1 Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park
Avenue, Bronx, NY 10461, USA
2 Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY 10461, USA
3 Cancer Research Center, Albert Einstein College of Medicine, 1300 Morris Park
Avenue, Bronx, NY 10461, USA
4 General Clinical Research Center, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY 10461, USA
Author for correspondence (e-mail: sanjvgupta{at}pol.net )
Accepted 12 April 2002
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Summary |
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Key words: Stem, Progenitor, Cells, Epithelial, Fetal, Human, Liver
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Introduction |
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The engraftment of transplanted cells in the liver drew significant
interest from the field of cell therapy
(Gupta et al., 1995;
Gupta et al., 1999
).
Transplanted cells regulate gene expression physiologically and can
proliferate in the liver (Gupta et al.,
1999b
; Grompe et al.,
1999
). However, despite the early promise of cell therapy
(Fox et al., 1998
), the
scarcity of donor human livers, the absence of proliferation in cultured
hepatocytes and the poor viability of hepatocytes after cryopreservation
impose restrictions. We hypothesized that use of fetal cells will overcome
these problems and help advance cell and gene therapy. Fetal liver cells are
largely diploid, whereas maturing hepatocytes exhibit increasing polyploidy,
which attenuates cell proliferation and eventually produces cell senescence
(Sigal et al., 1995a
;
Sigal et al., 1999
;
Gorla et al., 2001
).
Progenitor cells from the fetal rat liver can generate both hepatocytes and
bile duct cells in animals (Sigal et al.,
1995b
; Sandhu et al.,
2001
). These considerations suggested to us that the fetal
humanliver will be an appropriate source of stem/progenitor cells. The human
liver arises from the foregut endoderm after four weeks of gestation and
develops rapidly, such that bile is produced by 14 weeks. During this
gestational period, hepatic cells express hepatocyte markers, for example,
albumin, alphafetoprotein (AFP),
-1 microglobulin, glycogen,
glucose-6-phosphatase (G-6-P) and Hep-Par-1, and biliary markers, for example,
gamma glutamyl transpeptidase (GGT), dipeptidyl peptidase IV (DPPIV),
cytokeratin (CK)-19 and Das-1-monoclonal antibody-reactive antigen,
(Haruna et al., 1996
;
Badve et al., 2000
). Human
hepatoblasts express these markers throughout the second trimester (20-24
weeks), despite significant development of the fetal liver, which offered
opportunities to isolate and study large numbers of progenitor cells. Here we
demonstrate the survival of progenitor cells in long-term cultures, which
indicates that similar opportunities to investigate cellular mechanisms could
be developed by isolating progenitor cells from additional fetal organs.
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Materials and Methods |
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Cell isolation and culture
The liver was irrigated at 37°C with sterile buffer A (10 mM HEPES, 3
mM KCl, 130 mM NaCl, 1 mM NaH2PO4-H2O and 10
mM glucose, pH 7.4; unless specified chemicals were from Sigma Chemical Co.,
St. Louis, MO), followed by buffer B (5 mM CaCl2, 0.03%
collagenase, from Worthington Biochemical Corp., Lakewood, NJ). Liver was next
passed through wide bore syringe and incubated in buffer B for 30-40 minutes
at 37°C. Dissociated cells were collected periodically at 4°C, passed
through 80 µm dacron mesh, washed twice in buffer A and pelleted by
centrifugation at 500 g for 5 minutes at 4°C. The cell
pellet was resuspended in Dulbecco's minimal essential medium with 5 µg/ml
insulin, 5 µM hydrocortisone, 100 U/ml penicillin, 100 µg/ml
streptomycin and 250 ng/ml amphotericin B (DMEM; Life Technologies Inc.,
Rockville, MD). Cell viability and number were determined with 0.2% trypan
blue dye using Neubauer hemocytometer. 4x103
cells/cm2 tissue culture plastic were incubated with DMEM
containing 10% fetal bovine serum (FBS, Atlanta Biologicals Inc., Norcross,
GA) in 5% CO2. The medium was changed daily for 3 days after
removing non-adherent cells with phosphate buffered saline, pH 7.4 (PBS);
subsequently the media was changed every 3 days. Loosely adherent cells were
removed with trypsin-EDTA diluted 1:10 in PBS for 8-10 min at 37°C on each
occasion. For subpassaging, near-confluent cultures were split 1:3 using
half-strength trypsin-EDTA for 6-10 minutes at 37°C.
Cryopreservation
1x106 cells were frozen per 200 µl freezing mixture
(University of Wisconsin solution, FBS and dimethylsulfoxide in 7:2:1 ratios
(v/v), respectively) in cryofreezing containers (Nalge Nunc International,
Rochester, NY). Cells were stored at -80°C for one day and transferred to
liquid nitrogen.
