Metabolic activity of rat hepatocytes cultured on homologous acellular matrix and transplanted into Gunn rats (original) (raw)
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Hepatology, 2002
The treatment of inherited metabolic liver diseases by hepatocyte transplantation (HT) would be greatly facilitated if the transplanted normal hepatocytes could be induced to proliferate preferentially over the host liver cells. We hypothesized that preparative hepatic irradiation (HIR) should inhibit host hepatocyte proliferation in response to partial hepatectomy (PH). Normal nonirradiated hepatocytes transplanted in this setting should have a selective growth advantage over the host liver cells and should progressively repopulate the liver. To test this hypothesis, we transplanted 5 million hepatocytes from normal Wistar-Roman High Avoidance (RHA) rats into the livers of congeneic bilirubin-uridine 5-diphosphoglucuronate glucuronosyltransferase (UGT1A1)-deficient jaundiced Gunn rats by intrasplenic injection after one of the following treatments: (1) 68% PH, (2) HIR (50 Gy), or (3) HIR ؉ PH. In rats receiving either PH or HIR alone before HT, serum bilirubin concentrations declined by 25% to 30% in 28 weeks. In contrast, serum bilirubin levels were normalized completely in rats receiving HIR ؉ PH before HT. Massive repopulation of the Gunn rat liver by the UGT1A1-positive Wistar-RHA hepatocytes was shown by UGT1A1 enzyme assay, immunoblot analysis, and immunohistochemical staining of the recipient liver. High-performance liquid chromatography analysis of the bile collected from Gunn rats 5 months after PH, HIR, and HT showed normalization of the pigment profile, with bilirubin diglucuronide and monoglucuronide as the predominant pigments. In conclusion, a preparative regimen of HIR ؉ PH results in massive repopulation of the liver with functionally normal transplanted hepatocytes, resulting in complete correction of a metabolic deficiency. Noninvasive strategies to replace PH for providing proliferative stimuli to the transplanted cells should make this regimen valuable in augmenting the effects of HT for the treatment of liver diseases. (HEPATOLOGY 2002; 36:354-362.)
Journal of Pediatric Surgery, 1992
Orthotopic liver transplantation as treatment for hereditary enzyme deficiences in the absence of cirrhosis suffers from significant operative risks, complications, and donor shortages. Transplantation of isolated hepatocytes (HTX) may offer opportunities for the treatment of these diseases and retain the recipient liver. Hepatocytes transplanted into the portal vein, spleen, or omentum lack an ideal growing environment for cell proliferation and maintenance. Therefore, we investigated a method combining 75% recipient hepatectomy with direct injection of hepatocytes into the remaining 25% of liver parenchyma to provide proliferative stimuli and a stable environment during and following liver regeneration. Recipient Gunn rats (glucuronyltransferase deficiency and hyperbilirubinemia) underwent hepatectomy before HTX by direct injection of IO7 isolated hepatocytes into the remaining parenchyma. Inbred male Wistar and Gunn rats were used as normal and control hepatocyte donors and saline injection served as a sham transplant control. Isolation of donor hepatocytes was performed with a two-step collagenase digestive method (Seglen) with cell viability of 85% to 95%. Liver regeneration was complete by 2 weeks posttransplant. Four weeks following HTX, total serum bilirubin and qualitative bile analysis were performed. A significant decrease in total serum bilirubin levels was observed in Gunn rats receiving Wistar hepatocytes compared with those receiving Gunn hepatocytes and saline control. Bile analysis from HTX rats demonstrated a normal pattern containing bilirubin monoglucuronides and diglucuronides (conjugated bilirubin) in the rats receiving Wistar hepatocytes, whereas the control group receiving Gunn hepatocytes or saline injection demonstrated only unconjugated bilirubin. No differences in histological appearance were noted between groups. We conclude that partial hepatectomy may provide the required stimulus for proliferation of transplanted hepatocytes and the liver may provide the necessary environment for optimal hepatocyte function and bile excretion following transplantation.
