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Experimental Transplantation

TREATMENT OF SURGICALLY INDUCED ACUTE LIVER FAILURE BY TRANSPLANTATION OF CONDITIONALLY IMMORTALIZED HEPATOCYTES1,2

Nakamura, Junta3; Okamoto, Tomoyoshi3; Schumacher, Ingo K.3; Tabei, Isao3; Chowdhury, Namita Roy4; Chowdhury, Jayanta Roy4; Fox, Ira J.3,5

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Abstract

Acute liver failure is an uncommon clinical syndrome that affects previously healthy individuals. Orthotopic liver transplantation can improve the survival of comatose patients with acute liver failure and is currently the treatment of choice for patients with poor prognostic signs (1). Hepatocyte transplantation has been shown to improve survival in rats with surgically induced acute liver failure and has been considered a theoretical alternative to whole organ transplantation for patients with acutely decompensated liver function (2-4). Thus, transplanted hepatocytes could provide short-term hepatic function to support patients until their native livers recover from acute injury (5). Clinically, hepatocyte transplantation would require minimal intervention and would be unlikely to compromise native liver regeneration. Unfortunately, the lack of human livers available for whole organ or hepatocyte transplantation severely limits the use of either of these forms of therapy for the rescue of patients with acute liver failure (6).

A potential alternative source of liver cells for transplantation would be a clonal cell line. An hepatocyte cell line that could be engineered to revert from a transformed to a normal phenotype after transplantation would produce hepatocytes that could be grown in unlimited quantity and at far less cost than isolated hepatocytes. Cells derived from such a cell line would also have the advantages of uniformity, unlimited availability, and freedom from infectious pathogens.

We have created an hepatocyte cell line that can be used for transplantation by conditionally immortalizing rat hepatocytes using a thermolabile mutant simian virus 40 (SV40*) T antigen. The resulting hepatocyte clones proliferate at 33°C, the transforming gene permissive temperature, but stop growing and express relatively high levels of liver-specific proteins at the transforming gene nonpermissive temperatures, 37-39°C (7). In previous studies, we have shown that temperature-sensitive SV40 large T antigen (SV40ts)-conditionally immortalized hepatocytes integrate normally into hepatic cords upon transplantation into the liver and do not form tumors when transplanted into syngeneic rats or mice with severe combined immunodeficiency disease (7, 8). When transplanted into the spleens of portacavally shunted rats, the cells secrete bile, express albumin messenger RNA, and protect recipients from hyperammonemia-induced hepatic encephalopathy (8). We now report that SV40ts-conditionally immortalized hepatocytes can function as well as isolated primary hepatocytes in supporting life during hepatectomy-induced acute liver insufficiency when transplanted into the spleen or peritoneal cavity. We believe that these studies are the first to demonstrate that it is possible to transplant conditionally immortalized cells for the treatment of experimental liver injury. The proposed use of tightly regulated, conditionally immortalized cells is a novel and potentially reasonable alternative to the use of primary hepatocytes for transplantation. This work represents the initial step in developing an hepatocyte cell line that could partially alleviate the organ-donor shortage.

MATERIALS AND METHODS

Animals. Inbred male Lewis rats (200-300 g) were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and maintained in the Animal Resource Facility of the University of Nebraska College of Medicine. Rats were maintained on standard laboratory rat chow on a 12-hr/12-hr light/dark cycle. All procedures performed on rats were approved by the University of Nebraska Institutional Animal Care and Use Committee and thus were within the guidelines for humane care of laboratory animals.

