Transplanted reporter cells help in defining onset of hepatocyte proliferation during the life of F344 rats

Rana P. Sokhi, Pankaj Rajvanshi, and Sanjeev Gupta

Marion Bessin Liver Research Center, Cancer Research Center, and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).

To localize transplanted cells undergoing DNA synthesis, tissues were first stained for DPPIV activity, followed by immunostaining for bromodeoxyuridine (BrdU) incorporation. For this purpose, animals were given a 2-h pulse of 50 mg/kg BrdU (Boehringer Mannheim) before their death (15). BrdU incorporation was analyzed in the liver, as well as in the small intestine and spleen, which served as internal controls to ensure adequate delivery and utilization of BrdU in animals. Cryostat sections were prepared and fixed in chloroform and acetone as described above to detect DPPIV activity. Tissues were then blocked with Power Block universal blocking reagent (BioGenex, San Ramon, CA), which contains buffer, casein, and preservative, and subjected to immunostaining with a commercially available BrdU antibody system, according to the manufacturer's suggested protocol (Amersham, North Chicago, IL). The tissues were blocked with 2% nonimmune rabbit serum, and antibody binding was detected by a supersensitive multilink antibody containing the peroxidase reporter (BioGenex), followed by color development with a Vectastain kit (Vector Laboratories, Burlingame, CA). The tissues were counterstained with methyl green or hematoxylin as appropriate.

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.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Strategy for analyzing the fate of transplanted cells in young and old rats. Two experiments were conducted to cover various stages in the life of rats. A: in one study, hepatocytes were transplanted into 3-day-old suckling Fischer 344 (F344) rat pups. These animals were killed at the ages of 6 and 10 days (d) and 4, 6, and 42 wk after cell transplantation for tissue analysis. B: in the second study, hepatocytes were transplanted into 10-wk-old adult F344 rats. Animals were then killed within 1 wk of cell transplantation, as well as at 4, 12, 18, and 24 mo of age as indicated. At least 3 animals were killed at each time indicated. Rats were weaned at 4 wk of age and considered to have entered old age at 15 mo of age. Not shown are untreated animals, which served as controls for studies at various times.

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 chi 2 test and ANOVA as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (4K):
[in this window]
[in a new window]
 
Fig. 2.   Intrasplenic injection of hepatocytes followed by localization of transplanted cells in the liver of rat pups. Transplanted cells were localized by dipeptidyl peptidase IV (DPPIV) histochemistry, and tissues were counterstained lightly with hematoxylin. A: transplanted cells containing DPPIV-positive bile canaliculi (red; filled arrows) in the periportal area of the liver lobule in a 10-day-old pup. Extramedullary hematopoiesis was present in the liver at this time (open arrow at bottom). It is noteworthy that transplanted hepatocytes were arranged as single cells. B: sequential staining showing conjoint bile canaliculi in transplanted cells, which contain DPPIV activity (red; filled arrow), and host cells, which contain ATPase activity (brown; open arrow), indicated that transplanted cells integrated in the liver of 10-day-old pups. The microphotograph shows 4 host cells, in which cell nuclei are encircled by ATPase-positive bile canaliculi, along with 2 transplanted cells containing DPPIV-positive bile canaliculi (red). C: DPPIV staining of the liver from a 4-wk-old pup showing some proliferation of transplanted cells, which are now arranged in occasional small clusters (arrow), as well as single cells. D: liver of a 6-wk-old pup showing transplanted cells in larger clusters. The arrow at right points to a single transplanted cell. E: higher- magnification view showing scoring of the number of cells, which are indicated by arrows in D. The cell cluster at left contains 6 transplanted cells, whereas only 1 cell is present at right. F: liver from another animal at 6 wk after cell transplantation showing transplanted cells in an even larger cluster. This cluster contains 12 transplanted cells.

To demonstrate whether increased transplanted cell numbers were associated with DNA synthetic activity in the host liver, we undertook analysis of BrdU incorporation in hepatocyte-recipient animals. This analysis was undertaken in all animals at each time point studied, including at the ages of 6 and 10 days and 4, 6, and 42 wk. The spleen and intestines of each animal served as controls to demonstrate the efficacy of BrdU uptake and utilization (Fig. 3, A-C). There was excellent BrdU incorporation in splenic cells and intestinal crypt or intraepithelial cells. BrdU incorporation was apparent in the liver of animals at early times, up to 6 wk after cell transplantation, but was restricted to rare cells in the liver (1-2 cells in entire sections) at 42 wk after cell transplantation.


