Marion Bessin Liver Research Center, Cancer Research Center, and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Transplanted hepatocytes integrate in the liver parenchyma and exhibit gene expression patterns that are similar to adjacent host hepatocytes. To determine the fate of genetically marked hepatocytes in the context of hepatocellular proliferation throughout the rodent life span, we transplanted Fischer 344 (F344) rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. The proliferative activity in transplanted hepatocytes was studied in animals ranging in age from a few days to 2 yr. Transplanted hepatocytes proliferated during liver development between 1 and 6 wk of age, each dividing an estimated two to five times. DNA synthesis in occasional cells was demonstrated by localizing bromodeoxyuridine incorporation. There was no evidence for transplanted cell proliferation between 6 wk and 1 yr of age. Subsequently, transplanted cells proliferated again, with increased sizes of transplanted cell clusters at 18 and 24 mo of age. The proliferative activity of transplanted cells was greater in rats entering senescence compared with during postnatal liver development. In old rats, some liver lobules were composed entirely of transplanted cells. We conclude that hepatocyte proliferation in the livers of very young and old F344 rats is regulated in a temporally determined, biphasic manner. The findings will be relevant to mechanisms concerning liver development, senescence, and oncogenesis, as well as to cell and gene therapy.
hepatocyte transplantation; dipeptidyl peptidase IV; liver; regeneration; aging
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INTRODUCTION |
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THE CAPACITY OF THE LIVER to regenerate itself has long been established (24). In response to two-thirds partial hepatectomy, liver mass is restored by proliferative activity in hepatocytes (2, 22), whereas in other situations, such as toxic liver injury or exposure to carcinogenic regimens, additional liver cell types are recruited (1, 7, 9, 15, 31). Although the kinetics of hepatic regenerative responses have been well established in short-term studies, information concerning changes in hepatocyte cycling in the normal liver during prolonged periods is limited. During postnatal growth, liver mass increases more rapidly compared with the body mass in the first several weeks of life (21, 25), whereas in the normal adult liver, proliferative activity is minimal, with restriction of hepatocytes in G0/G1, so that evidence for DNA synthesis can be elicited in only a rare cell (6, 12, 18, 33). Although studies (3) using [3H]thymidine labeling have been interpreted to show a limited life span in hepatocytes, during which cells are proposed to migrate from periportal areas of the liver lobule to perivenous areas, other studies (15, 17, 18) have provided compelling evidence against this possibility. In old animals, evidence (5, 11, 29, 30) has been provided for perturbations in liver regeneration, with impaired capacity or delayed onset of hepatocellular proliferation. However, although biochemical changes accompanying these perturbations, such as decreased cell cycle-regulated gene expression, e.g., thymidine kinase, have been recognized (4, 5, 19, 34), assessing the significance of these changes has been difficult because of contradictory evidence (32). Also, in old Fischer 344 (F344) rats, various types of hepatic foci involving hepatocytes, as well as biliary cells, arise spontaneously (8), suggesting increased rather than decreased hepatic proliferative activity, at least in this strain of rats.
Recent work (14) involving liver repopulation with
transplanted hepatocytes suggested to us that transplanted hepatocytes could serve as convenient reporters to analyze cell proliferation changes during the animal life span. In this respect, development of
assays utilizing transplantation of F344 rat hepatocytes into the liver
of syngeneic dipeptidyl peptidase IV-deficient (DPPIV) F344 rats was
particularly noteworthy. Studies (15-17, 26)
demonstrated that transplanted hepatocytes entered liver plates and
became integrated with adjacent host hepatocytes in the liver. This
included development of conjoint plasma membrane structures, retention of normal gene expression patterns, and DNA synthetic responses, such
as following acute carbon tetrachloride injury, which are similar to
those of adjacent host hepatocytes (15-17,
26). Therefore, we hypothesized that analysis of the
fate of transplanted cells in the DPPIV
F344 rat during various
stages of the rodent life will provide new information concerning cell
proliferation in the liver.