Hepatocyte preparation and culture. Hepatocytes were harvested using a modification of the two-step collagenase perfusion technique introduced by Berry and Friend and adapted by Seglen (9, 10). Rats were anesthetized with methoxyflurane (Pitman Moore, Mundelein, IL) and given 1000 units of heparin by intraperitoneal injection. After a midline laparotomy was performed, an 18G catheter (Travenol Laboratories, Deerfield, IL) was inserted into the portal vein and connected to perfusate tubing. The inferior vena cava was cut and the liver perfused at 25 ml/min. The perfusion was carried out with Seglen's initial buffer at 37°C for 8-10 min and then switched to a solution of identical composition enriched with 2.5 mM calcium chloride and 0.04% collagenase (type IV, Sigma, St. Louis, MO). The collagenase perfusion was carried out at 37°C for another 8-10 min. The liver was excised, the capsule was removed, and with gentle shaking the cells were dispersed into ice-cold Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, NY). The cell suspension was filtered through a 250-μm mesh and centrifuged at 4°C at 500 rpm for 2 min. The cell pellet was resuspended in DMEM, filtered through a 100-μm mesh, and washed 2 times. Cell viability was determined by trypan blue exclusion and varied from 70 to 95%. Hepatocytes for conditional immortalization were then suspended in DMEM containing 4% fetal calf serum (Gibco), 0.2 mM dexamethasone (Sigma), 1% antibiotic-antimycotic solution (Gibco) and plated on tissue culture flasks (Nunc, Naperville, IL) at 6×106 cells per T75 flask. Cells were incubated at 37°C in a 95% air/5% CO2 atmosphere. Hepatocytes for primary hepatocyte transplantation were washed twice in DMEM and then transplanted.

Creation of conditionally immortalized rat hepatocytes. The conditionally immortalized hepatocyte cell line used for these studies has been previously described (8). Briefly, a Ψ-2 cell line (pZipSVtsA58) that produces an ecotropic, recombinant retrovirus containing the genes encoding a temperature-sensitive mutant of the SV40 large T antigen (tsA58) and neomycin phosphotransferase was kindly provided by P.S. Jat (Ludwig Institute of Cancer Research, London, UK) (11). This producer line provides a viral titer of 5×104 neomycin-resistant colony forming units/ml when assayed on NIH 3T3 cells. Viral supernatant was harvested from confluent plates of producer cells 18 hr after the addition of fresh medium and filtered through a 0.45-mm filter. Eighteen hours after harvesting, primary Lewis rat hepatocytes were transduced with 3 ml of Ψ-2 pZipSVtsA58 retrovirus supernatant per T75 flask in the presence of 12 mg/ml polybrene (Aldrich Chemical Co., Milwaukee, WI) at 37°C twice for 4 hr. The virus supernatant was then aspirated, and the cultures were incubated in DMEM containing 4% fetal calf serum, 0.2 mM dexamethasone, and 1% antibiotic-antimycotic solution at 33°C. Transformed hepatocyte colonies emerged within 3 weeks. After 5-6 weeks, individual colonies were isolated using cloning rings and expanded by culturing at 33°C. Cultured at 33°C, the immortalized hepatocytes were smaller than primary hepatocytes, grew in monolayers, doubled in number approximately every 40 hr, and required low serum concentrations for growth. When cultured at 37°C or 39°C, the cells regained the morphologic characteristics of differentiated hepatocytes and ceased to proliferate within a 48-hr study period. One clone, L2A2, was selected for use in transplant experiments based on growth characteristics and morphology.

Induction of acute liver failure. Acute liver failure was induced by 90% hepatectomy as described previously (12, 13). A midline laparotomy was performed and 90% hepatectomy was done by removing the median and left lateral liver lobes by ligation. The inferior portion of the right lobe was then ligated flush to the vena cava and removed. The superior portion of the right lobe was drawn anteriorly and to the left to free its posterior attachment to the diaphragm. The lobe was then removed with two ligatures to clear the parenchyma posterior to the vena cava without causing caval compression, leaving only the caudate lobe. After confirming hemostasis, 1 ml of normal saline was injected intravenously and 20 mg/kg gentamicin (SoloPak Laboratories Inc., Elk Grove Village, IL) was administered intraperitoneally. The mean liver mass removed was 7.3±0.8 g. Rats were kept in an incubator that provided an environmental temperature greater than 28°C after surgery and were allowed free access to food and tap water supplemented with 5% dextrose. Core body temperature was maintained above 36°C within 2 hr of surgery for all animals.