View larger version (3K):
[in this window]
[in a new window]
 
Fig. 3.   Bromodeoxyuridine (BrdU) incorporation in the liver of pups subjected to cell transplantation. A: negative control splenic tissue showing no BrdU immunostaining when the primary antibody was omitted from the reaction. B: spleen from a 10-day-old pup after cell transplantation with extensive BrdU incorporation in splenic cells (arrow), indicating successful incorporation of the label. C: small intestine from the animal in B, showing BrdU incorporation in scattered crypt cells, as well as intraepithelial lymphocytes (arrow). D: liver from a 10-day-old pup showing clusters of parenchymal cells with BrdU incorporation (arrow with feathery tail). Several adjacent transplanted hepatocytes (arrows point to 4 transplanted cells) did not stain for BrdU. E: showing BrdU incorporation in 3 transplanted cells with DPPIV-positive domains in a 10-day-old animal (arrows). Three more transplanted cells are present on upper left with no BrdU staining. F: BrdU incorporation in occasional transplanted cells containing DPPIV activity (arrow), as well as host hepatocytes in a 4-wk-old animal (curved arrow). G: shows absence of BrdU incorporation in an animal at 42 wk of age. Transplanted cells are present in a periportal area. The tissue was exposed for a longer time compared with other conditions to ensure that BrdU incorporation was not missed, which accounts for greater background. The tissues were reacted first for DPPIV activity to localize transplanted cells followed by BrdU immunostaining and counterstained lightly with hematoxylin or methyl green (C).

At early times, such as 3 and 7 days after cell transplantation, BrdU incorporation was observed most readily in hematopoietic cells in the liver (Fig. 3, D and E). Among liver epithelial cells, BrdU incorporation was observed in groups of hepatocytes (both in periportal regions and elsewhere), single hepatocytes scattered throughout the liver lobule, and occasional bile duct cells. The magnitude of BrdU incorporation at both 3 and 7 days after cell transplantation was low with our 2-h pulses of BrdU and ranged from 1 to 10 BrdU-positive cells per high power field (×200), which constituted <1% of the cells. The evidence of this proliferative activity, as shown by our BrdU pulses, became less obvious by 4 wk and subsided completely by 6 wk of age (Fig. 3F). Similarly, the number of transplanted cells containing BrdU that were captured by the BrdU pulse was limited, so that only rare cells were encountered with BrdU incorporation at 7 days and 4 wk after cell transplantation. No transplanted cells containing BrdU were observed at either 6 or 42 wk after cell transplantation (Fig. 3G).

Analysis of cell cluster sizes more clearly defined the magnitude of transplanted cell proliferation in animals. The detailed findings are presented in Table 1, which demonstrates the distribution of transplanted cells in small or large cell clusters. The data were normalized for percent cell distributions to allow comparisons between the two sets of our studies. In the first study concerning cell transplantation into pups, most cells were distributed in small cell clusters at the earliest time, i.e., 3 days after cell transplantation (6 days of age). Subsequently, starting at as early as 7 days after cell transplantation (10 days of age), larger transplanted cell clusters became more prevalent, although cell proliferation was relatively modest throughout this period of liver development up to 6 wk, as indicated by a shift to cell clusters containing 4-15 cells each, but not to larger cluster sizes. Nonetheless, increases in the size of transplanted cell clusters continued for up to 6 wk, corresponding to the period of rapid liver growth. There was no further increase in the cluster sizes of transplanted cells at the ages of 6 and 42 wk after cell transplantation, which corresponds with young adult life, as indicated in Fig. 1.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Distribution of transplanted cells according to cluster sizes in the liver of younger DPPIV- rats

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.


View larger version (3K):
[in this window]
[in a new window]
 
Fig. 4.   Localization of transplanted hepatocytes after cell transplantation in 10-wk-old rats. A: low-power view of the liver of a 16-wk-old animal with transplanted cells containing DPPIV activity scattered in periportal areas. B: liver from a 12-mo-old animal without significant increase in transplanted cell clusters. C: liver from an 18-mo-old rat showing a large cluster of transplanted cells. D: higher magnification view of the transplanted cell cluster with numbering of individual cells in the cluster. As shown, this cluster contained at least 20 cells in the 2-dimensional analysis. E: liver from a 24-mo-old rat showing further increase in cluster sizes of transplanted cells. F: repopulation of virtually an entire liver lobule by transplanted cells in a 24-mo-old rat. P, portal areas; C, central vein. G: proliferation of endogenous bile duct cells in a 24-mo-old rat (curved arrow at right). The straight arrow points to discrete transplanted cells in an adjacent portal area. Biliary proliferation was unrelated to cell transplantation and was also observed in old F344 rats not subjected to hepatocyte transplantation.

