Prolonged Survival of Porcine Hepatocytes in Cynomolgus Monkeys

Abstract

Background & Aims: Management of patients with liver failure can be a significant medical challenge, and transplantation of the liver is the only definitive therapy. Whole liver allotransplantation is limited by a shortage of human donors and the risks of the surgery in those most ill. Transplants consisting of xenogeneic hepatocytes might overcome these problems, and work in rodents indicates that such transplants can correct some metabolic deficiencies and can prevent the complications and mortality associated with hepatic failure. As a prelude to clinical application, we tested the feasibility of hepatocyte xenotransplantation in nonhuman primates. Methods: One to 2 billion hepatocytes from outbred swine were transplanted into the spleens of cynomolgus monkeys using conventional immunosuppression to control rejection. Duration of graft function was determined based on assay for porcine albumin. Results: Following a single infusion, xenogeneic hepatocytes functioned for more than 80 days and, following re-transplantation, for more than 253 days. Engraftment in the spleen was confirmed 40 days after transplantation by asialoglycoprotein receptor-directed nuclear scanning. The humoral immune response to the transplanted porcine cells had no discernible impact on the survival of the grafts. Conclusions: Xenotransplantation of hepatocytes should be explored as a readily available, minimally invasive form of therapy for hepatic failure. Background & Aims: Management of patients with liver failure can be a significant medical challenge, and transplantation of the liver is the only definitive therapy. Whole liver allotransplantation is limited by a shortage of human donors and the risks of the surgery in those most ill. Transplants consisting of xenogeneic hepatocytes might overcome these problems, and work in rodents indicates that such transplants can correct some metabolic deficiencies and can prevent the complications and mortality associated with hepatic failure. As a prelude to clinical application, we tested the feasibility of hepatocyte xenotransplantation in nonhuman primates. Methods: One to 2 billion hepatocytes from outbred swine were transplanted into the spleens of cynomolgus monkeys using conventional immunosuppression to control rejection. Duration of graft function was determined based on assay for porcine albumin. Results: Following a single infusion, xenogeneic hepatocytes functioned for more than 80 days and, following re-transplantation, for more than 253 days. Engraftment in the spleen was confirmed 40 days after transplantation by asialoglycoprotein receptor-directed nuclear scanning. The humoral immune response to the transplanted porcine cells had no discernible impact on the survival of the grafts. Conclusions: Xenotransplantation of hepatocytes should be explored as a readily available, minimally invasive form of therapy for hepatic failure. See editorial on page 450. See editorial on page 450. Management of patients with hepatic failure is quite complex and expensive. Hepatic failure causes impaired coagulation, altered consciousness and cerebral function, and a heightened risk of multiple organ system failure and sepsis.1Durand F. Valla D. Assessment of the prognosis of cirrhosis: Child-Pugh versus MELD.J Hepatol. 2005; 42: 100-107Abstract Full Text Full Text PDF Scopus (467) Google Scholar Although pulmonary, cardiac, and renal failure can be supported with ventilators, ventricular assist devices, and dialysis, no extracorporeal device effectively replaces hepatic function. Thus, even transient hepatic failure, if severe, must be treated by liver transplantation. However, use of whole liver transplantation is limited by a severe shortage of donors and by the risks associated with major surgery in those most severely ill.2Merion R.M. When is a patient too well and when is a patient too sick for a liver transplant?.Liver Transplant. 2004; 10: S69-S73Crossref PubMed Scopus (85) Google Scholar To address these challenges, we as well as others have examined transplantation of xenogeneic hepatocytes. Xenotransplantation of hepatocytes could address many of the challenges of treating hepatic failure.3Fox I.J. Hepatocyte transplantation.Am J Transplant. 2004; 4: 7-13Crossref PubMed Scopus (125) Google Scholar The transplant procedure, which involves injection of hepatocytes into the liver or spleen, is far less intrusive than transplantation of the whole liver and could be performed on severely ill patients with relatively low risk and as an outpatient procedure in those less ill. Because the native liver is not removed, the transplanted hepatocytes need only improve some of the functions of the failing liver and need not replace all hepatic functions. Xenotransplantation of hepatocytes is not limited by the availability of donors, could be performed repeatedly if needed, and might even be more effective than allotransplantation for the treatment of viral hepatitis, because xenogeneic hepatocytes do not appear susceptible to infection by human hepatitis viruses.4Platt J.L. New directions for organ transplantation.Nature. 