Preparation of transplanted cells. Primary hepatocytes were prepared as described and transplanted fresh. Immortalized hepatocytes were grown at 33°C and recovered using 0.25% trypsin solution (Life Technologies Inc., Grand Island, NY) and washed twice with DMEM before transplantation. Nucleated bone marrow cells were harvested from femurs of Lewis rats as previously described (14), washed twice in DMEM, and counted using a Coulter counter (Coulter Electronics Inc., Hialeah, FL). Cell homogenates were prepared by sonication for 3 min on ice (Sonicator Cell Disruptor model W220F, Heat-Systems-Ultrasonics, Inc., Long Island, NY), and a portion of the sonicated samples was examined by light microscopy to confirm the loss of cell integrity.

Hepatocyte transplantation. All animals were anesthetized using methoxyflurane. For intrasplenic hepatocyte transplantation, a small surgical incision was made in the animal's flank and the spleen was exposed. One day before the hepatectomy, 10×106 primary or SV40ts-conditionally transformed hepatocytes, suspended in 0.5 ml of DMEM, were injected into the inferior pole of the spleen using a 26-gauge needle. The blood flow in the splenic artery and vein were temporarily occluded to avoid immediate passage of cells out of the spleen and into the portal vein during transplantation. At the end of the injection, the injection site was also ligated to prevent cell leakage and bleeding.

Intraperitoneal hepatocyte transplantation was performed immediately after the hepatectomy. For these studies, 200×106 cells or equivalent cell homogenate, suspended in 3 ml of DMEM, were injected into the lower peritoneal cavity to avoid leakage. Transplanted animals were sacrificed at 4 days, 1 week, and 2 weeks after transplantation to assess liver regeneration and evidence of hepatocyte engraftment. Spleen, peritoneum, and liver specimens were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. Serial tissue sections were cut and stained with hematoxylin and eosin.

The experimental design resulted in eight animal groupings. All animals underwent 90% hepatectomy and were divided into the following groups:

  • Group 1 (G1): no treatment (n=6) or intraperitoneal injection of DMEM (n=9).
  • Group 2 (G2): intrasplenic transplantation of 10×106 primary Lewis rat hepatocytes 1 day before hepatectomy (n=6).
  • Group 3 (G3): intrasplenic transplantation of 10×106 SV40ts-conditionally immortalized Lewis rat hepatocytes 1 day before hepatectomy (n=8).
  • Group 4 (G4): intraperitoneal transplantation of 200×106 primary Lewis rat hepatocytes (n=10).
  • Group 5 (G5): intraperitoneal transplantation of 200×106 SV40ts-conditionally immortalized Lewis rat hepatocytes (n=10).
  • Group 6 (G6): intraperitoneal transplantation of 200×106 Lewis rat bone marrow cells (n=7).
  • Group 7 (G7): intraperitoneal transplantation of a cellular homogenate of 200×106 primary Lewis rat hepatocytes (n=9).
  • Group 8 (G8): intraperitoneal transplantation of a cellular homogenate of 200×106 SV40-conditionally immortalized Lewis rat hepatocytes (n=10).

Plasma glucose monitoring. Plasma glucose levels were monitored daily using blood obtained from the tail for the first 3 days postoperatively. After whole blood centrifugation, plasma glucose level was measured using a Glucose Analyzer 2R (Beckman Instruments, Palo Alto, CA).

Statistics. Survival rates were analyzed using the Wilcoxon method, and time-point comparisons were made using the z test. Plasma glucose levels were compared using Mann-Whitney's U test. Values are expressed as mean ± standard deviation.

