Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells.

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.

Transmission of hepatitis B infection from hepatitis B core antibody--positive liver allografts is prevented by lamivudine therapy.

Donor shortage has led to the use of hepatitis B core antibody (anti-HBc)--positive (anti-HBc(+)) liver allografts for patients in need of relatively urgent orthotopic liver transplantation (OLT). Because anti-HBc(+) allografts transmit hepatitis B virus (HBV) infection at a high rate, effective prophylaxis is required. We assessed the effectiveness of lamivudine in preventing HBV transmission by anti-HBc(+) allografts. Between March 1996 and March 2000 at Cedars-Sinai Medical Center (Los Angeles, CA), 15 of 169 patients (8.9%) received liver allografts from anti-HBc(+) donors. Six patients were hepatitis B surface antigen (HBsAg)(+) (group 1), and 9 patients were HBsAg negative (HBsAg(-); group 2) before OLT. All patients were administered lamivudine, 100 or 150 mg/d, orally after OLT. Patients who were HBsAg(+) before OLT also were administered hepatitis B immunoglobulin (HBIG) prophylaxis. Hepatitis B serological tests were performed on all patients, and HBV DNA was determined in liver tissues in 10 patients. All 15 patients remained HBsAg(-) at their last follow-up 2 to 40 months (mean, 17 months) post-OLT. All patients in group 1 had antibody to HBsAg (anti-HBs) titers greater than 250 mIU/mL post-OLT (mean follow-up, 20 months; range, 7 to 40 months). Of the 2 patients in group 1 who underwent liver biopsy after OLT, 1 patient had detectable hepatic HBV DNA despite being anti-HBs(+) and HBsAg(-). Among the patients in group 2, none acquired anti-HBc or HBsAg. Hepatic HBV DNA was undetectable in the 7 patients in group 2 who underwent liver biopsy after OLT. Anti-HBc(+) allografts can be safely used in patients who undergo OLT for chronic hepatitis B and susceptible transplant recipients if prophylaxis with combination HBIG and lamivudine or lamividine alone is administered after OLT, respectively. However, more data are needed to determine the efficacy of lamivudine monotherapy in preventing transmission of HBV infection from anti-HBc(+) liver allografts to susceptible recipients.

Radiographic findings of ischemic hepatitis in a cirrhotic patient.

We report the radiographic findings of ischemic hepatitis in a patient with cirrhosis. The abdominal ultrasound exam showed multiple hypoechoic nodules in the liver measuring up to 2 cm, suggestive of diffuse metastatic disease. Abdominal computed tomography (CT) scan revealed multiple hypodense masses throughout the liver with no enhancement. Liver biopsy revealed coagulative hepatocyte necrosis at the center of the regenerative nodules. Repeat CT scan obtained 5 months later showed complete resolution of the hypodense nodules. Ischemic necrosis of regenerative nodules should be differentiated from diffuse hepatic metastatic disease in the setting of ischemic hepatitis in cirrhotic patients.

Characterization of human xenoreactive antibodies in liver failure patients exposed to pig hepatocytes after bioartificial liver treatment: an ex vivo model of pig to human xenotransplantation.

BACKGROUND: There are limited experimental data on the nature of the humoral response elicited in humans against pig antigens. In this study, we have examined the xenoantibody (XAb) response in eight patients with acute liver failure exposed to pig hepatocytes after treatment with the bioartificial liver (BAL). METHODS: Patients' plasma samples obtained before and after BAL treatment were tested for IgM and IgG XAbs, IgG subclasses, and XAb cytotoxicity, using enzyme-linked immunosorbent assay and flow-cytometric assays. The characterization of pig aortic endothelial cell (PAEC) surface xenoantigens was analyzed by immunoprecipitation. RESULTS: We observed by day 10, a strong anti-pig IgG and IgM XAb response in patients undergoing two or more BAL treatments, with a significant increase in all the IgG subclasses; in contrast, XAb titers did not change if the patients received only one BAL treatment. The majority of the XAbs produced to porcine antigens were primarily specific for the alphaGal epitope. Both IgG and IgM XAbs were cytotoxic to PAECs, and the cytotoxic activity of IgG was associated with high levels of IgG1 and IgG3 subclasses, known to be efficient on complement activation. The characterization of porcine surface antigens demonstrated that IgM human XAbs, before and after BAL exposure, recognized xenoantigens on PAECs with similar molecular weights, suggesting that the same population of XAbs were present in the patients before and after exposure to pig antigens. CONCLUSIONS: Repetitive exposure of humans to porcine antigens after BAL treatment, results in a strong IgG and IgM XAb responses that are primarily directed against the alphaGal epitope. These XAbs are cytotoxic to PAECs and the IgG toxicity correlates with high IgG1 and IgG3 levels. Our data also suggest that no new XAb specificity emerges after porcine exposure.

