1 Marion Bessin Liver Research Center, 2 Cancer Research Center, Departments of 3 Medicine, 4 Pediatrics, 5 Microbiology and Immunology, and 6 Epidemiology and Social Medicine, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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To understand regulation of transplanted hepatocyte proliferation in the normal liver, we used genetically marked rat or mouse cells. Hosts were subjected to liver injury by carbon tetrachloride (CCl4), to liver regeneration by a two-thirds partial hepatectomy, and to hepatocellular DNA synthesis by infusion of hepatocyte growth factor for comparative analysis. Transplanted hepatocytes were documented to integrate in periportal areas of the liver. In response to CCl4 treatments after cell transplantation, the transplanted hepatocyte mass increased incrementally, with the kinetics and magnitude of DNA synthesis being similar to those of host hepatocytes. In contrast, when cells were transplanted 24 h after CCl4 administration, transplanted hepatocytes appeared to be injured and most cells were rapidly cleared. When hepatocyte growth factor was infused into the portal circulation either subsequent to or before cell transplantation and engraftment, transplanted cell mass did not increase, although DNA synthesis rates increased in cultured primary hepatocytes as well as in intact mouse and rat livers. These data suggested that procedures causing selective ablation of host hepatocytes will be most effective in inducing transplanted cell proliferation in the normal liver. The number of transplanted hepatocytes was not increased in the liver by hepatocyte growth factor administration. Repopulation of the liver with genetically marked hepatocytes can provide effective reporters for studying liver growth control in the intact animal.
carbon tetrachloride; hepatocyte transplantation; liver regeneration
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INTRODUCTION |
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THE REGENERATIVE POTENTIAL of the liver has long been recognized (24), although there has been considerable debate concerning the relative role of hepatocytes and putative progenitor (or stem) cells in this process under various circumstances (1, 4, 22, 35-37). Recent studies (2, 3, 31) indicated that progenitor liver cells can be isolated, with the potential to differentiate either completely or partially along the hepatocyte lineage in suitable microenvironments. On the other hand, when host hepatocytes are chronically and extensively depleted, such as in the albumin-urokinase-type plasminogen activator transgenic (Alb-uPA) or fumarylacetoacetate hydrolase (FAH)-deficient mice, transplanted hepatocytes can repopulate the entire liver without involving progenitor cell activation (27, 30). Indeed, serial cell transplantation studies with FAH-deficient mice proved that hepatocytes isolated from adult mouse donors can replicate indefinitely during repopulation of the diseased liver (27). These findings suggest that, in appropriate pathobiological settings, such as fulminant hepatic failure due to extensive loss of parenchymal liver cells, transplanted hepatocytes should proliferate significantly if at a selective advantage to do so and, consequently, help improve survival (13). However, whether similar concepts concerning liver repopulation could be applied to proliferation of transplanted hepatocytes in the normal liver has not been well established.
To establish mechanisms concerning proliferation of transplanted
hepatocytes in the normal liver, we performed a series of studies in
rodent models. Our general hypothesis was that in the presence of
suitable mitogenic signals, transplanted hepatocytes will proliferate
in the normal liver. We further considered that, on integration into
the hepatic parenchyma, transplanted cells would participate
physiologically in ongoing processes in the liver similar to those
affecting host hepatocytes. We utilized recently developed genetic
reporter systems for our studies, including a transgenic hepatitis B
virus (HBV) cell transplantation system in which transgenic cells
expressing HBV surface antigen (HBsAg) are transplanted into congenic
mice (10, 11, 14) and the dipeptidyl peptidase IV-deficient
(DPPIV) F-344 rat system in which F-344 rat hepatocytes are
transplanted into syngeneic mutant animals (12, 13, 28, 29). The former
system allows estimates of the transplanted hepatocyte mass by serum
HBsAg measurements (11), whereas the latter system offered convenient
strategies for in situ localization and dual-label studies to analyze
proliferation in transplanted cells (12, 28). Previous work by
Rajvanshi et al. (28) established that transplanted
hepatocytes are distributed in periportal areas (zone 1) of the rodent
liver, allowing consideration of strategies to spare transplanted cells
from ablative injury.