RESULTS

Survival of rats after 90% hepatectomy and intrasplenic hepatocyte transplantation. Rats were divided into three groups for these studies. Animals underwent 90% hepatectomy only (G1; n=6); 90% hepatectomy and transplantation of 10×106 primary hepatocytes 1 day before hepatectomy (G2; n=6); or 90% hepatectomy and transplantation of 10×106 SV40-conditionally immortalized hepatocytes 1 day before hepatectomy (G3; n=8). All rats that underwent 90% hepatectomy only died within 96 hr, whereas 50% of animals that received either primary or SV40-conditionally immortalized hepatocyte transplants survived for more than 96 hr (Fig. 1A). All animals surviving for greater than 96 hr survived for the duration of the experiments (>100 days) or until they were euthanized. The difference in survival among groups was statistically significant (P<0.05 for G1 vs. G2 or G3). Plasma glucose levels indicated that all rats that survived maintained glucose levels near normal (plasma glucose>75 mg/dl). In contrast, all nonsurviving rats became persistently hypoglycemic after hepatectomy (plasma glucose<50 mg/dl) (Fig. 2, A and B).

Histologic examination of liver and spleen tissue in rats after 90% hepatectomy and intrasplenic hepatocyte transplantation. Histological examination of liver tissue taken from surviving (mean remnant liver weight on day 5: 5.67±0.50 g) and nonsurviving (mean remnant liver weight on day 2: 0.723±0.09 g) rats that underwent 90% hepatectomy showed severe microvesicular steatosis and hepatocyte necrosis, mostly centrilobular (Fig. 3). Four days after hepatectomy, spleens taken from surviving rats that underwent intrasplenic transplantation with primary hepatocytes contained numerous large hepatocyte clusters (250-400 μm) in the middle and lower portion of the spleen (Fig. 4). Spleens from surviving rats transplanted with conditionally immortalized hepatocytes also contained numerous hepatocyte clusters, although individual clusters were smaller (150-200 μm) than those in spleens from animals transplanted with primary hepatocytes (Fig. 5). The number of transplant clusters decreased by day 7 after hepatectomy, and almost no transplanted hepatocytes, primary or immortalized, could be identified in the spleens of surviving rats 14 days after hepatectomy. Very few hepatocytes could be identified in nonsurviving rats from groups G2 or G3 despite hepatocyte transplantation.

Survival of rats following 90% hepatectomy and intraperitoneal cell transplantation. Animals underwent 90% hepatectomy and intraperitoneal transplantation of 200×106 primary hepatocytes at the time of hepatectomy (G4; n=10); 90% hepatectomy and transplantation of 200×106 SV40ts-conditionally immortalized hepatocytes at the time of hepatectomy (G5; n=10); or 90% hepatectomy and transplantation of 200×106 syngeneic nucleated whole bone marrow cells (G6; n=7). Eighty percent of animals that received either 200×106 primary Lewis rat hepatocytes or 200×106 SV40ts-conditionally immortalized intraperitoneal hepatocyte transplants survived for more than 96 hr (Fig. 1B). Again, all animals in these studies surviving for greater than 96 hr survived for the duration of the experiments (>100 days) or until they were euthanized. The difference in survival between the groups receiving primary versus conditionally immortalized hepatocytes was not statistically significant but was statistically different from that of animals receiving no treatment or DMEM only (P<0.01). Survival after intraperitoneal transplantation of an equivalent number of syngeneic nucleated bone marrow cells was 29% and was statistically different from that of animals receiving primary hepatocyte transplantation, conditionally immortalized hepatocyte transplantation, or no treatment or DMEM only (P<0.05 for G6 vs. G4 or G5; P<0.05 for G6 vs. G1). Plasma glucose levels again indicated that all rats that survived maintained glucose levels near normal, whereas nonsurviving rats became persistently hypoglycemic after hepatectomy (Fig. 2, C and D).

Histologic examination of peritoneal tissue in rats after 90% hepatectomy and intraperitoneal hepatocyte transplantation. No evidence of hepatocyte engraftment was evident histologically on peritoneal surfaces up to 4 days after hepatectomy from either the surviving or nonsurviving animals transplanted intraperitoneally with either primary or conditionally transformed hepatocytes. However, there was also no peritoneal reaction or fibrosis that would interfere with later whole organ transplantation.