Treatment of enzyme deficiency by hepatocyte transplantation in rats
Journal of Surgical Research, 1985
The inbred, homozygous Gunn rat exhibits unconjugated hyperbilirubinemia due to a hereditary absolute deficiency of bilirubin UDP-glucuronyltransferase activity. The mechanism of action of hepatocyte transplantation (HTX) in the treatment of enzyme deficiency has been investigated in this study. Gunn rats underwent HTX by the injection of isolated hepatocytes from a nondeficient donor rat (Wistar) into the spleen. A transient, statistically significant decrease in total plasma bilirubin (TB) levels was observed. Gunn rats receiving Gunn hepatocytes did not show such a decrease. Histological examination 2-3 months post-HTX of the recipient spleens showed the absence of grafted hepatocytes in the first group and graft survival in the second. Bile specimens from sublethal irradiated Gunn rats, collected 6 days after HTX with viable Wistar hepatocytes, all contained bilirubin mono-and diglucuronides. Control groups consisting of Gunn rats receiving nonviable Wistar hepatocytes or Gunn hepatocytes, and sham-operated Gunn rats did not excrete bilirubin glucuronides in bile. It was also demonstrated that bilirubin UDP-glucuronyltransferase activity, which appeared in Gunn rats after HTX with Wistar hepatocytes, was only transient. It is concluded that the decrease of TB in the Gunn rat after HTX with nondeficient hepatocytes is explained by the appearance of the enzyme, which was absent in the recipient animal. Viable, nondeficient hepatocytes are required for the elicited bilirubin conjugation. Rejection of the transplanted hepatocytes abolishes this effect. o 1985 Academic Press, Inc.
Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats
Hepatology, 1998
To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n ؍ 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n ؍ 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor 1 (TGF-1) levels. Group 3 (n ؍ 16) rats received intrasplenic injection of isolated hepatocytes (2.5 ؋ 10 7 cells/rat) followed by total hepatectomy after 3 days. Group 4 (n ؍ 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 ؎ 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 ؎ 8.5 vs. 15.5 ؎ 4.8 hrs, P F .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-1 levels in rats rendered anhepatic. (HEPATOLOGY 1998;28: 1365-1370.) Transplantation of isolated hepatocytes (HcTx) has been shown to ameliorate, at least partially, specific enzymatic defects 1-4 and provide temporary support to animals with experimentally induced liver failure. 5-10 Preliminary results with HcTx in humans are also encouraging. 11-13 These studies indicate that transplanted hepatocytes can assume intact whole liver functions. However, whereas in the case of a genetic liver defect, the appearance of a missing gene product can be attributed solely to HcTx, the mechanism by which transplanted hepatocytes support recipients with physical liver injury (viral, toxic, ischemic) remains obscure. The observed beneficial effects, e.g., improved blood chemistry and neurological status, can be ascribed to transplanted cell function, to improved function of the native liver, or both. We have recently studied this problem and shown, for the first time, that in rats with fulminant hepatic failure (FHF), ectopically (spleen) implanted syngeneic hepatocytes improved regeneration of the native liver. 14 In the present study, we describe a novel single-stage technique of total hepatectomy (TH) in the rat and show that in rats rendered anhepatic, intrasplenic HcTx prolonged survival, improved blood chemistry, and partially corrected imbalance between blood levels of hepatocyte growth factor (HGF) and transforming growth factor 1 (TGF-1). MATERIALS AND METHODS Animal studies were conducted according to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. Animals Adult male Sprague Dawley rats weighing 150 to 350 gm (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were used. Rats were acclimatized to our laboratory conditions for 1 week before use in the experiments. They were housed in a climate-controlled (21°C) room under a 12-hour light/dark cycle. Rats were given tap water and commercial rat chow (Rodent Chow 5001, Ralston Purina, St. Louis, MO) ad libitum. All operations were performed under methoxyflurane (Mallinckordt Veterinary Inc., Mundelein, IL) general anesthesia with use of sterile surgical technique. All animals were warmed externally (heating lamp) during surgery and then until recovery from anesthesia. Chemicals Unless otherwise noted, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Hepatocyte Isolation Donor hepatocytes were isolated from the livers of Sprague-Dawley rats (ϳ150g) by in situ two-step ethylenediaminetetraacetic acid/collagenase digestion, as described previously. 15 Hepatocytes were further purified by sedimentation on Percoll density gradient (Pharmacia Biotech., Piscataway, NJ). Final hepatocyte viability was always greater than 90%, as determined by trypan blue exclusion.