In situ splitting of the cadaveric liver for transplantation.

BACKGROUND: The shortage of cadaveric donor livers is the rate-limiting step in clinical liver transplantation. Split liver transplantation provides a means to expand the cadaveric donor pool. However, this concept has not reached its full potential because of inferior patient and graft survival and high complication rates when traditional ex vivo split techniques are used. Therefore we sought to evaluate the safety, applicability, and effectiveness of a new technique for split liver transplantation. METHODS: This study consists of 15 in situ split liver procurements, which resulted in 28 liver transplants. In situ splitting of selected livers from hemodynamically stable cadaveric donors was performed at the donor hospital without any additional work-up or equipment being needed. In situ liver splitting is accomplished in a manner identical to the living-donor procurement. This technique for liver splitting results in a left lateral segment graft (segments 2 and 3) and a right trisegmental graft (segments 1 and 4-8). This procedure required the use of the donor hospital operating room for an additional 1.5-2.5 hr and did not interfere with the procurement of 30 kidneys, 12 hearts, 7 lungs, and 9 pancreata from these same donors. RESULTS: The 6-month and 1-year actuarial patient survival rates were 92% and 92%, respectively, while the 6-month and 1-year actuarial graft survival rates were 86% and 86%, respectively. The 6-month and 1-year actuarial patient survival rate of patients who received a left lateral segment graft was 100% and 100%, respectively, while those who received a right trisegmental graft had 6-month and 1-year rates of 86% and 86%, respectively. The actuarial death-censored graft survival rates at 6 months and 1 year were 80% and 80%, respectively, for the left lateral segment grafts, and 93% and 93%, respectively, for the right trisegmental grafts. Alograft and patient survival was independent of United Network for Organ Sharing status at the time of liver transplantation. No patient developed a biliary stricture, required re-exploration for intra-abdominal hemorrhage, or suffered from portal vein, hepatic vein, or hepatic artery thrombosis CONCLUSIONS: In situ split liver transplantation can be accomplished without complications and provides results that are superior to those obtained previously with ex vivo methods. It abolishes ex vivo benching and prolonged ischemia times and provides two optimal grafts with hemostasis accomplished. This technique decreases pediatric waiting time and allows adult recipients to receive right-sided grafts safely. In situ splitting is the method of choice for expanding the cadaveric liver donor pool.

Orthotopic liver transplantation for primary sclerosing cholangitis. A 12-year single center experience.