Clonogenic assays
Autologous fetal liver cells from various cell cultures were irradiated to
80 Gray and attached overnight to tissue culture plastic at
3x104 cells/cm2 with 60 µl/cm2 of
rat-tail collagen overlayed for 4-6 hours. Collagen was extracted from the
tail of several F344 rats. The tails were broken at joints and tendons were
pulled out with a hemostat, disrupted and exposed to ultraviolet light for 24
hours. To solubilize, 1 g tendon was stirred for 48 hours in 300 ml acetic
acid (diluted 1:1000) followed by filtration through sterile cheesecloth and
storage at 4°C. Test cells in DMEM were placed on top of the feeder
cell-collagen overlay under limiting dilutions, and medium was replaced
regularly for 3 weeks. Colonies were stained with crystal violet and glycogen,
G-6-P, GGT, and DPPIV activities were analyzed histochemically, as described
previously (Ott et al.,
1999b).
Cell proliferation
Doubling times were determined in 1x104 cells plated per
cm2 under triplicate conditions followed 1, 3, 5 and 7 days later
by analysis of cell numbers during exponential cell growth, as recommended
previously (Wieder, 1999).
Cell ploidy was analyzed by flow cytometry, senescence-associated
ß-galactosidase (SABG) activity by histochemistry and p21 expression by
immunostaining, as described previously
(Sigal et al., 1999
;
Gorla et al., 2001
). Growth
factor responses were tested in 1x105 cells per 35 mm dish.
F344 rat hepatocytes were isolated by standard collagenase perfusion. Growth
factors were 10 ng/ml hHGF (Genentech Inc., South San Francisco, CA) and 20
ng/ml TGF-
and EGF (Sigma). 1 µCi [3H]thymidine was added
after 47 hours for 1 hour, and 3H-thymidine incorporation into DNA
was measured as described previously
(Gorla et al., 2001
).
Telomerase content
A commercial PCR TRAP assay was used according to the manufacturer's
instructions (Roche Molecular Biochemicals, Indianapolis, IN). HepG2 cells
served as positive controls. 2x105 cells were lysed in 200
µl, and 3 µl of the extract, corresponding to 3,000 cells, was used. All
reactions were in triplicate and repeated twice. The extracts were heated for
15 minutes at 65°C for inactivating telomerase activity.
Cytogenetic analysis
Cells under 50-60% confluency were exposed to 4 µg/ml colchicine for 12
hours, lysed in 0.075 M KCl for 30 minutes at room temperature, pelleted by
centrifugation and fixed in methanol and glacial acetic acid (3:1 v/v) for 20
minutes at 4°C. Metaphases were prepared on glass slides and stained with
Giemsa according to standard procedures. Image analysis used the Cytovision
Software (Applied Imaging, Santa Clara, CA).
Gene expression analysis
We used anti-human albumin (A6684, HSA-11 clone, Sigma), -1
antitrypsin (BioGenex Corp., San Ramon, CA), CK-19 (RPN 1165, Amersham
Pharmacia Biotech Inc., Piscataway, NJ), CK-8 (Vector Labs. Inc., Burlingame,
CA) and AFP (Sigma), orosomucoid, and plasminogen activator inhibitor, type-1
(PAI-1) (Accurate Chemical and Scientific Corp., Westbury, NY). Flow cytometry
used ethanol-fixed cells suspended in 1% bovine serum albumin (BSA) in PBS.
Primary antibodies were diluted 1:10 in DIFTAGS (Shandon Lipshaw, Pittsburgh,
PA) and then 1:100 in PBS with 2% goat serum and 1% BSA. Anti-albumin was
diluted in PBS alone. Cells were stained for 2 hours at 4°C and analyzed
by FITC-conjugated antibodies using FACScan (Becton Dickinson). For western
blotting, cells were lysed in 50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS and 0.1%
Triton-X at 4°C. Proteins in supernatant were measured by Bio-Rad assay
(Cambridge, MA) and resolved by 8-12% SDS-PAGE. PVDF membrane (Amersham) blots
were blocked with 5% non-fat milk in Tris-buffered saline containing Tween-20;
except for CK-19, which used 1% BSA in PBS-Tween-20. After primary antibody
incubation, a peroxidase-conjugated secondary antibody was used for enzymatic
chemiluminiscence (Amersham). For demonstrating novel proteins, cells were
pulsed with 1 µCi 35S-cysteine and 35S-methionine for
30 minutes and serum-free DMEM was chased for one hour. Cellular proteins were
extracted with 10% trichloroacetic acid, solubilized in sodium hydroxide,
resolved in 7.5% SDS-PAGE followed by autoradiography. Relevant bands excised
from Coomassie blue stained gels were analyzed by peptide mass
spectroscopy.
Gene transfer studies
The Adßgal vector was from the Genetic Engineering Core (Albert
Einstein College of Medicine, Bronx, NY) and recombinant retrovirus and
lentivirus were used as described previously
(Gagandeep et al., 1999;
Ott et al., 1998
;
Zahler et al., 2000
).
Transient transfections with luciferase plasmids expressing hepatitis B virus
(HBV) enhancer I and preS1 promoter or simian virus (SV)40 enhancer and
promoter were as described before (Ott et
al., 1999a
).