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MATERIALS AND METHODS |
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Animals.
Male F344 rats were obtained from the National Institutes of Health
(Bethesda, MD) to serve as hepatocyte donors. These rats weighed
250-300 g and were 12-16 wk old when used. DPPIV F344 rats
(10 wk old) were provided by the Special Animal Core of the Marion
Bessin Liver Research Center and served as cell recipients. Rats were
maintained in the Institute for Animal Studies at Albert Einstein
College of Medicine with unrestricted access to pelleted food and
water, under a 14:10-h light-dark cycle. Animals were used in multiple
groups comprising three to four rats each. The institutional Animal
Care and Use Committee approved the protocols in accordance with
standard guidelines.
Hepatocyte isolation. The liver was perfused with 0.025% (wt/vol) collagenase D (Boehringer Mannheim, Indianapolis, IN) using a two-step protocol as described previously (16, 26). Cell viability was tested by trypan blue dye exclusion, as well as by attachment to tissue culture plastic in RPMI 1640 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY).
Cell transplantation. Isolated hepatocytes were suspended in serum-free RPMI 1640 cell culture medium and injected into the spleen as described previously (16, 26). Intrasplenic injection of cells leads immediately to the deposition of the vast majority of transplanted cells into liver sinusoids (26). This is followed by engraftment of cells in liver plates after cell transplantation (16). Adult rats received 2 × 107 cells and suckling pups 1 × 105 cells each. After the abdomen was closed, each animal was monitored until recovery. The pups were returned to the dam after completion of the procedure.
Histological studies.
Tissues were obtained from animals and frozen in methylbutane cooled to
70°C on dry ice. Cryostat sections (5 µm thick) were made and
fixed in chloroform and acetone (1:1 vol/vol) at 4°C. Transplanted
cells were localized in the liver of DPPIV
F344 rats by histochemical
staining for DPPIV activity, according to previous methods (16,
26). Normal tissues from rats were included in reactions as
positive controls, and liver sections from DPPIV
rats, which were
not subjected to cell transplantation, served as negative controls.
Integration of transplanted cells in the liver parenchyma was analyzed
by localization of DPPIV activity, along with bile canalicular ATPase
activity, as reported previously (16, 26).
Morphometric analysis. To determine changes in the number of transplanted cells in the liver, at least 250 areas containing transplanted hepatocytes were analyzed serially in each animal. Multiple sections were made from the left lateral and median lobes of the liver with a depth of 40 µm between serial sections to avoid cutting the same cells again. Transplanted cells could be localized readily by inspection of DPPIV-positive bile canalicular domains. In some cells, bile canaliculi encircled the cells completely (see Fig. 2). In others, only parts of bile canaliculi were present. In this instance, the arrangement of bile canaliculi around individual nuclei allowed us to determine the cells to which the bile canalicular segments belonged. The number of transplanted cells contained in each cell cluster was determined as a parameter to demonstrate the magnitude of cell proliferation. Transplanted cells were scored for the number of cells per cluster as follows: 1-3, 4-6, 7-9, 10-12 , 13-15, and >16 cells each.
Experimental design.
Two studies were undertaken (Fig. 1). In
one study, 18 suckling DPPIV pups were transplanted with cells, and
animals were followed for up to 42 wk. Animals in this group were
killed at the ages of 6 and 10 days and 4, 6, and 42 wk after cell
transplantation. In a second experiment, fifteen 10-wk-old
DPPIV
rats were transplanted with cells, and animals were followed
for up to 24 mo of age. Animals in this group were killed at 7 days
after cell transplantation and at 4, 12, 18, and 24 mo of age.
Untreated animals were included in additional studies as controls.
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Statistics.
Data are expressed as means ± SD. SigmaStat 2.0 software (Jandel,
San Rafael, CA) was used for data analysis. The significance of
differences was determined by 2 test and ANOVA as appropriate.