1998; 392: 11-17Crossref PubMed Scopus (5) Google Scholar Hepatocyte xenotransplantation has been performed with some success in small animals. Gunsalus et al transplanted porcine hepatocytes into the livers of Watanabe rabbits, which have severe hypercholesterolemia due to a defect in LDL receptors.5Gunsalus J.R. Brady D.A. Coulter S.M. Gray B.M. Edge A.S.B. Reduction of serum cholesterol in Watanabe rabbits by xenogeneic hepatocellular transplantation.Nat Med. 1997; 3: 48-53Crossref PubMed Scopus (94) Google Scholar Following the procedure, the serum cholesterol of the rabbits decreased to a level substantially below that of untreated controls for the 3 months the animals were studied. We transplanted porcine hepatocytes into the spleens of rats with liver failure from chemical-induced cirrhosis.6Nagata H. Ito M. Cai J. Edge A. Platt J.L. Fox I.J. Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation.Gastroenterology. 2003; 124: 422-431Abstract Full Text PDF PubMed Scopus (131) Google Scholar The rats with hepatocyte xenografts had improved indices of coagulation, less encephalopathy, and survived longer than cirrhotic rats that did not receive hepatocyte xenografts. Given these encouraging results in rodents, and as a prelude to clinical trials, we conducted a limited series of experiments in nonhuman primates to determine whether xenogeneic hepatocytes could function for extended periods and whether there might be unforeseen barriers to success. Male cynomolgus monkeys weighing between 5–9 kg were used as transplant recipients. The animals were kept in the animal care facility at the University of Nebraska Medical Center. Long-dwelling single-lumen venous catheters were inserted into the internal jugular vein and threaded to the level of the superior vena cava in all primate transplant recipients. Central lines were tunneled subcutaneously to exit through the skin in the interscapular region of the animal’s back. Jackets were used to protect the indwelling central IV line. Donor hepatocytes were isolated from female piglets 6–8 weeks of age (8–15 kg) obtained from Larson Farms Inc. (Sargeant, MN) and kept in the animal care facility at the Mayo Clinic. Eight-week-old male severe combine immune-deficient (SCID) mice (Taconic, Germantown, NY) were transplanted with recovered porcine hepatocytes to retrospectively assess their capacity to engraft in the liver of animals where rejection has been shown not to affect graft survival. All of the studies were approved by the University of Nebraska Institutional Animal Care and Use Committee and thus within the guidelines for humane care of laboratory animals. Piglets were fasted for 24 hours prior to surgery and sedated by intramuscular administration of 2.5 mg/kg tiletamine hydrochloride and 2.5 mg/kg zolazepam hydrochloride (Telazol, Fort Dodge Animal Health, Fort Dodge, IA), 2 mg/kg xylazine hydrochloride 2 (X-Ject E, Burns Veterinary Supply, Inc., Westbury, NY), and 0.06 mg/kg glycopyrrolate (American Regent Laboratories, Inc., Shirley, NY). After endotracheal intubation, general anesthesia was initiated and maintained using isoflurane. One thousand units of heparin was administered to the piglet, and the portal vein was dissected and cannulated. Hepatocytes were harvested using a modification of the 2-step collagenase perfusion technique of Seglen et al.7Seglen P.O. Jervell K.F. A simple perfusion technique applied to glucocorticoid regulation of tryptophan oxygenase turnover and bile production in the isolated rat liver.Hoppe Seylers Z Physiol Chem. 1969; 350: 308-316Crossref PubMed Scopus (50) Google Scholar, 8Nishitai R. Plummer T.B. Platt J.L. Detection of albumin synthesis in transplanted porcine hepatocytes in mice.Liver Transplant. 