Cell transplantation
Cells passaged 3, 5 and 8 times were attached to microcarrier beads
(Cytodex 3TM, Amersham Pharmacia) by incubating 1x106
cells per 1 ml of swollen beads overnight at 37°C. 1x106
cells from three separate livers were injected intraperitoneally (i.p.),
subcutaneously (s.c.) or intrasplenically into severe combined
immunodeficiency mice in the Balb/c background (SCID) as described earlier
(Gupta et al., 1999a). Ten
days after intrasplenic cell transplantation, one group of mice (n=7)
was treated with three doses at 10 day intervals of 1.45 ml/kg carbon
tetrachloride in mineral oil (1:1 v/v).
Immunohistochemistry
Glycogen, DPPIV, GGT and G-6-P activities were stained as described
previously (Ott et al.,
1999b). To colocalize glycogen and CK-19, cells were fixed with 4%
paraformaldehyde and endogenous peroxidase, then quenched with 3%
H2O2 in methanol for 30 minutes. After glycogen staining
(24), cells were blocked with 10% goat serum for 30 minutes at 37°C and
incubated with 20 nanogram CK-19 antibody (A53-B/A2, Santa Cruz
Biotechnologies, Santa Cruz, CA) for 90 minutes at 37°C. Antibody binding
was localized by biotinylated goat anti-mouse IgG (Sigma) using the
avidin-biotin complex (Vector) and diaminobenzidine (DAKO). Cryosections were
probed with anti-human albumin (Sigma) and
-1 microglobulin after
blocking peroxidase with Power BlockTM (BioGenex), and antibody binding
was detected with a peroxidase system.
In situ hybridization
5 µm cryosections were fixed in paraformaldehyde, and paraffin-embedded
sections were dewaxed. Slides were rinsed in 2xSSC for 30 minutes at
37°C, denatured in 70% formamide for 2 minutes at 80°C and hybridized
with a digoxigenin-labeled total human DNA probe (Oncor, P5080-DG.5, Vysis
Inc., Downers Grove, IL) after denaturing for 5 minutes at 80°C.
Hybridization was performed overnight at 37°C followed by washes in 50%
formamide and 2xSSC. Sections were incubated with
alkaline-phosphatase-conjugated anti-digoxigenin (Roche, 1093274) for 1 hour
at room temperature, and the color was developed with either BCIP/NBT or Fast
Red substrate (Sigma B5655 and F4523, respectively). Bile canalicular ATPase
and G-6-P were colocalized histochemically as described previously
(Gupta et al., 1995;
Gupta et al., 1999
). To
determine the number of transplanted cells in the recipient liver, tissues
subjected to in situ hybridization were analyzed by morphometric methods. The
liver volume of several mice was determined. The number of transplanted cells
was counted in defined areas of liver sections and converted into
cells/mm3, as the tissue thickness was already known
(Rajvanshi et al., 1996
). To
estimate transplanted cell fractions surviving in the mouse liver after
various intervals, we used the number of transplanted cells 2 hours after cell
transplantation as the denominator.
Electron microscopy
Tissues were fixed in 2.5% glutaraldehyde in cacodylate buffer, post-fixed
in osmium tetroxide and stained with 1% uranyl acetate before embedding in
plastic. Ultrathin sections were examined under a Phillips transmission
microscope.
Statistics
Data are expressed as means±s.d. The significance was analyzed by
the Student's t-test or Mann-Whitney rank correlation tests with the
SigmaStat software (Jandel Scientific, San Rafael, CA). P values <0.05 were
considered significant.
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Results |
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|
Fetal progenitor liver cells exhibited unique morphological and gene
expression profiles
Early characterization of the initial cell isolate showed that virtually
half of the cells expressed the biliary marker, GGT, along with hepatocyte
markers G-6-P, glycogen and DPPIV (Fig.
1A). These cells showed typical epithelial morphology. During
prolonged primary culture, non-adherent or loosely adherent cells, including
hematopoietic cells, became depleted. In this situation over 2 to 4 weeks,
epithelial cells proliferated in dishes with copious cytoplasm, forming a
prominent rounded or oval shaped nuclei and complex cytoplasmic organization
on ultrastructural analysis (Fig.
1B). These cells exhibited a tendency to form heaps and retained
expression of hepatocytic genes, such as G-6-P and glycogen, and biliary
genes, such as DPPIV and GGT (Fig.