Our major goal here was to determine how transplanted hepatocytes responded to proliferative stimuli in the normal liver. The experimental perturbations were to ablate liver cells in a targeted fashion by carbon tetrachloride (CCl4), which depletes perivenous hepatocytes (zone 3 of the liver lobule) (23), to perform a partial hepatectomy, which induces highly synchronous DNA synthesis in the remnant liver (16, 24, 38), and to administer hepatocyte growth factor (HGF), a potent hepatic mitogen (24, 25, 32) that is released after both partial hepatectomy and toxic liver injury such as by CCl4 (20, 21).
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MATERIALS AND METHODS |
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Animals. Male F-344 rats serving as
hepatocyte donors were obtained from the National Institutes of Health
(Bethesda, MD) and weighed 250-300 g (12-16 wk old) when
used. C57BL/6J mouse recipients weighing 15-20 g (6-8 wk old)
were from Jackson Laboratories (Bar Harbor, ME). Syngeneic,
DPPIV F-344 recipient rats (160-180 g) and congenic G26 HBV
transgenic donor mice (25-30 g) were provided by the Special
Animals Core of the Marion Bessin Liver Research Center (Bronx, NY).
The studies used animals in multiple groups composed of at least three
to four animals each under protocols approved by the institutional
Animal Care and Use Committee in accordance with the
Guide for the Care and Use of Laboratory
Animals [DHEW Publication No. (NIH) 85-23, revised
1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD
20505].
Cell transplantation by intrasplenic injection in rodents was performed as previously described (14, 28). The animals were anesthetized with inhaled ether (Fisher Scientific, Fairlawn, NJ). Rats received 2 × 107 cells and mice received 2 × 106 cells suspended in RPMI 1640 medium. A two-thirds partial hepatectomy in rats was performed according to Higgins and Anderson (16). A partial hepatectomy in mice, which required leaving a collar of tissue adjacent to the gallbladder bed and bile duct, was performed as previously described (38). After the abdomen was closed, the animals were monitored closely until recovery. To induce hepatic injury, 1.45 ml/kg of CCl4 was administered intramuscularly once after dilution to either 1:1 or 1:2.5 (vol/vol) in mineral oil for rats and mice, respectively (Sigma, St. Louis, MO). To analyze changes in individual animals, serial wedge liver biopsies were performed via a midline laparotomy in recipients of CCl4 as previously described (29).
For growth factor-induced hepatic DNA synthesis, we used recombinant
human (h) HGF (lot 18181-25, Genentech, South San Francisco, CA). The
hHGF was >90% two chain as shown by densitometry of reduced, purified protein by SDS-PAGE (25). To administer HGF, Alzet osmotic
pumps (Alza, Palo Alto, CA) were primed overnight, and injection ports
were inserted into the spleen of animals with P-60 tubing (VWR
Scientific, West Chester, PA). The tubing was secured by an encircling
ligature around the splenic lower pole while the proximal end was
tunneled subcutaneously to connect with the pump implanted on the back
of the animals. For mice, Alzet 2001 pumps were used, with infusion
rates of 1 µl/h, and for rats, Alzet 2ML1 pumps were used, with
infusion rates of 10 µl/h. The hHGF was diluted in 0.5 M NaCl and
mixed with an equal amount of dextran sulfate as previously described
(32). The dose of hHGF administered was 2.4 mg · kg1 · day
1
for 7 days.
Hepatocyte isolation and culture. Hepatocytes were isolated from donor animals with a two-step perfusion with 0.025% (wt/vol) collagenase D (Boehringer Mannheim, Indianapolis, IN) as previously described (14, 28). The cell viability was tested by trypan blue exclusion as well as by attachment to tissue culture plastic in RPMI 1640 medium containing 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY). To document mitogenic activity of hHGF, hepatocytes were cultured on rat tail collagen-coated dishes at 2.5 × 105 cells/cm2 with and without 20 ng/ml of hHGF for 48 h. DNA synthesis was measured by incubating hepatocytes with [3H]thymidine (specific activity 50-70 mCi/mmol; ICN, Irvine, CA) for 1 h. The cells were then lysed in 0.3 M sodium hydroxide, TCA-precipitable DNA was extracted, and [3H]thymidine incorporation was determined along with microfluorometric quantitation of DNA in aliquots as previously described (9).
