Pankaj Rajvanshi

Efficacy and safety of repeated hepatocyte transplantation for significant liver repopulation in rodents

BACKGROUND & AIMS Significant liver repopulation by hepatocyte transplantation will advance clinical applications. The aim of this study was to test the hypothesis that translocation of transplanted cells into liver plates will allow repeated cell transplantation for increasing the transplanted hepatocyte mass. METHODS Hepatocytes were transplanted via spleen from either F344 rats into syngeneic recipients deficient in dipeptidyl peptidase IV or from transgenic hepatitis B surface antigen-producing G26 mice with hepatitis B virus into nontransgenic congeneic recipients. Portosystemic shunting was shown by radiological methods. RESULTS Repeated hepatocyte transplantation led to progressively increased liver repopulation. Transplantation of 1.75 x 10(8) hepatocytes in three divided doses repopulated more than an estimated 5% of the host rat liver, with 3.8 x 10(6) +/- 0.1 x 10(6) transplanted cells/cm3 liver. This was a tenfold or threefold mean increase in transplanted cell number compared with recipients of 2.0 x 10(7) or 7.5 x 10(7) cells transplanted in single sessions, respectively (P < 0.001). Repeated hepatocyte transplantation interfered with neither cell integrations in liver parenchyma nor secretory function of transplanted cells. Portal hypertension, portasystemic collaterals, or intrahepatic shunting were not observed in cell recipients. CONCLUSIONS Repeated transplantation of hepatocytes in large numbers is safe and effective and should advance strategies for liver repopulation.

Transplanted hepatocytes proliferate differently after CCl4 treatment and hepatocyte growth factor infusion

To understand regulation of transplanted hepatocyte proliferation in the normal liver, we used genetically marked rat or mouse cells. Hosts were subjected to liver injury by carbon tetrachloride (CCl4), to liver regeneration by a two-thirds partial hepatectomy, and to hepatocellular DNA synthesis by infusion of hepatocyte growth factor for comparative analysis. Transplanted hepatocytes were documented to integrate in periportal areas of the liver. In response to CCl4 treatments after cell transplantation, the transplanted hepatocyte mass increased incrementally, with the kinetics and magnitude of DNA synthesis being similar to those of host hepatocytes. In contrast, when cells were transplanted 24 h after CCl4 administration, transplanted hepatocytes appeared to be injured and most cells were rapidly cleared. When hepatocyte growth factor was infused into the portal circulation either subsequent to or before cell transplantation and engraftment, transplanted cell mass did not increase, although DNA synthesis rates increased in cultured primary hepatocytes as well as in intact mouse and rat livers. These data suggested that procedures causing selective ablation of host hepatocytes will be most effective in inducing transplanted cell proliferation in the normal liver. The number of transplanted hepatocytes was not increased in the liver by hepatocyte growth factor administration. Repopulation of the liver with genetically marked hepatocytes can provide effective reporters for studying liver growth control in the intact animal.To understand regulation of transplanted hepatocyte proliferation in the normal liver, we used genetically marked rat or mouse cells. Hosts were subjected to liver injury by carbon tetrachloride (CCl4), to liver regeneration by a two-thirds partial hepatectomy, and to hepatocellular DNA synthesis by infusion of hepatocyte growth factor for comparative analysis. Transplanted hepatocytes were documented to integrate in periportal areas of the liver. In response to CCl4 treatments after cell transplantation, the transplanted hepatocyte mass increased incrementally, with the kinetics and magnitude of DNA synthesis being similar to those of host hepatocytes. In contrast, when cells were transplanted 24 h after CCl4 administration, transplanted hepatocytes appeared to be injured and most cells were rapidly cleared. When hepatocyte growth factor was infused into the portal circulation either subsequent to or before cell transplantation and engraftment, transplanted cell mass did not increase, although DNA synthesis rates increased in cultured primary hepatocytes as well as in intact mouse and rat livers. These data suggested that procedures causing selective ablation of host hepatocytes will be most effective in inducing transplanted cell proliferation in the normal liver. The number of transplanted hepatocytes was not increased in the liver by hepatocyte growth factor administration. Repopulation of the liver with genetically marked hepatocytes can provide effective reporters for studying liver growth control in the intact animal.