1C). Remarkably, during serial subpassaging in long-term culture
over several months, cell morphology altered from an obviously hepatocyte-like
shape in primary cultures to cells that were flatter and spindlier. These
findings were observed in cells isolated from different livers and from cells
cultured at different times following cryopreservation from the same liver. To
determine whether liver-type function was still expressed in these subpassaged
cells, we initially analyzed in situ expression of liver genes. These studies
demonstrated that despite 2, 3, 5, 8 or more passages, fetal cells expressed
G-6-P, GGT, glycogen and DPPIV in patterns similar to cells in primary
cultures (Fig. 1D). Expression
of hepatocyte (glycogen, G-6-P) and biliary markers (GGT, DPPIV) in large
number of these cells suggested that many cells were coexpressing these
markers, which is in agreement with bilineage gene expression. To verify this
possibility, coexpression of glycogen and CK-19, which is expressed in mature
bile duct cells, was examined in cultured cells
(Fig. 1E,F). These experiments
showed that 5-10% of cultured cells coexpressed glycogen and CK-19. The
prevalence of cells in these cultures with expression of additional biliary
markers, such as GGT and DPPIV, was far greater (see below), which presumably
was in agreement with the usual expression of CK-19 only in mature bile duct
cells.
|
To further analyze gene expression in cultured cells, western blots were
used (Fig. 2). Primary cells
expressed genes observed in hepatocytes, for example, albumin, AFP, ASGR,
orosomucoid and -1 microglobulin, as well as biliary genes, for
example, CK-19. Moreover, cells displayed additional markers found in
hepatoblasts and oval cells, such as CK-8 and PAI-1. The presence of PAI-1 in
our cells was suggested initially by metabolic labeling with [35S].
This showed abundant expression of a
43 kDa protein in cell lysates and
medium. Peptide mass spectroscopy and western blotting verified that the
protein was PAI-1 (Fig. 2C).
The overall pattern of how various liver genes were expressed in cultured
cells is summarized in Table 2.
It is noteworthy that most markers were expressed during long-term culture of
cells, although the overall magnitude of gene expression altered somewhat in
late cell passages.
|
|
Analysis of cell proliferation and selected
cell-senescence-associated parameters
Analysis of subpassaging capacity showed that the cells could be
subpassaged repeatedly, up to 15-16 times over 8-10 months. To determine the
fraction of cells capable of clonogenic growth, we studied colony formation
under limiting dilution conditions after depleting non-adherent cells over 3
weeks in P0 cultures and subpassaging cells once (P1). The studies utilized
cells from two fetal livers (#7899 and #12700). When cells were plated on
tissue culture plastic alone, no cell colonies formed. However, after fetal
cells were plated on top of irradiated autologous cells (the same cells as
those being tested), which served as feeders, single cell colonies appeared
with efficiencies ranging from 5 to 30%
(Fig. 3A). These cell colonies
exhibited the properties of the original cells plated, as shown by liver gene
expression, with the presence of G-6-P, glycogen, DPPIV and GGT in substantial
proportions of cells (>80-90%). As our culture conditions were devoid of
supplemental growth factors, this clonogenic ability presumably reflected
either intrinsic properties of cells or release of paracrine growth factors in
culture conditions. To examine whether cultured cells were responsive to
supplemental growth factors, we studied HGF, EGF and TGF, which are
hepatic growth factors, in primary (P0) fetal cells and fetal cells
subpassaged three (P3) or 10 times (P10) (donor livers were #21198, #32598 and
#7899). Primary rat hepatocytes were used as controls to establish growth
factor activity in DNA synthesis assays. After exposure to HGF, TGF
and
EGF, rat hepatocytes showed four- to 12-fold greater DNA synthesis,
P<0.001 (Fig. 3B).
In contrast, DNA synthesis was unchanged in P0 fetal cells exposed to these
growth factors. However, subpassaged progenitor cells were responsive to
TGF
and to EGF, but not to HGF, with up to 2.4-fold greater DNA
synthesis, P<0.01. These findings were verified in an additional
experiment and were in agreement with the alteration in either cell autonomous
behavior or composition of cell cultures with respect to the release of
specific growth factors by other cells.
|
Analysis of cells isolated from two separate livers showed that
cell-doubling times ranged from 59-70 hours during extensive subpassaging
of cells for up to 12 passages, with longer doubling times observed in late
passages (donor livers #9899 and 12700). The number of cells recovered from
confluent dishes remained virtually constant during long-term culture,
although after plating 5x105 cells per cm2 in 100
mm cell culture dishes, early passages required 2-3 weeks, and later passages
required 6-8 weeks for cells to become confluent in culture dishes.
Nonetheless, we did not observe a greater prevalence of cells with polyploidy,
immunostainable p21 expression or appearance of SABG in late cell passages.
Moreover, we found telomerase activity to be two- to five-fold above controls,
P<0.01, in fetal cells. Telomerase activity remained detectable,
despite 16 subpassages (in cells isolated from liver #43098), which represent
approximately 50 or more population doublings. Detailed cytogenetic analysis
of primary (P0), intermediate (P3-5) and late passages (P10-13) showed cells
with only normal chromosomal complement and structures. These findings
suggested that our cell isolation and culture procedures provided genetically
normal cell populations with extensive replication capacity.