DNA synthesis measurements in tissues. To determine the magnitude and kinetics of liver regeneration with the procedures used, groups of three mice and three rats each were given 0.5 µCi [3H]thymidine/g body wt intraperitoneally 1 h before the animals were killed at various times after the interventions. The tissues were subjected to autoradiography with NTB-2 emulsion (Eastman Kodak, Rochester, NY) for 4 wk as previously described (11). To determine labeling indexes, two independent observers counted 10,000-15,000 hepatocytes/section in a blinded manner.
In some animals, H3 histone mRNA expression, which correlates with DNA synthesis, was analyzed with previously described specific probes (38). A 280-base pair fragment was used from the coding sequences of human albumin cDNA (plasmid pHSA1), which binds mouse albumin mRNAs (38). To generate antisense and sense RNA probes labeled with digoxigenin-11-UTP, commercial kits were used according to the manufacturer's instructions (Boehringer Mannheim). In situ hybridization was on 5-µm-thick paraformaldehyde-fixed cryostat sections under stringent conditions (11, 14, 38). After hybridizations, probe binding was localized with an anti-digoxigenin-alkaline phosphatase conjugate according to the manufacturer's instructions (Boehringer Mannheim).
For localization of DNA synthesis in transplanted cells, animals were given a 2-h pulse of 50 mg/kg of bromodeoxyuridine (BrdU; Boehringer Mannheim). To detect BrdU incorporation, cryostat sections were subjected to immunostaining with a commercially available antibody system (Amersham, North Chicago, IL). The tissues were blocked with 2% rabbit serum, and antibody binding was detected by a supersensitive multilink antibody system with the peroxidase reporter (BioGenex Laboratories, San Ramon, CA), followed by color development with a Vectastain kit (Vector Laboratories, Burlingame, CA).
Localization of transplanted cells. To localize transgenic G26 HBV hepatocytes, in situ hybridization was performed on cryostat tissue sections with 35S-dUTP-labeled HBsAg probes as previously described (11, 14). The sections were autoradiographed at 4°C with NTB-2 emulsion (Eastman Kodak) for several days before being developed.
To localize F-344 rat hepatocytes in DPPIV animals,
histochemical staining was performed as previously reported (12, 28, 29). Integrations of transplanted cells in the liver parenchyma were
demonstrated by colocalization of DPPIV activity and bile canalicular
ATPase activity. To colocalize DNA synthesis in
transplanted cells, tissues were first subjected to DPPIV staining, and
this was followed by immunostaining for BrdU incorporation. Tissues were counterstained with methyl green or hematoxylin as appropriate.
Morphometric analysis. To determine changes in cell number in the tissues, wedge biopsies taken at 4 wk after cell transplantation from three individual rats before initiation of CCl4 treatment and final tissue samples obtained at 36 wk after cell transplantation were analyzed with histochemical staining for DPPIV activity. The number of cells per portal area was determined in individual tissue sections for quantitative morphometry. A minimum of 50 portal areas were analyzed in each tissue. For additional analysis, changes in cluster sizes were determined by analyzing a minimum of 250 consecutive areas with transplanted hepatocytes in each tissue. For this purpose, transplanted cells in consecutive areas were scored for the number of cells per cluster: 1-3, 4-6, 7-9, 10-12, 13-15, and >16 cells each. Similar analysis was conducted to determine whether the cell number changed in animals subjected to other mitogenic treatments.
Serological assays. Blood was sampled
at 3- to 7-day intervals from the tails of animals, and the sera were
stored at 20°C for analysis. Serum HBsAg was measured with a
commercially available radioimmunoassay (AUSRIA II, Abbott
Laboratories, North Chicago, IL). HBsAg standards were included in a
known range of concentrations as previously described (11, 14). The
baseline serum HBsAg levels were used to normalize subsequent serum
HBsAg levels in individual animals for comparisons.