Transplanted hepatocytes engraft, survive, and proliferate in the liver of rats with carbon tetrachloride‐induced cirrhosis

Repopulation of the cirrhotic liver with disease‐resistant hepatocytes could offer novel therapies, as well as systems for biological studies. Establishing whether transplanted hepatocytes can engraft, survive, and proliferate in the cirrhotic liver is a critical demonstration. Dipeptidyl peptidase IV‐deficient F344 rats were used to localize transplanted hepatocytes isolated from the liver of syngeneic normal F344 rats. Cirrhosis was induced by administration of carbon tetrachloride with phenobarbitone and these drugs were withdrawn prior to cell transplantation. Cirrhotic rats showed characteristic hepatic histology, as well as significant portosystemic shunting. When hepatocytes were transplanted via the spleen, cells were distributed immediately in periportal areas, fibrous septa, and regenerative nodules of the cirrhotic liver. Although some transplanted cells translocated into pulmonary capillaries, this was not deleterious. At 1 week, transplanted cells were fully integrated in the liver parenchyma, along with expression of glucose‐6‐phosphatase and glycogen as reporters of hepatic function. Transplanted cells proliferated in the liver of cirrhotic animals and survived indefinitely. At 1 year, transplanted hepatocytes formed large clusters containing several‐fold more cells than normal control animals, which was in agreement with increased cell turnover in the cirrhotic rat liver. The findings indicate that the cirrhotic liver can be repopulated with functionally intact hepatocytes that are capable of proliferating. Liver repopulation using disease‐resistant hepatocytes will be applicable in chronic conditions, such as viral hepatitis or Wilsons disease. Copyright

Integration and proliferation of transplanted cells in hepatic parenchyma following D-galactosamine-induced acute injury in F344 rats

To determine whether liver repopulation with cell transplantation could be of therapeutic value in acute hepatic failure, it is necessary to establish the fate of transplanted hepatocytes. This study used dipeptidyl peptidase IV‐deficient F344 rats as recipients to analyse the engraftment and proliferation of transplanted hepatocytes. Syngeneic hepatocytes were transplanted intrasplenically 24–30 h after induction of liver injury by D‐galactosamine (GalN). Portosystemic shunting was analysed with 99m‐Tc‐labelled albumin microspheres. GalN‐treated rats showed characteristic hepatic necrosis, inflammation, gamma‐glutamyl transpeptidase activation, and regenerative activity, without increased portosystemic shunting (>99% 99m‐Tc activity was in the liver in normal and GalN‐treated rats). Transplanted cells entered hepatic sinusoids promptly and were observed in liver plates at 48 h. The number of transplanted cells increased in GalN‐treated rats by approximately seven‐fold (range two‐ to 12‐fold), along with evidence for DNA synthesis between 3 and 14 days after cell transplantation and greater prevalence of larger transplanted cell clusters. These findings indicate that the liver can be safely repopulated in animals with acute liver failure, although the time required for regenesis of plasma membrane structures and proliferation in transplanted hepatocytes will need to be considered in developing therapeutic strategies. Copyright

Differentiation-specific regulation of transgene expression in a diploid epithelial cell line derived from the normal F344 rat liver.