Also, it was noteworthy that our cells were highly viable following release from culture dishes and serial subpassaging, including, after repeated cryopreservation, >80% of cells attaching to culture dishes following thawing and producing long-term cultures (Table 3).
|
Differentiation of fetal cells into mature hepatocytes
To establish the differentiation potential of progenitor cells in vivo,
cells were first transplanted intraperitoneally in SCID mice. To localize
transplanted cells in the peritoneal cavity, cells subpassaged 3, 5 and 8
times from two separate livers (#21198, #32598) were attached to microcarrier
beads. Within 3 weeks, vascularized conglomerates developed and transplanted
fetal cells that formed confluent masses
(Fig. 4A). The presence of
transplanted cells was verified by in situ hybridization using a
human-specific DNA probe that visualized cell nuclei
(Fig. 4B,C). Transplanted cells
expressed G-6-P, glycogen, -1 microglobulin and albumin
(Fig. 4D-G). Electron
microscopy of microcarriers recovered after 3 weeks showed characteristic
hepatocytic morphology in transplanted cells, with microvilli on the apical
surface, rounded nuclei, prominent nucleoli and bile canaliculi
(Fig. 4H).
|
Also, no tumors were formed in scid mice 3 (n=6) and 5 months (n=4) after s.c. or i.p. injection of P8 subpassaged cells from these two livers, which was again in agreement with the absence of cell transformation under our culture conditions.
Progenitor cells expressed introduced genes and engrafted into the
mouse liver
Incubation of cells with Adßgal-containing bacterial ß
galactosidase (LacZ) gene transduced 100% cells. The use of amphotropic
retrovirus or lentivirus vectors to express lacZ or green fluorescence
protein, respectively, transduced 5-40% cells, with gene expression during
five subsequent subpassages. The cells correctly regulated transfected HBV
sequences (luciferase expression versus mock-treated cells was three- to
four-fold greater, P<0.05), although SV40 sequences showed eight-
to 16-fold greater luciferase activity, P<0.05.
After intrasplenic transplantation, primary P0 cells, as well as P5 and P8 subpassaged cells, survived in the SCID mouse liver. As expected, transplanted cells were in portal spaces 1 hour and 1 day after cell transplantation. Subsequently, transplanted cells entered liver plates and became integrated in the liver parenchyma (Fig. 5A-E). Moreover, transplanted cells showed proliferative capacity in animals treated with CCl4 (Fig. 5F,G). Detailed morphometric analysis of cell engraftment in multiple animals showed that transplanted cells constituted 1.5% of the mouse liver after 1 hour (Table 4). Subsequently, 14 to 55% of these transplanted cells engrafted in the liver, which reconstituted <1% of the host mouse liver. However, after CCl4, transplanted cells proliferated and their numbers increased significantly (control animals, n=3, 6.3±4.2x103 cells versus 1.6±1.1x104 cells per liver in CCl4-treated mice, n=7, p=0.02, t-test). This represented up to approximately an approximately five-fold increase in transplanted cell numbers, which was in agreement with more than one round of cell division in transplanted cells following CCl4 injury.
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Discussion |
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Replication of progenitor cells in culture, with retention of
differentiation capacity, would be helpful in a variety of biological studies.
Our progenitor cells were obviously not derived from single cells.
Nonetheless, these cells showed significant clonogenic capacity, which
indicates that single-cell-derived colonies can possibly be expanded for
further analysis. It is noteworthy that the normal adult liver, which contains
replicatively quiescent cells, is devoid of telomerase activity
(Tahara et al., 1995). By
contrast, our cells expressed telomerase, which could be beneficial for
continued cell division (Kobayashi et al.,
2000
). It was noteworthy that after
50 population doublings,
our cells showed decreasing proliferation. By contrast, mature hepatocytes are
difficult to maintain and expand in cell culture (Reid and Jefferson, 1994;
Block et al., 1996
;
Runge et al., 2000
). We did
not incorporate hepatic growth factors (except those present in fetal bovine
serum), lipids and extracellular matrix components, which are required for
culturing mature hepatocytes (Reid and Jefferson, 1994). Also, unlike murine
embryonic liver cells, our progenitor cells survived and proliferated without
requiring feeder cells (Rogler,
1997
). Specific manipulations, including the release of cells with
low trypsin/EDTA concentrations, were aimed at limiting cell membrane injury
and selective removal of loosely adherent cells
(Herring et al., 1983
). It is
noteworthy that hydrocortisone inhibits proliferation of fibroblasts and
erythroid/granulocyte-macrophage hematopoietic progenitor cells, whereas
insulin promotes hepatocyte attachment
(Hoshi et al., 1987
;
Papoff et al., 1998
); these
observations were reflected in our culture conditions. Although mature
hepatocytes dedifferentiate in culture with rapid loss of tissue-specific
genes, such as albumin, ASGR, etc., (Reid and Jefferson, 1994), our fetal
epithelial cells expressed liver genes despite extensive culture. Moreover,
our cells correctly regulated HBV enhancer/promoter, which requires the
presence of multiple hepatic transcription factors
(Ott et al., 1999a
).
Furthermore, the mitogenic responsiveness of our progenitor cells to
TGF
and EGF was in agreement with oval cell responses, as shown
previously with F344 rat-derived cells
(Gupta et al., 1992
).