Statistics. Data are expressed as
means ± SD. SigmaStat 2.0 software was used for data analysis
(Jandel, San Rafael, CA). The significance of variances was analyzed as
appropriate by Student's t-test,
2 test, Mann-Whitney rank sum
test, and ANOVA.
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RESULTS |
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Characteristics of isolated hepatocytes. The viability of isolated rat and mouse hepatocytes ranged from 85 to 90% as assessed by trypan blue dye exclusion. The cells promptly attached to tissue culture plastic (>60% attachment after 1 h) and assumed normal parenchymal morphology in culture. In addition, both mouse and rat hepatocytes responded to hHGF, with DNA synthesis rates increasing by four- to sixfold in comparison with unstimulated control cells at 48 h (P < 0.001). These data verified the integrity of isolated cell preparations and indicated that our hHGF was bioactive.
Magnitude of liver regeneration
induced. To document that various maneuvers induced
liver regeneration, we studied C57Bl/6J mouse and DPPIV F-344
rat hosts before proceeding with subsequent experiments.
The kinetics of DNA synthesis after a two-thirds partial hepatectomy in
rats was similar to a previous experience, with an early peak at 24 h,
followed by rapid attenuation of the regenerative response (Fig.
1A).
In contrast, after CCl4
administration, the initial peak was delayed and DNA synthesis was
characterized by a relatively lower magnitude as well as a longer
persistence compared with partial hepatectomy in both mice and rats.
Also, after partial hepatectomy, DNA synthesis was apparent in
hepatocytes essentially throughout the liver lobule, whereas
CCl4 treatment resulted in DNA
synthesis in the periportal areas because perivenous hepatocytes were
destroyed by the toxin (Fig. 1B).
The distribution of hepatocytes containing either
[3H]thymidine or BrdU
was similar to the distribution of hepatocytes expressing histone H3
mRNA (Fig. 1C) as also previously
observed (38). In the dose used,
CCl4 depleted hepatocytes from
only ~30 to 50% of the liver lobule (Fig. 1,
D-F),
which most likely accounted for the difference in the magnitude of DNA
synthesis compared with that resulting from a two-thirds partial
hepatectomy. The hepatic susceptibility to
CCl4 is determined by the activity of specific cytochrome P-450 isoforms
in individual cells, with perivenous hepatocytes being most sensitive
(21).
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After hHGF infusion in the animals, we analyzed DNA synthesis rates at 60 h after commencing the infusion, which has previously been shown to be effective in intact animals (32). Studies were performed in three mice per group and showed significantly increased BrdU incorporation compared with untreated control animals (Fig. 1, G and H). The overall hepatocyte labeling index approached 42 ± 6/1,000 cells, which was less than that observed with either a two-thirds partial hepatectomy or CCl4 treatment, although this compared favorably with DNA synthesis after a 35% partial hepatectomy in a previous study (38).
Effect of CCl4-induced liver regeneration on transplanted cell proliferation. The strategy was based on the fact that intrasplenic transplantation deposits cells in periportal locations (28). The hypothesis was that the susceptibility of perivenous hepatocytes to CCl4 would not extend to transplanted cells located in periportal areas and that transplanted cells would participate in the ensuing proliferative response of host hepatocytes in nonablated areas.
The experimental strategy was to let transplanted cells become fully integrated into the liver parenchyma over a period of several weeks (Fig. 2). Although cell engraftment may require less time, we wished to ensure that transplanted cells, which were not fractionated to exclude cytochrome P-450-expressing cells, were resistant to CCl4 injury, and the time required for this was unknown. The animals were then given three doses of CCl4 at 4-wk intervals to permit complete recovery from the preceding injury. Subsequently, the animals were observed for a total of 36 wk from the time of cell transplantation.
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DISCUSSION |
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Our findings provide novel insights into mechanisms regulating proliferation of transplanted hepatocytes in the healthy liver. The ability of transplanted hepatocytes to integrate in the liver parenchyma and assume normal polarity in the liver plate suggested to us that the cells should respond appropriately to physiological stimuli (12). We were impressed by the capacity of transplanted hepatocytes to undergo repeated proliferation in the setting of recurrent liver injury, although cell proliferation appeared to be highly regulated each time and occurred in a manner sufficient to replace the lost hepatocyte mass.