2002; 8: 972-974Crossref PubMed Scopus (7) Google Scholar The liver was perfused with 5 L of oxygenated Perfusate I (143 mM sodium chloride, 6.7 mM potassium chloride, 10 mM HEPES, and 2.5 mM EGTA, pH 7.4, 37°C) while being dissected free of surrounding tissues. Once removed, the liver was placed in a sterile bowl and maintained in a water bath at 37°C while being perfused with 2 L of Perfusate II (67 mmol/L sodium chloride, 6.7 mmol/L potassium chloride, 100 mmol/L HEPES, 4.8 mmol/L calcium chloride dihydrate, 1.0% bovine serum albumin [Sigma-Aldrich, St. Louis, MO], and 0.1% collagenase [Collagenase D, Roche, Indianapolis, IN, pH 7.4, 37°C] at a rate of 1.2 L/min and recirculated for 20 minutes). Following perfusion, the capsule of the liver was torn, and the parenchymal cells were separated from connective tissues using a plastic comb in ice-cold Williams’ E medium containing 10% heat-inactivated fetal calf serum (HyClone, Logan, UT). The connective tissues were separated using a Buchner funnel (Coors Porcelain Buchner, Fisher Scientific, Pittsburgh, PA). The hepatocyte suspension was filtered through nylon gauze with a pore size of 100 μm. Hepatocytes were washed 3 times with Williams’ E medium containing 10% heat-inactivated fetal calf serum and separated by centrifugation at 50g. Isolated hepatocytes were resuspended in ViaSpan (Bristol-Myers Squibb, Princeton, NJ) supplemented with penicillin G (200,000 U/L, Calbiochem, La Jolla, CA), regular insulin (40 U/L, Humulin R, Eli Lilly, Indianapolis, IN), dexamethasone (16 mg/L), and reduced glutathione (0.922 g/L, Calbiochem). Hepatocytes were shipped to the University of Nebraska Medical Center by overnight delivery. The time from isolation to transplantation was approximately 20 hours in all cases. Viability of hepatocytes was determined by exclusion of trypan blue. As another measure of cell quality, plating efficacy of hepatocytes was assessed and determined by plating 1 × 106 cells on 60-mm Petri dishes and counting adherent cells after 24 hours. To retrospectively assess capacity for engraftment, 1 million porcine hepatocytes from cell preparations were also suspended in 50 μL of RPMI (GIBCO, Invitrogen Corporation, Carlsbad, CA) and transplanted into the spleens of 8-week-old male SCID mice. Porcine serum albumin levels in mice were measured for 2 weeks after transplantation. A subcostal incision was made 1–2 cm caudal to the last rib on the left side. Hepatocytes were washed and resuspended in sodium alginate (Kelgin LV, Monsanto, St. Louis, MO) in a 1:4 (packed cell volume: alginate volume) ratio producing a 1% alginate concentration for transplantation. The alginate matrix enhances hepatocyte engraftment in the spleen by inhibiting immediate translocation of cells into the portal circulation. Porcine hepatocytes, suspended as a packed volume of 15–30 mL in Dulbecco’s Modified Eagle Medium, were injected directly into the parenchyma of the spleen using a 22-gauge needle over approximately 20 minutes. The needle was redirected into different portions of the splenic parenchyma to distribute the cells more completely. was obtained by Hepatocyte asialoglycoprotein was following transplantation to assess engraftment of porcine hepatocytes in the spleens of transplanted primates. All and were from Louis, was from hydrochloride was as by et D.A. D. of and their use for the synthesis of Chem. PubMed Scopus Google Scholar were from NY). pH was from a by and and was as M. J. H. Fox I.J. J. for of the asialoglycoprotein and hepatic Chem. 2003; PubMed Scopus Google Scholar was placed in a and mL of a of in was by mL of a of 1 in 2 After a of of was and the was to for minutes. The was using in or sodium When approximately of the applied to the the was isolated on a using pH as the procedure with a of One of was injected into the central venous of of the whole was made with a The was in an The were at and after A × matrix was used for all The was on a using the for of and in of Health, Scholar a and at the of All were into and a of the was made to and The in function of hepatocytes was determined by porcine albumin in was into Medical Corporation, and the separated by Porcine albumin was by assay albumin and Westbury, NY) were used as the and on Porcine albumin and primate in containing 20 and 1% bovine serum were applied to of the in for 1 The of untreated primates was in and used as a control and as a for porcine albumin Porcine albumin was using albumin Laboratories, Inc., was used as the for the and the of the measured at using a Inc., The for of albumin by was and The immune of with Medical Corporation, mg/kg IV on days and IV on days and and the IV on days and after and immune with after transplantation to IV on and 3 months a with a of that effectively in for by or with to 40 was used on 1 animal at 3 on the of A. M. Reduction of by of 2 and to 2004; PubMed Scopus Google Scholar, A. H. A. R. D. of with an 2002; PubMed Scopus Google Scholar NJ) was at 5 mg/kg a for 2 weeks in animals of infection based on a low cell and serum levels were determined by using with II or were in to a and The 5 for and for to were determined by on isolated cells from swine for the an procedure, a with serum or at or 10% concentration was by a using or to that with nonhuman primate was on a using The control in assay was from animals to porcine which after and of to and M. levels in and cynomolgus 2002; PubMed Scopus Google Scholar, D. M. J. J.L. A. of by of nuclear with of 2004; PubMed Scopus Google Scholar monkeys with hepatic function transplantation. 