Survival of rats after 90% hepatectomy and intraperitoneal transplantation of cellular homogenates. Animals underwent either 90% hepatectomy and intraperitoneal transplantation of a cellular homogenate from 200×106 primary hepatocytes at the time of hepatectomy (G7; n=9); or 90% hepatectomy and transplantation of a cellular homogenate of 200×106 SV40ts-conditionally immortalized hepatocytes at the time of hepatectomy (G8; n=10). Thirty-three percent of animals that received the homogenate from 200×106 primary Lewis rat hepatocytes and 40% of animals that received the homogenate from 200×106 SV40ts-conditionally immortalized hepatocytes survived for more than 96 hr (Fig. 1B). The difference in survival after 96 hr between the groups receiving homogenate from primary versus conditionally immortalized hepatocytes was not statistically significant. Survival was statistically different from that of animals receiving no treatment or media only (P<0.05) and animals receiving 200×106 primary or conditionally immortalized hepatocytes (P<0.01) but not from that of animals receiving 200×106 syngeneic bone marrow cells intraperitoneally.

DISCUSSION

The efficacy of hepatocyte transplantation in altering pathologic processes has been extensively studied in rodents. Several laboratories have demonstrated that transplantation of normal isolated hepatocytes into the spleen, liver, or peritoneal cavity of syngeneic or allogeneic laboratory animals can correct liver-based metabolic deficiencies (15-18) or improve survival after chemically or surgically induced acute liver failure (19-22). In addition, when hepatocytes from adult mice are transplanted into the livers of mice with DNA that contains a transgene that damages hepatocytes, 80% of their native livers are replaced with donor hepatocytes (23). Thus, theoretically, hepatocyte transplantation, could provide an alternative to orthotopic liver transplantation for many patients.

A major limitation to the application of this form of therapy is the present inability to isolate an adequate number of transplantable hepatocytes for clinical use. Cadaver donor livers with traumatic damage, excess macrovesicular fat, and residual segments from reduced liver transplants could provide a source of hepatocytes for transplantation. However, if the liver of a 70-kg adult contains approximately 3×1011 hepatocytes, reconstitution of 10% of the normal host hepatocyte mass would require transplantation of approximately 6×1010 hepatocytes, assuming 50% engraftment and cell survival. Using isolation techniques available at present, hepatocytes from more than one donor may be required to provide a sufficient number of cells for transplantation. In addition, because organ availability is critical, preserved cells will most likely be required for the treatment of acute liver failure. Unfortunately, the viability of cryopreserved hepatocytes can be quite variable and has been reported to decrease by as much as 30-60% after recovery (24, 25).

Because of the limited availability of human livers for either whole organ or hepatocyte transplantation, we have developed an hepatocyte cell line to examine whether conditionally immortalized hepatocytes could be used for transplantation in the treatment of liver insufficiencies. These present studies demonstrate that SV40-conditionally immortalized hepatocytes function as well as primary hepatocytes to reduce the mortality associated with surgically induced acute liver failure in rats.

None of the currently used animal models for testing the efficacy of hepatocyte transplantation in treating acute liver failure is free of problems. The literature contains numerous inconsistencies regarding the amount of hepatotoxin needed to induce reversible hepatic failure in rats (19-22, 26). In addition, lack of reproducibility using hepatotoxins in our own laboratory led to the use of the 90% hepatectomy model for our present studies. However, even in this model, in which 5% dextrose is given perioperatively to prevent early mortality from hypoglycemia, we and others (12) have been able to improve survival to nearly 50% by giving animals 20% glucose perioperatively (data not shown). Whether any of the techniques for inducing reversible acute liver failure in animals adequately parallels the clinical situation is questionable because none of them seems to significantly inhibit hepatic regeneration. This may explain why transplantation of cell homogenates (27, 28), nonhepatic cells (29), and hepatocytes with a cell volume equivalent to less than 1% of liver mass (2) appears to be mildly effective at altering survival in rodents with either chemically or surgically induced acute liver failure. In fact, the mechanism by which cell transplantation or homogenate infusion may affect survival in this animal model may be enhancement of liver regeneration (30).