Although indefinite cell replication has been induced in somatic cells by
expressing the SV40 T antigen or the catalytic subunit of telomerase, it is
unresolved whether genetic transformation will induce greater susceptibility
for cancer (Kobayashi et al.,
2000; Farwell et al.,
2000
). By contrast, our progenitor cells were genetically
unperturbed despite more than 40 to 50 population doublings over 16-18
subpassages. Such proliferation capacity in our cells indicates that cells
isolated from a single fetal liver could potentially generate billions or even
trillions of cells; whereas only 1-10 billion hepatocytes are required for
treating an adult person and proportionately fewer cells will be necessary for
treating a child. Therefore, expansion of progenitor cells in culture will
facilitate novel clinical applications and help alleviate organ shortages. If
highly efficient permanent gene transfer, such as those using lentiviral or
retroviral vectors, were combined with effective strategies to repopulate the
liver extensively, ex vivo liver gene therapy will once again become
attractive. In this context, integration and differentiation of our fetal
liver cells in the parenchyma of the mouse liver indicate that use of such
cells will be appropriate for liver repopulation. Our data shown here indicate
that significant proportions of transplanted cells were lost in mice shortly
after transplantation. These findings were not surprising because a large
fraction of transplanted cells sequestered in portal vein radicles and hepatic
sinusoids undergoes phagocytotic clearance, even in syngeneic recipients
(Gupta et al., 1999a
). These
cell losses constitute removal of approximately 70-80% of all transplanted
cells, as also observed in our studies shown here. However, it is unclear at
present whether human cells are at a survival disadvantage in the mouse liver
compared with rodent cells. Interspecies differences in growth factors,
extracellular matrix components, cell-cell interactions or other factors,
could potentially regulate survival of human hepatocytes in the mouse liver.
Of course, these findings do not exclude the possibility that our cells will
show far superior engraftment in the human liver. Nonetheless, it will be of
great interest to establish how engraftment of human cells in the mouse liver
may be improved, because this will be relevant for developing novel models of
human disease, as well as establishing reproducible bioassays to test the
properties of human hepatocytes before use in cell or gene therapy. In
addition, under suitable situations, transplanted hepatocytes proliferate
significantly in rodents, and the mouse liver can be repopulated virtually
completely with transplanted cells (Grompe
et al., 1999
). In this respect, proliferation of our fetal cells
in the mouse liver following CCl4-induced hepatotoxicity was in
agreement with the properties of rodent hepatocytes
(Gupta et al., 1999a
).
Therefore, the clinical implications of our findings should be obvious for
cell and gene therapy, especially when coupled with our data showing excellent
recovery of cells following cryopreservation, which should greatly facilitate
banking of cells for use at short notice.
The intraperitoneal bioassay used here was effective in demonstrating differentiation of progenitor cells into hepatocytes. This in vivo assay should be helpful for analyzing progenitor cell subpopulations, including analyzing progenitor cells for quality controls prior to clinical use. The availability of human progenitor cells capable of extensive proliferation, such as ours, will facilitate development of bioartificial liver (BAL) devices, which are being tested for liver failure, but are limited to porcine hepatocytes or less effective cell lines. Seeding of BAL devices with primary adult hepatocytes has been limited by their inability to proliferate. Additional applications of human progenitor liver cells concern development of novel models for pathophysiological studies, drug discovery systems and drug toxicity studies.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alison, M. (1998). Liver stem cells: a two compartment system. Curr. Opin. Cell Biol. 19,710 -715.
Badve, S., Logdberg, L., Sokhi, R., Sigal, S. H., Botros, N., Chae, S., Das, K. M. and Gupta, S. (2000). An antigen reacting with Das-1 monoclonal antibody is ontogenically regulated in diverse organs including liver and indicates sharing of developmental mechanisms among cell lineages. Pathobiology 68, 76-86.[Medline]
Bisgaard, H. C., Santoni-Rugiu, E., Nagy, P. and Thorgeirsson, S. S. (1998). Modulation of the plasminogen activator/plasmin system in rat liver regenerating by recruitment of oval cells. Lab. Invest. 78,237 -246.[Medline]
Block, G. D., Locker, J., Bowen, W. C., Petersen, B. E., Katyal, S., Strom, S. C., Riley, T., Howard, T. A. and Michalopoulos, G. K. (1996). Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium. J. Cell Biol. 132,1133 -1149.[Abstract]
Coleman, W. B., McCullough, K. D., Esch, G. L., Faris, R. A., Hixson, D. C., Smith, G. J. and Grisham, J. W. (1997). Evaluation of the differentiation potential of WB-F344 rat liver epithelial stem-like cells in vivo. Differentiation to hepatocytes after transplantation into dipeptidylpeptidase-IV-deficient rat liver. Am. J. Pathol. 151,353 -359.[Abstract]
Dabeva, M. D., Hwang, S. G., Vasa, S. R. G., Hurston, E.,
Novikoff, P. M., Hixson, D. C., Gupta, S. and Shafritz, D. A.
(1997). Activation, proliferation, and differentiation of
progenitor cells into hepatocytes in the D-galactosamine model of liver
regeneration. Proc. Natl. Acad. Sci. USA
94,7356
-7361.
