaction mixture containing 2.5 mmol/L MgCl2, 0.4 µmol/L of each primer, 0.2 mmol/L of each dNTP, and 1 unit of Platinum Taq polymerase (Gibco BRL, Grand Island, NY). Amplification conditions included a 3-minute incubation at 94°C, followed by 32 cycles of 1 minute at 94°C, 1 minute at 58°C, and 1 minute at 72°C, and a final termination step of 7 minutes at 72°C. The product was resolved on a 2% agarose gel.
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Rat liver at ED 14 contains immature epithelial cells and a large
number of hematopoietic cells at different stages
of differentiation. We have analyzed cells isolated from ED
14–15 fetal liver for expression of AFP, albumin, G-6P, -GT, CK-19,
CK-14, OV-6, and DPPIV. The percentage of FLEC in the total cellular
suspension was approximately 15%, determined as the number of cells
expressing AFP mRNA (Figure 1A)
or
albumin mRNA (Figure 1B)
. None of the isolated ED 14 liver cells
expressed G-6P, CK-14, OV-6, or DPPIV (data not shown). However,
expression of -GT and CK-19 was clearly observed and increased on ED
15 (Figure 1, C and D)
.
|
.
Coexpression studies using immunohistochemistry for detection of CK-19
and ISH for AFP mRNA showed that most FLEC expressed only AFP mRNA
(Figure 2C)
, some cells expressed both AFP mRNA and CK-19 (Figure 2C)
,
and a third population of cells expressed only CK-19 (Figure 2D)
. Dual
label immunohistochemistry for CK-19 and ISH for albumin mRNA confirmed
this observation, as most FLEC expressed only albumin mRNA, some cells
expressed only CK-19, and a third, smaller group of cells coexpressed
albumin mRNA and CK-19 (Figure 2, E and F)
. Dual labeling of isolated
12-day fetal liver cells revealed the same heterogeneity (Figure 2, G and H)
.
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Proliferation and Differentiation of FLEC in the Liver of Adult Syngeneic Animals
To follow the proliferation and differentiation of FLEC, we transplanted isolated ED 14 cells into the liver of normal and Rs-treated rats subjected to two-thirds PH. Differentiation of the cells was monitored morphologically and phenotypically, using DPPIV expression to detect transplanted cells. DPPIV cannot be detected in the liver before ED 16–17 by enzyme histochemistry (our findings), immunoblot,49 or indirect immunofluorescence50 methods. Therefore, detection of this enzyme served as a marker for both proliferation and lineage progression of transplanted FLEC. In ED 16 liver and thereafter, fetal hepatocytes show diffuse membranous staining for DPPIV. However, in adult liver, hepatocytes show a distinctive and unique expression pattern for DPPIV; it is localized to the apical (bile canalicular) domain of the plasma membrane. On the other hand, mature BDEC still show diffuse membranous staining for DPPIV, so that these two phenotypically distinct liver epithelial cell types can be readily distinguished.
One week after transplantation of FLEC into the liver of normal adult
rats, cells scattered throughout the parenchyma were diffusely stained
for DPPIV (Figure 3A)
. This suggested
that the cells were not fully differentiated. Two weeks after
transplantation, cells in the parenchyma (zones 2 and 3) acquired an
hepatocytic morphology with canalicular expression of DPPIV (Figure 3B)
, whereas others in the regions of bile ducts (zone 1)
differentiated into biliary epithelial cells (Figure 3C)
. One month
after transplantation, larger clusters of DPPIV+
cells, on average 2–7 per cm2
in random tissue
sections, formed morphologically fully differentiated Hc (Figure 3D)
.
In addition, 1 to 2 clusters per cm2
exhibited a
fully differentiated bile duct epithelial cell morphology (data not
shown).
|
. Two
weeks after transplantation, large foci of DPPIV+
hepatocytes were already present in the liver parenchyma (Figure 4B)
,
and once again, DPPIV+ bile duct structures were
identified in portal regions (Figure 4C)
. One month after
transplantation of FLEC into Rs-treated rats, numerous foci of
DPPIV+ mature hepatocyte were observed
(50–120/cm2
in random tissue sections). These
foci were beginning to fuse into very large
DPPIV+ structures, taking over a substantial
portion of the liver parenchyma (Figure 4D)
. Although the number of
hepatocytic clusters increased considerably in Rs-treated compared to
untreated liver after PH and FLEC transplantation, the number of bile
duct structures remained unchanged (1–3/cm2
in
random tissue sections).