In view of the limited information provided by BrdU incorporation analysis in the previous study, we did not undertake this analysis in older rats and focused only on the analysis of cell cluster sizes, which are summarized in Table 2. Results from these studies in older rats showed that the cluster sizes of transplanted cells did not increase significantly between ~4 and 12 mo of age, which was similar to the lack of significant transplanted cell proliferation between 6 and 42 wk of age in the first study. However, subsequent to the age of 12 mo, transplanted cells began to proliferate more extensively, in an age-dependent fashion. The number of large transplanted cell clusters increased significantly at both 18 and 24 mo of age compared with the preceding period between 4 and 12 mo of age in these animals. Moreover, the number of large cell clusters containing 16 or more transplanted cells was quite prominent at the age of 24 mo, constituting an average of 10% of all cell clusters at this time (Table 2). However, as shown above (Fig. 4F), an occasional liver lobule was constituted entirely by transplanted cells at even 18 mo of age. At 18 and 24 mo of age, it was not uncommon to encounter cell clusters containing 50-60 transplanted cells each in our uniplanar analysis. At 24 mo, >50% of all large clusters contained 50 or more transplanted cells. Extrapolation of these numbers to a sphere (vol; <FR><NU>4</NU><DE>3</DE></FR>pi r3, where r = radius, or c3 × 0.0169, where c = circumference) would indicate that each of these clusters contained hundreds, and even thousands, of transplanted cells, where entire lobules were constituted by transplanted cells. Approximately one-third of all transplanted hepatocytes were still in clusters of one to three cells each at 24 mo of age. However, in comparison, a much larger transplanted cell fraction, ranging 40-90%, was arranged in one- to three-cell clusters at all other stages of life (P < 0.05, chi 2 test).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Distribution of transplanted cells according to cluster sizes in the liver of older DPPIV- rats


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alison, M, Golding M, Lalani EN, Nagy P, Thorgeirsson S, and Sarraf C. Wholesale hepatocytic differentiation in the rat from ductular oval cells, the progeny of biliary stem cells. J Hepatol 26: 343-352, 1997[ISI][Medline].

2.   Alison, MR, and Wright NA. The Biology of Epithelial Cell Populations. Oxford, UK: Oxford Univ. Press, 1985.

3.   Arber, N, Zajicek G, and Ariel I. The streaming liver. II. Hepatocyte life history. Liver 8: 80-87, 1988[ISI][Medline].

4.   Beyer, HS, Ellefson M, Sherman R, and Zieve L. Aging alters ornithine decarboxylase and decreases polyamines in regenerating rat liver but putrescine replacement has no effect. J Lab Clin Med 119: 38-47, 1992[ISI][Medline].

5.   Beyer, HS, Sherman R, and Zieve L. Aging is associated with reduced liver regeneration and diminished thymidine kinase mRNA content and enzyme activity in the rat. J Lab Clin Med 117: 101-108, 1991[ISI][Medline].

6.   Bucher, NLR Regeneration of the mammalian liver. Int Rev Cytol 15: 245-300, 1963[ISI].

7.   Dabeva, M, Hurston E, and Shafritz DA. Transcription factor and liver-specific mRNA expression in facultative epithelial progenitor cells of liver and pancreas. Am J Pathol 147: 1633-1648, 1995[Abstract].

8.   Eustis, SL, Boorman GA, Harada T, and Popp JA. Liver. In: Pathology of the Fischer Rat, edited by Boorman GA, Eustis SL, Elwell MR, Montgomery CA, Jr, and MacKenzie WF.. San Diego: Academic, 1990, p. 71-94.

9.   Fausto, N. Liver stem cells. In: Liver: Biology and Pathobiology, edited by Arias IM, Boyer JM, Fausto N, Jacoby WB, Schachter DA, and Shafritz DA.. New York: Raven, 1994, p. 1501-1518.

10.   Fox, IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI, Dorko K, Sauter BV, and Strom SC. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 338: 1422, 1998[Free Full Text].

11.   Fry, M, Silber J, Loeb LA, and Martin GM. Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice. J Cell Physiol 118: 225-232, 1984[ISI][Medline].

12.   Grisham, JW. A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver; autoradiography with thymidine-H3. Cancer Res 22: 842-849, 1962[ISI].

13.   Grossman, M, Rader DJ, Muller DWM, Kolansky DM, Kozarsky K, Clark BJ, III, Stein EA, Lupien PJ, Bryan Brewer H, Jr, Raper SE, and Wilson JM. A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia. Nat Med 1: 1148-1154, 1995[ISI][Medline].