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RESULTS |
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Proliferation of transplanted hepatocytes in suckling rats. In these animals, transplanted cells were present in periportal areas throughout the duration of the studies, extending from 3 days to 42 wk after cell transplantation. In negative control recipients, in which no cells were transplanted, the liver showed a complete absence of DPPIV activity.
At the earliest times, such as 3 and 7 days after transplantation, cells were found in clusters of one or two cells (Fig. 2A). Extramedullary hematopoiesis was also apparent in the liver at this time. For transplanted cells to participate physiologically in events affecting the liver lobule, we considered it necessary to demonstrate that cells were integrated in the host liver parenchyma. To determine this, tissues from hepatocyte recipients were analyzed for the development of conjoint bile canalicular structures among adjacent host and transplanted hepatocytes. In suckling rats, transplanted cells were well integrated in the liver parenchyma by 7 days after transplantation and showed DPPIV and ATPase-containing hybrid bile canalicular domains, arising from transplanted and host hepatocytes, respectively, similar to previous demonstrations (Fig. 2B) (15-17, 26). At subsequent times, more transplanted cells were arranged in larger cell clusters. The number of transplanted cells in the liver increased at 4 wk, as well as at 6 wk (Fig. 2, C-E). These figures indicate how it was possible to determine the number of transplanted cells in each cluster. However, subsequently at 42 wk, there was no further discernible increase in the number of transplanted cells. In all conditions, transplanted cells were present in periportal areas and not in perivenous areas of the liver lobule. If transplanted cells had migrated from periportal areas toward perivenous areas, this should have been apparent in animals killed at later times, such as several weeks or months after cell transplantation.
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Proliferation of transplanted hepatocytes in older rats.
In young adult recipients described in Fig. 1B, transplanted
cells integrated in the liver parenchyma within 7 days and formed conjoint bile canaliculi, similar to the findings in suckling rats and
our previous experience (15-17, 26). Transplanted
cells were arranged in clusters containing either one or less than
three cells. Notably, the number of transplanted cells did not change subsequently at ~1 or 9 mo after cell transplantation (4 and 12 mo of
age, respectively), with transplanted cells still constituting mostly
small cell clusters (Fig.
4, A and
B). These findings were similar to those in 16- to 42-wk-old
suckling rats, as described above. However, 15 mo after cell
transplantation (18 mo of age), the number of transplanted cells began
to increase (Fig. 4, C and D). Interestingly, the
transplanted cell number increased further at the age of 24 mo (Fig.
4E). At these times, transplanted cells were still located
in periportal areas, although the number of transplanted cells
increased. Occasionally, transplanted cells constituted 80-90% of
the entire liver lobule at 18 or 24 mo of age (Fig. 4F).
Such a finding was infrequent, however, and total or near-total
replacement of the liver lobule was observed in only 0.1-0.5% of
the liver lobules. Moreover, at the age of 24 mo, there was evidence
for increased proliferation of endogenous biliary cells (Fig.
4G). Although most portal areas contained increased number
of biliary cells at this time, the degree of biliary proliferation
differed between portal areas. Despite extensive bile duct
proliferation and appearance of "nodules" constituted by
transplanted cells, we did not observe tumors in the liver of our
transplanted animals, which was similar to our experience in >10
retired DPPIV breeders, which were observed for up to 2 yr of age.
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DISCUSSION |
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These findings advance our knowledge with respect to the biology of transplanted hepatocytes and show that transplanted hepatocytes effectively serve as reporters for defining mechanisms in liver growth control. Our results showing a bimodal change in the number of transplanted cells, during early liver development and old age, establish unequivocally that hepatocytes undergo cell division during these early and late stages of life.