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-GT, or AFP mRNA. As shown in Figure 5
, DPPIV+
hepatocytes did not express CK-19 (Figure 5, A and B)
, -GT (Figure 5, C and D)
, or AFP mRNA (Figure 5, E and F)
, although proliferating
small epithelial cells in Rs-treated animals express all three of these
markers.47
The FLEC that differentiated into Hc were
functional and expressed very high levels of albumin mRNA (Figure 6, A and B)
, and G-6P (Figure 6, C and D)
; the latter is not expressed in ED 14 FLEC. Bile duct structures
formed by transplanted cells retained expression of CK-19 (not shown)
and also became positive for OV-6 (Figure 6, E and F)
, another marker
that is not expressed in ED 14 FLEC, but is expressed after ED 16 in
biliary epithelial lineage-committed cells, as well as in mature BDEC.
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Proliferation and Differentiation of Immature FLEC into Hc and BDEC Occurs Only in Liver Subjected to a Proliferative Stimulus
In experiments described above, we transplanted 14 day FLEC into normal or Rs-treated adult liver subjected to two-thirds PH. In control experiments, in which PH was not performed, we did not detect DPPIV+ cells (Hc or BDEC). In other experiments, animals were subjected to two-thirds PH 1 week after cell transplantation and were then kept for an additional 1, 2, or 4 weeks. All livers (those removed at the time of PH and the regenerated liver 1, 2, or 4 weeks after PH) were analyzed to detect DPPIV+ cells. Again, no DPPIV+ cells were found either in the liver removed at the time of PH (1 week after cell transplantation) or in the regenerated liver 1, 2, or 4 weeks after PH. Serial sections from the liver removed 1 week after cell transplantation were also analyzed for clusters of AFP mRNA expressing cells and results were again negative. From these data, we conclude that undifferentiated ED 14 FLEC do not proliferate and differentiate in quiescent liver.
That the proliferative status of the liver is crucial for the
proliferation and differentiation of transplanted cells was confirmed
by an experiment in which Rs-treated recipient animals received 4 doses
of triiodothyronine (T3) instead of PH (see Methods). Recently, we
reported that T3 is an alternate mitogen for transplanted adult
hepatocytes in Rs-treated liver.51
One month after
transplantation of FLEC, small clusters of DPPIV+
mature hepatocytes (Figure 7A)
or bile
duct structures (Figure 7B)
were detected in the liver of T3-treated
recipients. Although the proliferation of transplanted cells was modest
in T3-treated compared to PH treated rats (compare Figures 4D and 7A
),
this result showed clearly that the recipient liver needs to be
activated (subjected to a regenerative stimulus) for proliferation and
differentiation of immature fetal cells to occur.
|
, some male fetal cells remained in the
liver after transplantation in the absence of PH. However, their number
was substantially lower than that present in the liver after PH (Figure 8
, lanes 1–3 versus lanes 4–6). These cells did not
express DPPIV, suggesting that they did not undergo hepatocyte lineage
progression in the absence of a liver regenerative stimulus or,
alternatively, that they were not of epithelial origin.
|
To determine the proliferative capacity and lineage commitment of
the three subpopulations of FLEC described in Figure 2
, we studied
their differentiation in normal and Rs-treated regenerating liver. For
this purpose, we analyzed the recipient liver at early time points, ie,
1 and 2 weeks after cell transplantation, following expression and
coexpression of AFP mRNA and CK-19 in transplanted cells, which were
identified in serial sections by DPPIV enzyme activity.