Evarts, R. P., Nagy, P., Marsden, E. and Thorgeirsson, S. S. (1987). A precursor-product relationship exists between oval cells and hepatocytes in the rat liver. Carcinogenesis 8,1737 -1740.[Abstract]
Farber, E. (1956). Similarities in the sequence of early histologic changes induced in the liver of rats by ethionine, 2-acetylaminofluorene and 3-methyl-4-dimethylaminoazobenzene. Cancer Res. 16,142 -148.
Farwell, D. G., Shera, K. A., Koop, J. I., Bonnet, G. A.,
Matthews, C. P., Reuther, G. W., Coltrera, M. D., McDougall, J. K. and
Klingelhutz, A. J. (2000). Genetic and epigenetic changes in
human epithelial cells immortalized by telomerase. Am. J.
Pathol. 156,1537
-1547.
Fausto, N. (2000). Liver regeneration. J. Hepatol. 32 Suppl,19 -31.[Medline]
Fox, I. J., Chowdhury, J. R., Kaufman, S. S., Goertzen, T. C.,
Chowdhury, N. R., Warkentin, P. I., Dorko, K., Sauter, B. V. and Strom, S.
C. (1998). Treatment of the Crigler-Najjar syndrome type I
with hepatocyte transplantation. N. Engl. J. Med.
338,1422
-1426.
Gagandeep, S., Ott, M., Sokhi, R. and Gupta, S. (1999). Rapid clearance of syngeneic transplanted hepatocytes following transduction with E-1-deleted adenovirus indicates early host immune responses and offers novel ways for studying viral vector, target cell and host interactions. Gene Ther. 6, 729-736.[Medline]
Gorla, G. R., Malhi, H. and Gupta, S. (2001).
Polyploidy associated with oxidative DNA injury attenuates proliferative
potential of cells. J. Cell Sci.
114,2943
-2951.
Grompe, M., Laconi, E. and Shafritz, D. A. (1999). Therapeutic liver repopulation. Semin. Liver Dis. 19,7 -14.[Medline]
Gupta, S., LaBrecque, D. R. and Shafritz, D. A. (1992). Mitogenic effects of hepatic stimulator substance on cultured nonparenchymal liver epithelial cells. Hepatology 15,485 -491.[Medline]
Gupta, S., Rajvanshi, P. and Lee, C.-D. (1995).
Integration of transplanted hepatocytes in host liver plates demonstrated with
dipeptidyl peptidase IV deficient rats. Proc. Natl. Acad. Sci.
USA 92,5860
-5864.
Gupta, S., Rajvanshi, P., Aragona, E., Yerneni, P. R., Lee,
C.-D. and Burk, R. D. (1999). Transplanted hepatocytes
proliferate differently after CCl4 treatment and hepatocyte growth
factor infusion. Am. J. Physiol.
276,G629
-G638.
Gupta, S., Rajvanshi, P., Sokhi, R. P., Slehria, S., Yam, A., Kerr, A. and Novikoff, P. M. (1999a). Entry and integration of transplanted hepatocytes in liver plates occur by disruption of hepatic sinusoidal endothelium. Hepatology 29,509 -519.[Medline]
Gupta, S., Rajvanshi, P., Sokhi, R., Vaidya, S., Irani, A. N.
and Gorla, G. R. (1999b). Position-specific gene expression
in the liver lobule is directed by the microenvironment and not by the
previous cell differentiation state. J. Biol. Chem.
274,2157
-2165.
Haruna, Y., Saito, K., Spaulding, S., Nalesnik, M. A. and Gerber, M. A. (1996). Identification of bipotential progenitor cells in human liver development. Hepatology 23,476 -481.[Medline]
Herring, A. S., Roychowdhuri, R., Kelley, S. P. and Iype, P. T. (1983). Repeated establishment of diploid epithelial cell cultures from normal and partially hepatectomized rats. In Vitro 19,576 -588.[Medline]
Hoshi, H., Kan, M. and McKeehan, W. L. (1987). Direct analysis of growth factor requirements for isolated human fetal hepatocytes. In Vitro Cell. Dev. Biol. 23,723 -732.[Medline]
Kobayashi, N., Fujiwara, T., Westerman, K. A., Inoue, Y.,
Sakaguchi, M., Noguchi, H., Miyazaki, M., Cai, J., Tanaka, N., Fox, I. J. and
Leboulch, P. (2000). Prevention of acute liver failure in
rats with reversibly immortalized human hepatocytes.
Science 287,1258
-1262.
Lagasse, E., Connors, H., Al-Dhalimy, M., Reitsma, M., Dohse, M., Osborne, L., Wang, X., Finegold, M., Weissman, I. L. and Grompe, M. (2000). Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat. Med. 6,1229 -1234.[Medline]
Michalopoulos, G. K. and DeFrances, M. C.
(1997). Liver regeneration. Science
276, 60-66.