The results of our CCl4 treatment studies were particularly instructive and revealed several important features. Previous studies (27, 30) documented that transplanted hepatocytes can proliferate extensively in the setting of chronic depletion of host hepatocytes, such as in the Alb-uPA transgenic and FAH-deficient mice. Serial transplantation studies in FAH-deficient mice showed that transplanted hepatocytes can undergo multiple cell divisions while repopulating the chronically diseased liver (27). However, because transplanted cells did not previously undergo significant proliferation in the normal liver (10), it was unclear as to what mechanisms could be useful in amplifying the transplanted hepatocyte mass in the healthy liver. This consideration is especially relevant in cell therapy strategies concerning metabolic deficiency states such as familial hypercholesterolemia and Criggler-Najjar syndrome, which cause little or no liver injury and will benefit from amplification of the transplanted hepatocyte mass for superior results (1a, 7, 15, 17, 26).
Proliferation in transplanted cells commensurate with healing needed to restore liver mass after CCl4 suggests that targeted hepatic ablation should be effective in applications of hepatocyte transplantation. Of course, CCl4 has extensive general toxicities as well as a relatively long half-life, precluding application, as also indicated in our studies, by injury to hepatocytes transplanted 24 h after CCl4 administration. However, additional strategies might well become available in the future to ablate portions of the hepatic lobule safely. One such approach concerned transient expression of Alb-uPA gene with an adenoviral vector in host hepatocytes before cell transplantation (39). These studies were reported subsequent to the initiation and completion of our experiments.
The migration of transplanted hepatocytes toward perivenous areas of the liver after CCl4 treatment was in agreement with shifts in host liver plates during recovery and offer further evidence for the potential of hepatocyte transplantation systems for addressing fundamental questions in liver physiology. For instance, such systems could be helpful in addressing mechanisms underlying position-specific regulation of gene expression and other events in the liver lobule that are incompletely resolved (8). We interpret migration of transplanted hepatocytes toward perivenous areas to represent a "wound repair" mechanism because such an event was not observed in the uninjured normal liver. The ability of transplanted hepatocytes to repopulate the entire liver in Alb-uPA and FAH-deficient mice, with transplanted hepatocytes shown to be present in perivenous areas (27), also illustrates replacement of injured hepatocytes and not necessarily "streaming" of hepatocytes from periportal to perivenous areas in the liver lobule, in agreement with similar interpretations by others using different systems (19).
Repeated proliferation in hepatocytes isolated from the unperturbed
adult rat liver in our studies extend recent findings of extensive stem
cell-type proliferative capacity in serially transplanted mouse
hepatocytes (27). However, in contrast with our findings in
CCl4-induced liver injury, we were
intrigued to learn that transplanted hepatocytes did not proliferate in
the liver after growth factor infusion, even though this induced
significant hepatic DNA synthesis. Our data here are in agreement with
a different proliferative response of transplanted hepatocytes after a
partial hepatectomy compared with that after
CCl4 treatment. Although hHGF
infusion was ineffective in increasing the number of transplanted cells, we do not know whether more sustained hHGF treatment might have
been effective in this regard. Similarly, whether simultaneous stimulation with additional growth factors such as transforming growth
factor- (40) or cell priming with other manipulations will improve
results requires further analysis.
In conclusion, our data demonstrate that transplanted hepatocytes can be induced to proliferate significantly in the normal liver after selective injury to host hepatocytes, such as with CCl4. The role of hepatic ablative stimuli is particularly noteworthy in this respect compared with induction of DNA synthesis with growth factor stimulation. Finally, liver repopulation with genetically marked cells will permit the use of transplanted hepatocytes as powerful reporters for analyzing physiological mechanisms in liver regeneration.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ralph Schwall (Genentech, South San Francisco, CA) for the kind gift of human heptocyte growth factor.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants K08-DK-01909 and R01-DK-46952 (to S. Gupta) and P33-DK-41296 (to D. A. Shafritz, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY) and the Irma T. Hirschl Trust.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Gupta, Ullmann 625, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: sanjvgupta{at}pol.net).
Received 25 June 1998; accepted in final form 5 November 1998.
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