One week after transplantation of FLEC into normal adult rat liver,
scattered DPPIV+ cells in the parenchyma were
usually AFP mRNA+ and
CK-19- (Figure 9, A and B)
. Very infrequently, we found DPPIV+
cells that expressed a dual phenotype, ie, they expressed AFP mRNA and
CK-19 (Figure 9, C and D)
. In the periportal region, transplanted
DPPIV+ cells also expressed CK-19 but did not
express AFP mRNA (Figure 9, E and F)
. The proportion of expanding cells
with a single or dual phenotype reflected their relative abundance in
the isolated FLEC preparations. Two weeks after transplantation, AFP
mRNA expression was still positive, but reduced, in transplanted cells
in the parenchyma, and the cells were negative for CK-19 (Figure 9, G and H)
. DPPIV+/CK-19+ BDEC
did not express AFP mRNA (data not shown).
|
. However, CK-19 expression
was lower in transplanted cells than in endogenous biliary epithelial
cells (see arrowhead in Figure 10B
). Other DPPIV+
clusters (Figure 10C)
were comprised of a mixed cell population
(CK-19+ and CK-19-)
expressing AFP mRNA (Figure 10D
, arrows) and a few clusters of
transplanted cells expressed only AFP mRNA (arrowhead). Expression of
AFP mRNA was higher in unipotential than in bipotential cells (see
Figure 10D
). Two weeks after cell transplantation, overall expression
of AFP mRNA was reduced, although AFP mRNA was still expressed in both
transplanted hepatocytes and biliary epithelium, with the signal being
weaker in the latter (Figure 10E and F)
. AFP mRNA expression in
CK-19+ hepatocytes in the parenchyma (Figure 10G and H
, arrow) was lower than in hepatocytes expressing only AFP mRNA
(Figure 10G and H
, arrowheads).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preferential Proliferation and Differentiation of Committed FLEC in Normal Liver and Uncommitted FLEC in Rs-Treated Liver
Studying the antigenic profile of ED 12 fetal liver cells, Hixson et al52 found three major subpopulations: one expressing only HBD.1, another expressing only OC3, and a third expressing both markers. The authors suggested that all ED 12 cells are transitional and bipotential, and that they originate from a common pre-ED 12 precursor. That hepatic tissue of ED 12 is composed of bipotential epithelial cells that give rise to Hc and BDEC also has been suggested by others studying the differential expression of cytokeratins, AFP, albumin, cell surface markers, and kinetics of appearance of liver-specific markers in the developing rat embryo,7-9,29,30 kinetics of increased expression of one marker and loss of another marker in human embryos,16 and differential expression of these markers in cell lines under the influence of different promoting agents.8,11,13
From our study, we cannot conclude that there is a specific precursor/product relationship between the different subpopulations of FLEC, as we have observed all three subpopulations from ED 12 up to birth (data not shown). However, our data strongly suggest that commitment toward the hepatocytic or bile duct lineage occurs either very early during formation of the liver diverticulum (before ED 12), or that there is not a single FLEC precursor, as CK-19+ cells not expressing AFP may have a separate developmental origin. As demonstrated by cell transplantation, a substantial proportion of ED 14 FLEC are already committed to one or the other lineage, hepatocytic (AFP+/albumin+) and bile ductular (CK-19+). In normal regenerating liver, most of the AFP+ transplanted cells, which were scattered throughout the parenchyma, did not express CK-19. CK-19+ transplanted cells were found specifically in zone 1 of the liver lobule, as part of bile duct structures. Since on the same sections we observed AFP-/CK-19+ BDEC and AFP+/CK-19- Hc, it is highly unlikely that the differences found in gene expression patterns are due to technical factors or that the cells have lost their dual phenotype 1 week after transplantation. (The expression of AFP in the neonatal liver decreases gradually and shuts off only after 4 weeks of age.)
The third subpopulation of FLEC has a dual phenotype (AFP+/albumin+ and CK-19+) and the cells behave like bipotential progenitors of Hc and BDEC. Our studies provide direct evidence for differentiation of epithelial progenitor cells with dual markers into Hc and BDEC after transplantation into the regenerating liver of Rs-treated animals. These cells exhibit a significantly higher proliferative capacity than endogenous liver cells, taking over approximately 20% of the liver mass within 1 month after cell transplantation and PH.