Ott, M., Stockert, R. J., Ma, Q., Gagandeep, S. and Gupta,
S. (1998). Simultaneous upregulation of viral receptor
expression and DNA synthesis is required for increasing efficiency of
retroviral hepatic gene transfer. J. Biol. Chem.
273,11954
-11961.
Ott, M., Ma, Q., Li, B., Gagandeep, S., Rogler, L. E. and Gupta, S. (1999a). Regulation of hepatitis B virus expression in progenitor and differentiated cell-types: evidence for negative transcriptional control in nonpermissive cells. Gene Expression 8,175 -186.[Medline]
Ott, M., Rajvanshi, P., Sokhi, R., Alpini, G., Aragona, E., Dabeva, M., Shafritz, D. A. and Gupta, S. (1999b). Differentiation-specific regulation of transgene expression in a diploid epithelial cell line derived from the normal F344 rat liver. J. Pathol. 187,365 -373.[Medline]
Overturf, K., Al-Dhalimy, M., Ou, C.-N., Finegold, M. and Grompe, M. (1997). Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am. J. Pathol. 151,1273 -1280.[Abstract]
Papoff, P., Christensen, R. D., Harcum, J. and Li, Y.
(1998). In vitro effect of dexamethasone phosphate on
hematopoietic progenitor cells in preterm infants. Arch. Dis.
Child. Fetal. Neonatal. Ed. 78,F67
-F69.
Petersen, B. E., Bowen, W. C., Patrene, K. D., Mars, W. M.,
Sullivan, A. K., Murase, N., Boggs, S. S., Greenberger, J. S. and Goff, J.
P. (1999). Bone marrow as a potential source of hepatic oval
cells. Science 284,1168
-1170.
Rajvanshi, P., Kerr, A., Bhargava, K. K., Burk, R. D. and Gupta, S. (1996). Efficacy and safety of repeated hepatocyte transplantation for significant liver repopulation in rodents. Gastroenterology 111,1092 -1102.[Medline]
Reid, L. M. and Jefferson, D. M. (1984). Culturing hepatocytes and other differentiated cells. Hepatology 4,548 -559[Medline]
Rogler, L. E. (1997). Selective bipotential differentiation of mouse embryonic hepatoblasts in vitro. Am. J. Pathol. 150,591 -602.[Abstract]
Runge, D., Runge, D. M., Jager, D., Lubecki, K. A., Beer Stolz, D., Karathanasis, S., Kietzmann, T., Strom, S. C., Jungermann, K., Fleig, W. E. and Michalopoulos, G. K. (2000). Serum-free, long-term cultures of human hepatocytes: maintenance of cell morphology, transcription factors, and liver-specific functions. Biochem. Biophys. Res. Commun. 269,46 -53.[Medline]
Sandhu, J. S., Petkov, P. M., Dabeva, M. D. and Shafritz, D.
A. (2001). Stem cell properties and repopulation of the rat
by fetal liver epithelial progenitor cells. Am. J.
Pathol. 159,1323
-1334.
Sigal, S., Gupta, S., Gebhard, D. F., Jr, Holst, P., Neufeld, D. and Reid, L. M. (1995a). Evidence for a terminal differentiation process in the liver. Differentiation 59, 35-42.[Medline]
Sigal, S., Rajvanshi, P., Reid, L. M. and Gupta, S. (1995b). Demonstration of differentiation in hepatocyte progenitor cells using dipeptidyl peptidase IV deficient mutant rats. Cell Mol. Biol. Res. 41,39 -47.[Medline]
Sigal, S. H., Rajvanshi, P., Gorla, G. R., Saxena, R., Sokhi, R.
P., Gebhardt, D. F., Jr, Reid, L. M. and Gupta, S. (1999).
Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and
activates cell aging events. Am. J. Physiol.
276,G1260
-G1272.
Tahara, H., Nakanishi, T., Kitamoto, M., Nakashio, R., Shay, J. W., Tahara, E., Kajiyama, G. and Ide, T. (1995). Telomerase activity in human liver tissues: comparison between chronic liver disease and hepatocellular carcinomas. Cancer Res. 55,2734 -2736.[Abstract]
Theise, N. D., Badve, S., Saxena, R., Henegariu, O., Sell, S., Crawford, J. M. and Krause, D. S. (2000). Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31,235 -240.[Medline]
Wieder, R. (1999). Selection of methods to measure cell proliferation. In Cell Growth, Differentiation and Senescence. (A practical approach) (ed. G. P. Studzinski), pp.1 -32. New York: Oxford University Press.
Yasui, O., Miura, N., Terada, K., Kawarada, Y., Koyama, K. and Sugiyama, T. (1997). Isolation of oval cells from Long-Evans Cinnamon rats and their transformation into hepatocytes in vivo in the rat liver. Hepatology 25,329 -334.[Medline]
Zahler, M. H., Irani, A., Malhi, H., Reutens, A. T., Albanese, C., Bouzahzah, B., Joyce, D., Gupta, S. and Pestell, R. G. (2000). The application of a lentiviral vector for gene transfer in fetal human hepatocytes. J. Gene Med. 2, 186-193.[Medline]
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