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Background. Hepatocyte transplantation is an attractive treatment for various liver diseases. The intraportal route of transplantation is favored, but little information is available on the possible adverse effects in this technique. We investigated the influence of intraportal loads of hepatocytes on portal, pulmonary, and systemic hemodynamics in 13 pigs. Methods. Under general anesthesia, pigs were provided with an arterial line, a Swan-Ganz catheter, and two intraportal catheters, one for cell infusion and one for heparin infusion and portal pressure measurement. Pig hepatocytes were infused at a rate of 25 million cells/min. Results. The first six animals were used to develop the infusion technique. In the last seven animals, portal pressure increased linearly with cell load upon infusion of 400–2400×106 hepatocytes (r2=0.704;P 40 mmHg. Pulmonary arterial pressure increased by 11–62%. AST increased up to 10-fold, and platelets decreased by 22–58%. Hepatocytes-containing thrombi were present in segmental and in smaller portal branches. Hepatocytes were always identified in lung sinusoids 48 hr after infusion, and a small basal pulmonary infarction was found in one animal. Conclusions. These data suggest that up to 2.4% of total hepatocyte mass can be infused in this large animal model. However, the risk of significant thrombotic complications should be considered for clinical applications.

Background and aims: Reversible Ischemia/Reperfusion liver injury was used to induce engraftment and hepatic parenchymal differentiation of exogenous β2-microglubulin -/ Thy1+ bone marrow-derived cells. This method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, was further tested to correct the metabolic disorder in a rat model of congenital hyperbilirubinemia. Methods and results: Analysis by confocal laser microscopy of fluorescence-labelled cells and by immunohistochemistry for β2-microglubulin, 72 hours after intraportal delivery, demonstrated engraftment of infused cells in liver parenchyma of rats with Ischemia/Reperfusion, but not in control animals with non-injured liver. Transplantation of bone marrow-derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP-positive hepatocytes in Ischemia/Reperfusion liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP-negative bone marrow-derived cells into GFP-positive transgenic rats resulted in the appearance of GFP-negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow-derived cells into hyperbilirubinemic Gunn rats with deficient bilirubin conjugation after Ischemia/Reperfusion damage resulted in 30% decrease of serum bilirubin, in the appearance of bilirubin conjugates in bile and in the expression of normal UDP-glucuronyltransferase enzyme evaluated by PCR. Conclusions: Ischemia/Reperfusion injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow-derived cells. Transplantation of bone marrow-derived cells from non-affected animals resulted in the partial correction of hyperbilirubinemia in the Gunn rat.

Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained 1 × 107 rat hepatocytes. Human HepG2 cells were encapsulated using alginate/poly-l-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6–10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.

Rat hepatocytes in a collagen-gel sandwich configuration were exposed to silicon alkoxides in a gas phase, yielding a 0.05 to 0.15 microm porous silica layer on the gel surface. Cell viability was unaffected by the procedure. After 24 h, bilirubin conjugation, ammonia removal, urea synthesis, and diazepam metabolism were unaffected by the procedure. However, both the ammonia removal rate and diazepam metabolism were increased after 48 hr, whereas urea synthesis was unaffected. These data indicate that silica overlay allows efficient metabolic activity of collagen-gel entrapped hepatocytes. The fact that the KM of bilirubin conjugation was unaffected by the presence of the silica membrane suggests that the transport of albumin-bound substrates is not decreased. The enhancement in some metabolic activities found 48 h after the entrapment procedure may be the result of favorable changes in the hepatocyte microenvironment. These characteristics might be useful for the development of organotypical bioartificial liver devices.

Recently it was shown that a population of cells in the bone marrow-expressing hematopoietic stem cell antigens could differentiate into hepatocytes. However, explicitly committed hepatocyte progenitors, which exhibit highly differentiated liver functions, immediately upon isolation, have not yet been isolated from bone marrow. After studying common antigens on blast-like cells in fetal and adult regenerating cholestatic rat livers and human regenerating and malignant livers, we hypothesized that beta-2-microglobulin-negative (beta(2)m(-)) cells might represent dedifferentiated hepatocytes and/or their progenitors. Utilizing a two-step magnetic bead cell-sorting procedure, we show that in bone marrow from rat and human, beta(2)m(-)/Thy-1(+) cells consistently express liver-specific genes and functions. After intraportal infusion into rat livers, bone marrow-derived hepatocyte stem cells (BDHSC) integrated with hepatic cell plates and differentiated into mature hepatocytes. In a culture system simulating liver regeneration and containing cholestatic serum, these cells differentiated into mature hepatocytes and metabolized ammonia into urea. This differentiation was dependent on a yet nondescript humoral signal existing in the cholestatic serum. Transmission electron microscopy and three-dimensional digital reconstruction confirmed hepatocyte ultrastructure of cultured BDHSC.

The limited availability of human hepatocytes results in the use of animal cells in most bioartificial liver support devices. In the present work, clinically relevant liver specific metabolic activities were compared in rat, pig and human hepatocytes cultured on liver-derived biomatrix to optimize the expression of differentiated functions. Pig hepatocytes showed higher rates of diazepam metabolism (2.549+/-0.821 microg/h/million cells vs. 0.474+/-0.079 microg/h/million cells rats, p