1 to 2 billion porcine hepatocytes in all at the time of and plating at the time of were suspended in alginate prior to transplantation to a matrix that engraftment in the J. Ito M. H. D. J.R. Fox I.J. Treatment of liver failure in rats with cirrhosis by transplantation of 2002; PubMed Scopus Google Scholar and directly into the spleen by splenic The hepatocytes were into the spleen than into the liver because treatment of liver failure to cirrhosis is to engraftment in Ito M. J. Cai J. Fox I.J. Hepatocyte transplantation in rats with PubMed Scopus Google Scholar Hepatocytes were injected into the of the spleen than into a splenic because parenchymal injection results in H. Ito M. Edge A. Fox I.J. of hepatocyte hepatocyte engraftment in the 2003; PubMed Scopus Google Scholar All animals the transplant procedure monkeys were treated with 2 of and at the time of transplantation and were maintained on and of the immunosuppression for of the 3 in Xenotransplantation in and of transplant of cells transplanted albumin level × level × level × × × in a the transplanted hepatocytes function. As shown in the transplanted porcine hepatocytes functioned from days to over 80 days following a single of The on the from which is in of The 2 were treated with less immune and with and no complications from did ill from the transplant procedure or the immune The hepatocyte in the functioned for and the in the functioned for at 80 days. the 3 weeks after the concentration of porcine albumin in the in all the level of porcine albumin in the The level of porcine albumin was not as the level of porcine albumin has been shown to in mice after porcine hepatocyte R. Plummer T.B. Platt J.L. Detection of albumin synthesis in transplanted porcine hepatocytes in mice.Liver Transplant. 2002; 8: 972-974Crossref PubMed Scopus (7) Google Scholar was also to be the when of porcine hepatocytes that were transplanted into primates were also transplanted into immune-deficient mice to retrospectively assess porcine hepatocyte As a for the function of the hepatocyte we used asialoglycoprotein in 1 As 3 nuclear confirmed porcine hepatocyte engraftment in the spleen 40 days after transplantation in the 1 examined in porcine albumin levels in the was approximately 1% of that in the the time of the the porcine albumin level was than of the native albumin level in the the in engraftment and function of transplanted porcine hepatocytes based on the serum porcine albumin level in a hepatic of hepatocytes in the with hepatic function transplantation with 1 to 2 billion porcine hepatocytes. Hepatocytes were suspended in alginate prior to transplantation to a matrix that engraftment in the spleen and directly into the spleen by splenic After the concentration of porcine albumin in the as a measure of hepatocyte engraftment, As a for the engraftment and function of the hepatocyte asialoglycoprotein was performed in 1 One of was injected into the central venous of the and whole was performed with a to nuclear only in the by native hepatocytes and of in the After porcine hepatocyte infusion, nuclear in the liver and also in the of the spleen, porcine hepatocyte engraftment in the spleen 40 days after Because of hepatocyte xenotransplantation is that transplants might be performed to or function, we performed a of whether xenotransplantation could be performed repeatedly in the After the level of porcine albumin in the serum of the decreased to hepatocytes isolated from 2 outbred were One conducted days after the a in the level of swine albumin in the transplant was associated with a in the level of porcine albumin in the of immune-deficient mice transplanted with an of these hepatocytes not The performed on functioned for days to 253 after the animal with a graft from complications associated with of a central venous To the immune response to xenotransplantation of hepatocytes, we measured the levels of in the monkeys for is a made by and not by or J. between the and the in PubMed Scopus Google Scholar and and have been the most to R.M. of that human for in Google Scholar, Platt J.L. The of in rejection of 1998; PubMed Scopus Google Scholar levels of for and were low in 1 in the of in another and in the After the level of in 1 and significant in the 2 from animals and were also tested by using cells from a swine assay no and indicates that transplantation to to xenogeneic different from not Although the of significant in serum levels of to the these results that xenotransplantation of hepatocytes a less humoral immune response than that after organ Platt J.L. in for the and treatment of rejection and for 1997; PubMed Scopus Google Scholar studies in rodents that transplants consisting of isolated liver cells can correct metabolic deficiencies of the liver and can hepatic failure.3Fox I.J. Hepatocyte transplantation.Am J Transplant. 