To establish the differentiation potential of progenitor cells, non‐parenchymal epithelial cells from the F344 rat liver (FNRL cells) were studied. These cells reacted with the OV‐6 antibody marker of oval cells, but were negative for hepatocyte markers (albumin, transferrin, glycogen, glucose‐6‐phosphatase, H4 antigen), biliary markers (gamma glutamyl transpeptidase, cytokeratin‐19), and α‐fetoprotein, although exposure to sodium butyrate induced nascent albumin and α‐fetoprotein mRNA transcription. When stably transduced, FNRL cells expressed a retroviral promotor‐driven lacZ reporter in vitro, similar to transgene expression in hepatocyte‐derived HepG2 cells. However, lacZ expression in FNRL cells was rapidly extinguished in intact animals, whereas the reporter remained active in HepG2 cells. Transplanted FNRL cells showed copious glucose‐6‐phosphatase expression; however, the cell differentiation programme remained incomplete, despite two‐thirds partial hepatectomy, D‐galactosamine treatment or bile duct ligation. Interestingly, lacZ expression resumed in cultures of FNRL cells explanted from recipients. Moreover, lacZ expression was down‐regulated by γ‐interferon in FNRL cells, without affecting lacZ activity in HepG2 cells. The data indicate that although subpopulations of oval cells may not fully differentiate into mature hepatocytes, these cells might serve critical functions, such as glucose utilization, and help survival after liver injury. Also, introduced genes may be regulated in progenitor cells at multiple levels, including by interactions between regulatory sequences, differentiation‐specific cellular factors, and extracellular signals; in vivo studies are thus especially important for analysing gene regulation in progenitor cells. Copyright

Analysis of hepatocyte distribution and survival in vascular beds with cells marked by 99mTC or endogenous dipeptidyl peptidase IV activity

Knowledge of the kinetics of cell distribution in vascular beds will help optimize engraftment of transplanted hepatocytes. To noninvasively localize transplanted cells in vivo, we developed conditions for labeling rat hepatocytes with 99mTc-pertechnetate. The incorporated o9mTc was bound to intracellular proteins and did not impair cell viability. When 99mTc hepatocytes were intrasplenically injected into normal rats, cells entered liver sinusoids with time-activity curves demonstrating instantaneous cell translocations. 99mTc activity in removed organs was in liver or spleen, and lungs showed little activity. However, when cells were intrasplenically transplanted into rats with portasystemic collaterals, 99mTc appeared in both liver sinusoids and pulmonary alveolar capillaries. To further localize cells, we transplanted DPPIV+ F344 rat hepatocytes into syngeneic DPPIV-recipients. Histochemical staining for DPPIV activity demonstrated engraftment of intrasplenically transplanted cells in liver parenchyma. In contrast, when 99mTc hepatocytes were injected into a peripheral vein, cells were entrapped in pulmonary capillaries but were subsequently broken down with redistribution of 99mTc activity elsewhere. Intact DPPIV+ hepatocytes were identified in lungs, whereas only cell fragments were present in liver, spleen, or kidneys. These findings indicate that although the pulmonary vascular bed offers advantages of easy accessibility and a relatively large capacity, significant early cell destruction is an important limitation.

Human Serum Albumin Microspheres Approximate Initial Organ-Specific Biodistributions of Transplanted Hepatocytes and Are Effective Cell Surrogates for Safety Studies

Liver repopulation with transplanted hepatocytes will generate novel cell-based therapies, although translocation of transplanted cells into lungs through portasystemic shunts has the potential for embolic complications. To facilitate safety analysis of hepatocyte transplantation, we wished to obtain effective cell surrogates and analyzed biodistributions of similarly sized 99mTc-labeled human serum albumin microspheres and rat hepatocytes. Image analysis with dual 99mTc and 111In labels indicated that cells and microspheres were similarly distributed in the liver when injected into normal rats via the spleen. Also, their distributions were similar when injected via a femoral vein or the superior mesenteric vein with cells and microspheres localizing in lungs or liver, respectively. Upon intraportal injection in rats with portal hypertension, microspheres localized in both liver and lungs, consistent with portasystemic shunting. These data demonstrate that human serum albumin microspheres are effective cell surrogates for approximating the safety of hepatocyte transplantation and should be clinically useful.

Gallbladder vasculitis associated with type-1 cryoglobulinemia.