The present study makes no attempt to address these issues but simply demonstrates the equivalence of transplanting primary and conditionally immortalized hepatocytes. In these studies, intrasplenic transplantation of either cell type improved survival to a level equal to that obtained by changing the concentration of glucose in the drinking water of animals after 90% hepatectomy. Thus, transplantation of such a small number of cells may provide a very limited amount of metabolic support that does not even require the function of intact hepatocytes. This is supported by the fact that transplantation of hepatocyte homogenates and bone marrow cells was almost as effective at compensating for diminished liver function as either intrasplenic transplantation of liver cells or increasing the glucose in the drinking water to 20%.

Intrasplenic transplantation of either primary or conditionally immortalized hepatocytes produced numerous microscopic clumps of transplanted cells in the spleen 4 days after hepatectomy. The number of cells that could be identified histologically decreased over time after transplantation. This could have resulted from migration of transplanted cells out of the spleen into the liver or from failure of intrasplenic engraftment, which might happen as a consequence of the decrease in hepatatrophic factor production associated with completed liver regeneration (31). In either case, the intrasplenically transplanted, conditionally immortalized hepatocytes exhibited the same general pattern of engraftment at 4 days as did the primary hepatocytes, although the conditionally immortalized hepatocytes appeared morphologically different at that time. Based on previous work from our laboratory that demonstrated histologically normal looking hepatocytes 30 days after transplantation, it is likely that the transplanted, conditionally immortalized cells take longer than 4 days to completely revert to a more normal appearing phenotype (8).

Although the efficacy of free intraperitoneal hepatocyte transplantation is controversial, the peritoneal cavity is potentially capable of engrafting a large quantity hepatocytes at one time (28, 32). In this report, intraperitoneal transplantation of either primary or conditionally immortalized hepatocytes improved the survival of 90% hepatectomized rats to 80%, significantly greater than what could be obtained with intrasplenic hepatocyte transplantation. Thus, the peritoneal cavity allowed the transplantation of 20 times more hepatocytes than did the intrasplenic approach. However, unlike the situation associated with intrasplenic transplantation, no histological evidence could be obtained that could prove that the intraperitoneally transplanted cells had engrafted.

Although it has been suggested that vascularization is required for function of intraperitoneally transplanted hepatocytes, our present data indicate that this requirement can be overcome be transplanting approximately 10% of the liver cell mass. One could argue that survival was altered simply by the significantly increased mass of cells or cell products transplanted. Our data do not support this conclusion. In fact, we have shown in the Gunn rat that intraperitoneally transplanted hepatocytes functioned within 6 hr of injection (33) and continued to function for up to 6 weeks. Function deteriorated at that time presumably as a result of failure of long-term engraftment. Thus, clinical intraperitoneal transplantation could be performed to transplant a large number of cells quickly. It would by easier to perform and would be associated with fewer risks. In addition, our studies revealed no evidence of significant intraabdominal scarring after intraperitoneal hepatocyte transplantation that would inhibit later whole organ transplantation if needed.

In conclusion, the present study demonstrates that transplanted conditionally immortalized hepatocytes function as well as primary hepatocytes at improving the survival of rats with surgically induced acute liver failure. When these data are added to those from previously reported studies showing that conditionally immortalized hepatocytes engraft normally when transplanted in the liver, reverse ammonium acetate-induced hepatic encephalopathy in portacavally shunted rats, and correct liver-based metabolic deficiencies (8, 34, 35), it appears that conditionally immortalized hepatocytes may be able to replace the function of host hepatocytes for some liver disorders. These experiments also indicate that transplantation of hepatocytes into the peritoneal cavity may be effective, at least temporarily, at supporting patients with acute liver failure. Development of this technology will require production of hepatocyte cell lines with tighter regulation on the expression of the transforming gene than a simple temperature-sensitive mutation. Site-specific recombination could be used to excise the transforming gene from a cell line prior to transplantation (36), and additional safeguards could be provided by using a combination of inducible promoters and transactivating factors to tightly control the expression of the transforming gene or by the introduction of a “suicide gene,” such as the herpes simplex virusthymidine kinase gene, into such cells. These experiments represent the first step in developing a tightly regulated, conditionally immortalized hepatocyte cell line that could be of potential value in the clinical management of patients with acute liver failure.