2004; 4: 7-13Crossref PubMed Scopus (125) Google Scholar However, the clinical with hepatocyte transplantation is quite and has been by a of the most of which has been the limited availability of isolated human hepatocytes for transplantation. to organ is that human hepatocytes be in to more than a of the many patients that could from transplantation. the of hepatocytes that might be used for such as cells or I.J. Hepatocyte transplantation.Am J Transplant. 2004; 4: 7-13Crossref PubMed Scopus (125) Google Scholar in the xenogeneic hepatocytes of the most to an of cells for clinical we that porcine hepatocytes engraft and function for periods of months in nonhuman primates. The survival and function of the was using a immunosuppression not different from is used in of organ transplants and did not the use of with synthesis of or that of by J.L. modification of in and 2003; PubMed Scopus Google Scholar, A. M. J. J. M. M. of porcine renal survival in through the use of donors and the of Med. 2005; PubMed Scopus Google Scholar Although immunosuppression which only in use not prevent rejection of xenografts in for xenografts consisting of isolated hepatocytes. The used is than that used in clinical and need to be reduced if porcine hepatocyte transplantation is applied to However, the immune administration of only 2 of as to the to used Because there was no to the in is that 1 or more of the not be because of did not appear to affect graft should not be the of as a to xenotransplantation of the hepatocyte xenografts in to be by the of and graft survival was not by administration of a that to whole which perfused through of the their by the of of the be the to success of M. Platt J.L. of a Full Text PDF PubMed Scopus Google Scholar, M. Platt J.L. The to Full Text Full Text PDF PubMed Scopus Google Scholar Thus, isolated hepatocyte xenografts the xenogeneic that is most to by humoral hepatocytes appear to be to A. R. Plummer T.B. Platt J.L. of hepatocytes to 2005; Google Scholar studies also an in hepatocyte that in of hepatocyte those from human livers and outbred has not been a for cell engraftment and function after transplantation. is that simple of plating or production do not the of collagenase on the of isolated hepatocytes to engraft and function following transplantation. is to the with in which in transplant studies in immune-deficient rodents, the of cell has for the of after cell transplantation. we the xenotransplantation of hepatocytes in nonhuman primates. The we monkeys with hepatic function, an of the impact of the immune only a at the of for human liver such as in nonhuman the to which a hepatocyte hepatic function in to a whole liver the liver is to following hepatocyte transplantation. Thus, transplantation of hepatocytes need only an in clinical function and, in many need do only the of function of these based on of swine albumin in the serum of the graft survival because hepatocyte albumin when the native liver functions R. Plummer T.B. Platt J.L. Detection of albumin synthesis in transplanted porcine hepatocytes in mice.Liver Transplant. 2002; 8: 972-974Crossref PubMed Scopus (7) Google Scholar, R. Plummer T.B. Platt J.L. the survival and function of xenogeneic hepatocyte Transplant. 2005; PubMed Scopus Google Scholar, M. R. A. H. liver in mice metabolic to J 2004; Full Text Full Text PDF PubMed Scopus Google Scholar et al that treatment of patients with a device containing swine hepatocytes improved some indices of liver function and survival to organ transplantation in a of patients with liver J. B.M. J. M. M. D. La M. of a liver in treating liver of 2004; PubMed Scopus Google Scholar Because transplantation of hepatocytes into the liver or spleen is more and less than using an device and less invasive than whole liver hepatocyte xenotransplantation could the treatment for in which some liver function or can Although hepatocyte xenografts function for limited periods of of hepatocytes can be and function of a of that need to be hepatocyte transplantation can be used for the treatment of liver be by the of animal to the transplant and a that hepatocyte xenotransplantation might in I clinical for the treatment of human such as liver failure and of the liver in patients with J. between the and the in PubMed Scopus Google Scholar, R.M. of that human for in Google Scholar, Platt J.L. The of in rejection of 1998; PubMed Scopus Google Scholar on the to the on the to liver cell therapy has not been when hepatocyte transplantation improved in animals with liver failure, metabolic and that clinical studies of therapy in hypercholesterolemia with and in with cell in limited cell engraftment, and of transplanted cell as major PDF A for of PDF