FLEC as a Source for Liver Cell Transplantation
The results in the present study demonstrate that immature FLEC in the environment of adult regenerating liver can proliferate, differentiate, and express genes characteristic of adult hepatocytes/bile duct epithelial cells. This strongly suggests the potential use of these cells for transplantation and ex vivo gene therapy. A few attempts have been made to transplant ED 18 and older fetal liver cells into the spleen or on solid supports implanted intraperitoneally.53-56 In all these cases, fetal hepatocytes engraft, proliferate to some extent, and perform liver-specific biochemical functions. Isolated fetal hepatocytes from late gestation, when transplanted intraportally into Nagase analbuminemic rats, engraft, expand, and give partial correction of serum albumin when an hepatic regenerative stimulus (portal branch ligation) is also applied.57 Several studies also report successful engraftment and differentiation of early fetal liver tissue or cell suspensions after transplantation into ectopic sites.7,35,58 However, engrafted liver tissue masses at ectopic sites do not expand very much, and it is unlikely that such limited liver transplantation will have broad therapeutic application.
The present study suggests that immature FLEC may represent a preferred source of hepatic cells for transplantation compared to adult hepatocytes for the following reasons: 1) FLEC are small (10–12 µm) and their intraporatal injection is better tolerated than transplantation of mature hepatocytes (20–35 µm); 2) the number of injected cells we have used for the current experiments is ~5 times lower than the number of adult hepatocytes used for liver repopulation at the same efficiency in our previous study;38 3) due to their small volume, FLEC are not trapped in the periportal region, where the highest concentration of transplanted adult hepatocytes is observed,59 and they move easily through the sinusoids, reaching zone 3 of the liver lobule. This increases the seeding and repopulating efficiency of the transplanted FLEC compared to hepatocytes; 4) immature FLEC possess sufficiently high proliferative capacity that they can repopulate the normal regenerating liver in the absence of Rs treatment; and finally, 5) FLEC differentiate morphologically and phenotypically into both mature hepatocytes and bile duct epithelial cells, which is not observed after hepatocyte transplantation. Since early fetal liver epithelial progenitor cells selectively proliferate in the normal liver in response to a regenerative stimulus (or hepatic parenchymal loss), they differentiate into mature hepatocytes and bile duct epithelial cells, and they become incorporated into the host liver lobule as part of normal hepatocytic cords and bile duct structures, this suggests that fetal liver cell transplantation represents an attractive method to restore functional liver tissue.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institutes of Health grants RO1 DK17609, RO1 DK56496 and P30 DK41296 (all to D. A. S.) and the Gail I. Zuckerman Foundation for Research in Chronic Liver Diseases of Children (to M. D. D.).
Accepted for publication February 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein messenger RNA expression in the retrorsine model for hepatocyte transplantation. Cancer Res 1998, 58:5825-5834This article has been cited by other articles:
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 P. A. Gruppuso, S.-W. Tsai, J. M. Boylan, and J. A. Sanders Hepatic translation control in the late-gestation fetal rat Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R558 - R567. [Abstract] [Full Text] [PDF]
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J. S. Sandhu, P. M. Petkov, M. D. Dabeva, and D. A. Shafritz Stem Cell Properties and Repopulation of the Rat Liver by Fetal Liver Epithelial Progenitor Cells Am. J. Pathol., October 1, 2001; 159(4): 1323 - 1334. [Abstract] [Full Text]
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 M. L. Dumble, B. Knight, E. A. Quail, and G. C. T. Yeoh Hepatoblast-like Cells Populate the Adult p53 Knockout Mouse Liver: Evidence for a Hyperproliferative Maturation-arrested Stem Cell Compartment Cell Growth Differ., May 1, 2001; 12(5): 223 - 231. [Abstract] [Full Text] [PDF]
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 H. Zulewski, E. J. Abraham, M. J. Gerlach, P. B. Daniel, W. Moritz, B. Müller, M. Vallejo, M. K. Thomas, and J. F. Habener Multipotential Nestin-Positive Stem Cells Isolated From Adult Pancreatic Islets Differentiate Ex Vivo Into Pancreatic Endocrine, Exocrine, and Hepatic Phenotypes Diabetes, March 1, 2001; 50(3): 521 - 533. [Abstract] [Full Text]
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 A. Suzuki, Y.-w. Zheng, S. Kaneko, M. Onodera, K. Fukao, H. Nakauchi, and H. Taniguchi Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver J. Cell Biol., January 7, 2002; 156(1): 173 - 184. [Abstract] [Full Text] [PDF]
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