14.   Gupta, S, Gorla GR, and Irani AN. Hepatocyte transplantation: emerging insights into mechanisms of liver repopulation and their relevance to potential therapies. J Hepatol 30: 162-171, 1999[ISI][Medline].

15.   Gupta, S, Rajvanshi P, Aragona E, Yerneni PR, Lee C-D, and Burk RD. Transplanted hepatocytes proliferate differently after CCl4 treatment and hepatocyte growth factor infusion. Am J Physiol Gastrointest Liver Physiol 276: G629-G638, 1999[Abstract/Free Full Text].

16.   Gupta, S, Rajvanshi P, and Lee C-D. Integration of transplanted hepatocytes in host liver plates demonstrated with dipeptidyl peptidase IV deficient rats. Proc Natl Acad Sci USA 92: 5860-5864, 1995[Abstract/Free Full Text].

17.   Gupta, S, Rajvanshi P, Sokhi R, Vaidya S, Irani AN, and Gorla GR. 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, 1999[Abstract/Free Full Text].

18.   Kennedy, S, Rettinger S, Flye MW, and Ponder KP. Experiments in transgenic mice show that hepatocytes are the source for postnatal liver growth and do not stream. Hepatology 22: 160-168, 1995[ISI][Medline].

19.   Kozurkova, M, Misurova E, and Kropacova K. Aging and radiation induced alterations of histones in regenerating rat liver. Mech Ageing Dev 72: 37-48, 1993[ISI][Medline].

20.   Laconi, E, Oren R, Mukhopadhyay DK, Hurston E, Laconi S, Pani P, Dabeva M, and Shafritz DA. Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am J Pathol 153: 319-329, 1998[Abstract/Free Full Text].

21.   LeBouton, AV. Growth, mitosis and morphogenesis of the simple liver acinus in neonatal rats. Dev Biol 41: 22-30, 1974[ISI][Medline].

22.   Michalopoulos, GK, and DeFrances MC. Liver regeneration. Science 276: 60-66, 1997[Abstract/Free Full Text].

23.   Mignon, A, Guidotti JE, Mitchell C, Fabre M, Wernet A, de La Coste A, Soubrane O, Gilgenkrantz H, and Kahn A. Selective repopulation of normal mouse liver by Fas/CD95-resistant hepatocytes. Nat Med 10: 1185-1188, 1998.

24.   Overturf, K, Al-Dhalimy M, Ou C-N, Finegold M, and Grompe M. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 151: 1273-1280, 1997[Abstract].

25.   Panduro, A, Shalaby F, and Shafritz DA. Changing patterns of transcriptional and post-transcriptional control of liver-specific gene expression during rat development. Genes Dev 1: 1172-1182, 1987[Abstract].

26.   Rajvanshi, P, Kerr A, Bhargava KK, Burk RD, and Gupta S. Studies of liver repopulation using the dipeptidyl peptidase IV deficient rat and other rodent recipients: cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule. Hepatology 23: 482-496, 1996[ISI][Medline].

27.   Rhim, JA, Sandgren EP, Degen JL, Palmiter RD, and Brinster RL. Replacement of diseased mouse liver by hepatic cell transplantation. Science 263: 1149-1152, 1994[ISI][Medline].

28.   Ross, MH. Aging, nutrition and hepatic enzyme activity patterns in the rat. J Nutr 97: 563-602, 1969.

29.   Sanz, N, Diez-Fernandez C, Alvarez AM, Fernandez-Simon L, and Cascales M. Age-related changes on parameters of experimentally-induced liver injury and regeneration. Toxicol Appl Pharmacol 154: 40-49, 1999[ISI][Medline].

30.  Schapiro H, Hotta SS, Outten WE, and Klein AW. The effect of aging on rat liver regeneration. Experientia 38: 1075-1076.

31.   Schmucker, DL. Aging and the liver: an update. J Gerontol Suppl 53: B315-B320, 1998.

32.   Sell, S. Liver stem cells. Mod Pathol 7: 105-112, 1994[ISI][Medline].

33.   Sigal, SH, Rajvanshi P, Gorla GR, Saxena R, Sokhi RP, Gebhardt DF, Jr, Reid LM, and Gupta S. Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. Am J Physiol Gastrointest Liver Physiol 276: G1260-G1272, 1999[Abstract/Free Full Text].

34.   Tsukamoto, I, Nakata R, and Kojo S. Effect of ageing on rat liver regeneration after partial hepatectomy. Biochem Mol Biol Int 30: 773-778, 1993[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 279(3):G631-G640
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society