Acknowledgments. The authors thank Dr. Kashinath Patil, Department of Preventive and Societal Medicine, University of Nebraska Medical Center, for assistance with the statistical analysis.

F1-1
Figure 1:
(A) Survival of rats after 90% hepatectomy and intrasplenic transplantation with 10 million hepatocytes 1 day before hepatectomy. Survival at 72 hr postoperatively for the control group (G1, -·-·-), the primary hepatocyte transplant group (G2, ·····), and the conditionally immortalized hepatocyte transplant group (G3, -) was 0%, 50%, and 50%, respectively (P<0.05 for G1 vs. G2 or G3; nonsignificant for G2 vs. G3). (B) Survival of rats after 90% hepatectomy and intraperitoneal transplantation with 200 million cells or cell equivalents at the time of hepatectomy. Survival at 96 hr postoperatively for the control group (G1, -·-·-), the primary hepatocyte transplant group (G4, ·····), the conditionally immortalized hepatocyte transplant group (G5, -), the bone-marrow cell transplant group (G6, -·-), the primary hepatocyte homogenate transplant group (G7, -----), and the conditionally immortalized hepatocyte homogenate transplant group (G8, ---) was 0%, 80%, 80%, 29%, 33%, and 40%, respectively (P<0.01 for G1 vs. G4 or G5; P<0.05 for G1 vs. G7 or G8; nonsignificant for G4 vs. G5 and for G7 vs. G8).
F2-1
Figure 2:
Plasma glucose levels after 90% hepatectomy and transplantation. (A) Survivors among the intrasplenic transplantation groups. (B) Nonsurvivors among the intrasplenic transplantation groups. (C) Survivors among the intraperitoneal transplantation groups. (D) Nonsurvivors among the intraperitoneal transplantation groups. Symbols indicate the control group (G1, □), the primary hepatocyte transplant group (G2 and G4, ○), the conditionally immortalized hepatocyte transplant group (G3 and G5, •), the bonemarrow cell transplant group (G6, ▪), the primary hepatocyte homogenate transplant group (G7, ▵), and the conditionally immortalized hepatocyte homogenate transplant group (G8, ▴). Data indicate that nonsurvivors were hypoglycemic whereas survivors remained euglycemic. This trend did not depend on the type of intervention that each experimental animal received. Data shown are the mean ± SD.
F3-1
Figure 3:
Histologic sections of remnant liver tissue (caudate lobe) taken 3 days after hepatectomy (hemotoxylin and eosin, ×200). Severe microvesicular steatosis and necrosis of hepatocytes is seen, particularly in the centrilobular areas. No differences were noted in liver histology between survivor and nonsurvivor animals at day 3 after hepatectomy.
F4-1
Figure 4:
Histologic sections demonstrating transplanted primary hepatocytes in the spleen (G2) 4 days after hepatectomy (hematoxylin and eosin, ×200). Numerous large hepatocyte clusters were found in the middle and lower portions of the spleen.
F5-1
Figure 5:
Histologic sections demonstrating transplanted conditionally immortalized hepatocytes in the spleen (G3) 4 days after hepatectomy (hematoxylin and eosin, ×200). Numerous hepatocyte clusters were found in the middle and lower portions of the spleen; however, rosette-like individual clusters are smaller than those found after primary hepatocyte transplantation.

Footnotes

Presented, in part, at the 22nd Annual American Society of Transplant Surgeons Meeting, Dallas, May 29-31, 1996.

This work was supported in part by National Institutes of Health grants AI31641, DK48794 (I.J.F.), DK39137 (N.R.C.), and DK46057(J.R.C.).

Abbreviations: DMEM, Dulbecco's modified Eagle medium; SV40, simian virus 40; SV40ts, temperature-sensitive SV40 large T antigen.

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