In view of the generally limited BrdU incorporation in the developing liver under our conditions, we did not undertake detailed morphometric analysis of this phenomenon and placed greater reliance on analysis of cell cluster sizes, which provided direct insight into whether daughter cells were produced. Our results concerning BrdU incorporation in the liver are not comparable with previous studies, in which longer pulses of BrdU or [3H]thymidine may have been administered. The disadvantages and limitations of long pulses of nucleotides for DNA labeling, including inhibition of DNA synthesis with large amounts of nucleotides, have been reviewed extensively (2). Although [3H]thymidine labeling is useful in establishing the kinetics of DNA synthesis, difficulties in disposal of contaminated carcasses have precluded our performing intact animal studies with [3H]thymidine. Moreover, although labeled nucleotides may be incorporated in cells undergoing DNA synthesis, there is no certainty that such cells will divide to generate daughter cells, because a cell fraction may give rise to polyploid cells without dividing (33). Therefore, use of transplanted cells was particularly effective in demonstrating changes in cell numbers over prolonged periods of time. Photoreconstruction methods were used previously (28) to demonstrate that thin liver sections can provide accurate and verifiable information on the number of hepatocytes in a given volume of liver tissue. Careful morphometric analysis using serial sections showed that even a tangential cut does not alter the probability of obtaining accurate global cell counts in liver sections. Of course, our analysis demonstrated a "steady state" without distinguishing between potential cell losses and replacement of such lost cells by proliferation in the remainder. Measurement of spontaneous cell losses in tissues, as might occur normally by apoptosis, is an uphill task because no methods are currently available to quantitate apoptosis rates precisely. Although we and others (33) used terminal deoxynucleotidyltransferase-mediated, dUTP nick end-labeling assays for demonstrating apoptosis in liver tissue, this semiqualitative assay underestimates the magnitude of apoptosis rates.
Our data indicated that during liver development in the first 6 wk of life, transplanted hepatocytes divided between at least two and five times, because one to three transplanted cells generated clusters of four to fifteen cells each. These findings are compatible with the known kinetics of postnatal liver growth in rats, where the period of most rapid liver growth is encountered between 2 and 6 wk of age, although further liver maturation continues between the ages of 6 and 18 wk, when liver gene expression patterns associated with mature hepatocytes become established (25). Although it was previously considered (21) that the number of hepatocytes may increase slightly after birth, the major change was believed to concern increased mass of hepatocytes due to cellular hypertrophy, along with morphological differentiation of cells and the organ. If host hepatocytes share the proliferative activity of transplanted hepatocytes in the developing liver, with two to five divisions each, cell proliferation will be the major mechanism to account for the ~10-fold increase in liver weight from birth to maturity (25). The ability of transplanted hepatocytes to enter liver plates and become integrated with adjacent host hepatocytes, along with the retention of physiologically regulated patterns of gene expression would strengthen such a possibility (7, 16, 26). Also, transplanted hepatocytes and adjacent host hepatocytes exhibit similar kinetics and magnitude of proliferative activity in the presence of a suitable proliferative stimulus, such as carbon tetrachloride, which destroys perivenous hepatocytes without affecting host and transplanted hepatocytes in periportal locations (15). Of course, when the proliferative capacity of transplanted hepatocytes becomes dissociated from that of host hepatocytes, due to a variety of reasons (such as use of toxic genes, DNA alkylating drugs, radiation, and partial hepatectomy; Refs. 14, 20, 23, 24, 27, and 33), transplanted hepatocytes are expected to behave differently. However, this was not the case in our studies reported here.
In contrast with active cell proliferation during postnatal liver development, we found an absence of transplanted cell proliferation in the livers of adult F344 rats up to 1 yr of age. The absence of significant proliferative activity in the adult rodent liver has been documented extensively and is in agreement with our findings (2, 6, 12, 33). On the other hand, increased proliferative activity in older F344 rats is most intriguing, although perhaps not necessarily surprising. The liver in the older F344 rat is known to undergo significant changes, including development of hepatocellular foci, focal hepatocellular hyperplasia, and bile duct hyperplasia (8). Previous experience indicated that the prevalence of hepatocellular foci increases with age in F344 rats, so that foci are encountered in virtually all animals by the age of 18 mo. Although these hepatic foci may be classified morphologically into basophilic, eosinophilic, clear, vacuolated, and mixed types, the pathophysiological basis regulating their appearance is unknown. Similarly, mechanisms regulating the onset of focal hepatocellular and bile duct hyperplasia are undefined.