A patient with type I cryoglobulinemia and monoclonal gammopathy of uncertain significance was found to have acute gallbladder vasculitis. The most prominent manifestation was upper abdominal pain in the setting of normal liver tests. An abdominal ultrasound demonstrated a thickened gallbladder wall, along with gallstones. HIDA scanning showed a nonfunctioning gallbladder with an edematous and thickened wall. There was characteristic leukocytoclastic vasculitis affecting the gallbladder. The patient recovered uneventfully subsequent to cholecystectomy. Gallbladder vasculitis should be considered in patients with unexplained upper abdominal pain and systemic vasculitis.

Transcatheter hepatocyte transplantation: preclinical studies of anatomic consequences in the portal vascular bed.

RATIONALE AND OBJECTIVES Intrasplenic transplantation deposits hepatocytes in host hepatic sinusoids with amelioration of chronic liver failure and genetic deficiency states. Because portal resistance can be altered by intrasinusoidal transplanted cells, we examined whether hepatocyte recipients would develop deleterious portal hypertension or portosystemic collaterals. METHODS Syngeneic hepatocytes in suspension were transplanted into recipient rats by transcatheter injection into the splenic parenchyma. Subjects included recipients of 2 x 10(7) hepatocytes representing approximately 3% of the host hepatic mass, recipients of 7.5 x 10(7) hepatocytes representing approximately 12.5% of the host hepatic mass, normal control rats, and positive control rats with portal hypertension induced by partial portal vein constriction. Portal pressures were recorded with a sensitive transducer, portosystemic collaterals were demonstrated with direct splenoportography, and survival of transplanted cells was determined with an endogenous dipeptidyl peptidase IV reporter gene. RESULTS In normal rats, the portal pressure was 6.25 +/- 1.9 mm Hg with no portosystemic collaterals. By contrast, portal pressures were significantly increased in portal vein-constricted rats, 20.7 +/- 3.9 mm Hg (P < 0.001), with extensive portosystemic collaterals. In hepatocyte recipients, portal hypertension observed during transcatheter cell injection but proved transient. When animals were examined up to 16 weeks after hepatocyte transplantation, portal pressures were in the normal range (after 2 x 10(7) cells, 7.5 x 2.6 mm Hg; after 7.5 x 10(7) cells, 9.5 +/- 4.2 mm Hg, P = not significant). No portosystemic collaterals developed in hepatocyte recipients at various times up to 8 months after transplantation. Transplanted hepatocytes expressing the reporter gene were present in recipients with assimilation in host hepatic cords. CONCLUSION Despite injection of a massive number of cells, transcatheter hepatocyte transplantation was devoid of any significant portal vascular alterations or toxicity in recipients. These findings are consistent with assimilation of transplanted hepatocytes into host hepatic cords and will facilitate therapeutic applications in metabolic diseases or acute liver failure.

20. ORTHOTOPIC LIVER TRANSPLANTATION

Publisher Summary Orthotopic liver transplantation (OLT) requires replacement of the liver by a donor organ. In this case, the transplanted liver is placed in the space made available following removal of the native liver. Trimming of the donor organ may be necessary if the recipient individual is of small body size. The most common sources of donor livers are brain-dead individuals on life-support. Organ transplantation would no doubt flourish were it possible to identify universal organ donors. When an organ is removed from a donor, it is necessary to preserve it in the best possible condition until transplantation. A guiding principle in organ preservation is avoidance of warm ischemia-reperfusion to prevent tissue injury. Another principle is to decrease the metabolic activity and oxidative damage in the organ. Blood is flushed out of the organ to prevent vascular occlusion by thrombus formation, followed by infusion of cold preservation solution. Surgery for OLT proceeds in three distinct stages: the dissection phase, anhepatic phase, and the reperfusion phase. Briefly, it is during the dissection phase, that the host liver is mobilized and vascular structures are prepared for resection and reanastomosis with the donor liver.