Amplification of Engrafted Hepatocytes by Preparative Manipulation of the Host Liver
Artificial Organs, 2001
Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated
Cell Transplantation, 1998
Ⅺ Abstract -Ex vivo gene therapy, in which hepatocytes are harvested from mutants, retrovirally transduced with a normal gene and transplanted back into the donor, has been used for correction of inherited metabolic defects of liver. Major drawbacks of this method include limited availability of autologous hepatocytes, inefficient retroviral transduction of primary hepatocytes, and the limited number of hepatocytes that can be transplanted safely. To obviate these problems, we transduced primary hepatocytes derived from inbred bilirubin-UDP-glucuronosyltransferase (BUGT)-deficient Gunn rats by infection with a recombinant retrovirus expressing temperature-sensitive mutant SV40 large T antigen ( ts T). The immortalized cells were then transduced with a second recombinant retrovirus expressing human B-UGT, and a clone expressing high levels of the enzyme was expanded by culturing at permissive temperature (33°C). At 37°C, ts T antigen was degraded and the cells expressed UGT activity toward bilirubin at a level approximately twice that present in normal rat liver homogenates. For seeding the cells into the liver bed, 1 ؋ 10 7 cells were injected into the spleens of syngeneic Gunn rats five times at 10-day intervals. Excretion of bilirubin glucuronides in bile was demonstrated by HPLC analysis and serum bilirubin levels were reduced by 27 to 52% in 40 days after the first transplantation and remained so throughout the duration of the study (120 days). None of the transplanted Gunn rats or SCID mice transplanted with the immortalized cells developed tumors.
Mechanisms of reparative regeneration of the pathologically changed liver
Bulletin of Experimental Biology and Medicine, 1973
Restoration of the normal parenchyma during reparative regeneration of the liver was studied by histological, cytological, biochemical, and immunochemical methods in rats with experimental cirrhosis and hepatitis. Analysis of the mitotic activity of the hepatocytes, the dynamics of the change in the number of binuclear cells, and the change in size and ploidy of the mononuclear hepatocytes revealed some general and specific rules governing reparative regeneration and the reversibility of the pathological changes in these conditions. Somatic polyploidization is suggested as playing an essential role in the reparative regeneration of the pathologically changed liver.
Repeated intraportal hepatocyte transplantation in analbuminemic rats
Cell Transplantation, 1995
The optimal site for implantation of isolated hepatocytes has not been established. We have developed a novel technique which allows repeated infusion of hepatocytes into the portal system via an indwelling catheter. Seven Nagase Analbuminemic rats (NAR) underwent single intraportal infusion of 2 × 10 7 isolated normal albumin-producing rat hepatocytes. Another seven NAR rats underwent placement of indwelling catheters into the portal venous system via the gastroduodenal vein. Each of them received six batches of 5 × 106 normal albumin producing hepatocytes. Seven control NAR rats were infused rel~eatedly (intraportally) with saline only. Plasma albumin (ELISA) showed significant increase in experimental animals and was more pronounced (p < 0.05) in rats transplanted repeatedly than in those given a single dose of cells. Imraunohistochemical staining of the liver sections confirmed the presence of transplanted albumin producing hepatocytes. Rats transplanted with a single large batch of isolated hepatocytes showed liver tissue damage, whereas those subjected to repeated cell infusions had normal liver histology. We have developed a novel intraportal transplantation method which allows successful engraftment of a large number of isolated hepatocytes.