Nonetheless, increased proliferation of transplanted hepatocytes subsequent to 12 mo of age in our F344 rats was in agreement with their participation in these unique processes and indicated again the potential of using transplanted hepatocytes as reporters for analyzing mechanisms in liver growth control. The proliferative rate of hepatocytes in our old rats often appeared to be greater than during postnatal growth, as indicated by the appearance of transplanted cell clusters containing far more transplanted cells, including 50-100 cells, or more, in individual clusters. For instance, 1 transplanted cell must divide 7 times to generate 128 daughter cells and 3 transplanted cells must divide 5 times to generate 96 daughter cells. We restricted our analysis to cell cluster size determination in a single tissue plane, rather than undertaking a more complex volumetric determination of transplanted cells using spheres as a model. As indicated above, this approach has previously been validated by the work of Ross (28). Our aim was to obtain comparative information concerning alterations in the proliferative rates of transplanted cells (and by extension in host hepatocytes) at various stages of life. We were much less interested in establishing the absolute number of transplanted cells in the liver. However, to place the magnitude of proliferative activity in the liver of old F344 rats into perspective, it might be worth considering that in the albumin-urokinase-type plasminogen activator transgenic mouse system, ~12 rounds of cell division in transplanted cells were estimated to be sufficient for repopulating the entire liver (27).
Our findings should be particularly relevant to the analysis of physiological perturbations during aging. There has been great difficulty in analyzing age-related perturbations in the intact liver and in establishing unequivocal associations between hepatocyte structure and function in the intact old animal (30). The findings here established that old F344 rats developed extensive heterogeneity in respect to the proliferative activity of hepatocytes, with significant differences in cell proliferation in a given lobule. For instance, Fig. 4G shows that extensive biliary proliferation has occurred without proliferation in hepatocytes, as well as among different liver lobules. Use of whole liver homogenates or lysates for the analysis of genetic or other events in this situation could well result in confounding. Therefore, incorporation of hepatocyte transplantation systems in experimental studies should represent a suitable strategy for defining the proliferative state of the liver. Similarly, hepatocyte transplantation systems could be gainfully employed for defining oncogenetic events in animals. If cells are transplanted before oncogenic manipulations, it might be possible to determine whether transplanted cells participate in oncogenesis. Alternatively, cell transplantation into animals that have already completed an oncogenic regimen could help exclude the role of specific transplanted cell types in oncogenesis. Although use of transgenic mice, with restriction of transgene expression to only some hepatocytes in the liver lobule, offers another approach (18), cell transplantation systems can be applied to studies in additional animal species and offer creative possibilities.
The results should be of interest in liver-directed cell and gene therapy. The ability of hepatocytes to engraft in the liver of 3-day-old rat pups suggests that cells could be transplanted into babies in the neonatal or infantile periods, possibly even in the third trimester in utero. A number of genetic disorders will be amenable to such interventions (14). Proliferation of transplanted cells in the developing liver may enhance the final therapeutic effect. In older individuals, an absence of progressive increase in liver repopulation is in agreement with our findings concerning lack of transplanted cell proliferation in young adult animals, which has limited therapeutic results of cell and gene therapies in genetic disorders (10, 13). Of course, our findings of increased cell proliferation in old F344 rats are not transferable to humans because this appears to be a unique feature of this animal strain. Nonetheless, life-long survival of transplanted cells in the liver will be desirable for treating patients, especially when permanent correction or amelioration of genetic disorders is necessary. Finally, our findings indicate that transplanted cells retain significant potential for proliferation throughout the lives of animals, which should facilitate consideration of strategies aimed at amplifying the transplanted cell mass.
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ACKNOWLEDGEMENTS |
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The work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-46952 and P33-DK-41296 and by an award from the Irma T. Hirschl Trust (S. Gupta).
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FOOTNOTES |
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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).
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.
Received 29 July 1999; accepted in final form 21 March 2000.
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