OBJECTIVE: The purpose of this study was to analyze a single center's 12-year experience with 127 orthotopic liver transplantations (OLT) for primary sclerosing cholangitis (PSC). SUMMARY BACKGROUND DATA: Primary sclerosing cholangitis is a chronic cholestatic liver disease of unknown origin that occurs most commonly in young men and is associated frequently (70-80%) with inflammatory bowel disease (IBD). Patients with PSC also are at risk for the development of cholangiocarcinoma (CCA) and those with IBD for colon carcinoma. Although the course of PSC is variable, it frequently is progressive, leading to cirrhosis and requirement for OLT. METHODS: The medical records of 127 consecutive patients undergoing OLT for PSC from July 1, 1984, to May 30, 1996, were reviewed. Actuarial patient and graft survival was determined at 1,2, and 5 years. The incidence and outcome of patients with CCA, recurrent sclerosing cholangitis, and post-transplant colon carcinoma was determined. Results were analyzed by way of stepwise Cox regression to determine the statistical strength of independent associations between pretransplant covariates and patient survival. The median follow-up period was 3.01 years. Incidental cholangiocarcinoma (ICCA) was defined as a tumor < 1 cm in size that was discovered at the time of pathologic sectioning of the explanted liver. RESULTS: Ninety-two patients (72%) had associated IBD. Seventy-nine (62%) had undergone previous biliary tract surgery. One hundred seven patients (84%) received a single graft, whereas 20 patients (16%) required 22 retransplants. Patients received either cyclosporine- (n = 76) or tacrolimus- (n = 51) based immunosuppression. The 1-, 2-, and 5-year actuarial patient survivals were 90%, 86%, and 85%, respectively, whereas graft survival was 82%, 77%, and 72%, respectively. The presence of previous biliary surgery had no effect on patient survival. Ten patients (8%) had ICCA and their survival was not significantly different from patients without ICCA (100%, 83%, and 83% at 1, 2, and 5 years, respectively). Four patients were known to have CCA at the time of OLT, all recurred within 6 months, and had a significantly worse outcome (p < 0.0001). Recurrent sclerosing cholangitis developed in 11 patients (8.6%). The patient and graft survival in this group was not different from those in whom recurrence did not develop (patient; 100%, 90%, and 90%; graft: 80%, 70%, and 52%). Thirty patients (23%) underwent colectomy after liver transplantation for dysplasia-carcinoma or symptomatic colitis. Of the nine covariates entered into the Cox multivariate regression analysis, only common bile duct frozen section biopsy specimen showing CCA was predictive of a survival disadvantage. CONCLUSIONS: Liver transplantation provides excellent patient and graft survival rates for patients affected with PSC independent of pretransplant biliary tract surgery. Incidental cholangiocarcinoma does not affect patient survival significantly. However, known CCA or common duct frozen section biopsy specimen or both showing CCA are associated with poor recipient survival, and OLT should be proscribed in these cases. Recurrent PSC occurs in approximately 9% of cases but does not affect patient survival. Post-transplant colectomy does not affect patient survival adversely.

Use of a novel bioartificial liver in a patient with acute liver insufficiency.

We have developed a bioartificial liver support system (BAL) using porcine hepatocytes attached to microcarriers and placed on the outer surface of hollow fibers. The BAL system was attached to a plasmapheresis device that was then used to treat the plasma of a patient with acute liver failure. Our aim was to test the efficacy and safety of this system after a single short treatment period. A patient with alcohol-induced, severe, acute liver failure manifested by coagulopathy, rising plasma ammonia level, and deteriorating mental status was studied. The procedure was well tolerated by the patient, who remained hemodynamically stable throughout the treatment period. A marked increase in coagulation factor V, VII, VIII, and IX activities, a decrease in serum ammonia level (120 to 32 mumol/L), a twofold increase in all serum amino acids except for aminobutyric acid, and an improvement in mental status were noted after a 6-hour treatment period. This preliminary report of the first use of this novel BAL system in conjunction with plasmapheresis appears promising. A clinical study is now in progress to prove its efficacy.

Development of bioartificial liver: bilirubin conjugation in Gunn rats.

We developed a novel bioartificial liver using microcarrier-attached hepatocytes and a bioreactor. The bioreactor consists of an intracapillary chamber made up of porous (0.2 micron) cellulose acetate hollow fibers enclosed in a polycarbonate module forming an extracapillary chamber. Cryopreserved (-80 degrees C, 3 weeks) microcarrier-attached hepatocytes (3-4 X 10(7] were inoculated into the extracapillary chamber. The bioartificial liver, containing either microcarrier hepatocytes (n = 6) or microcarriers alone (n = 5); was attached to adult Gunn rats via cannulas in the aorta and inferior vena cava. Bile samples were collected before and at hourly intervals after attachment to the bioartificial liver. The same animal model was used to demonstrate de novo conjugation of free bilirubin by the bioartificial liver (n = 3) following systemic administration of unconjugated [3H]bilirubin. Bile was analyzed for bilirubin mono- and diglucuronides by high-performance liquid chromatography and confirmed by digestion with beta-glucuronidase and cochromatography with normal rat bile. A progressive increase in the concentration of bilirubin monoglucuronide and bilirubin diglucuronide in bile was seen as early as 30 min and lasted up to 4 hr following attachment to a bioartificial liver containing microcarrier hepatocytes (3.53 +/- 0.68 to 8.07 +/- 0.85 microM; P less than 0.01). Approximately 22% of the radiolabeled bilirubin excreted in bile was seen in the form of bilirubin conjugates, no radioactivity was seen in the free bilirubin fraction. In conclusion, we developed a bioartificial liver using microcarrier hepatocytes which carry out an important differentiated liver function which results in partial correction of a specific metabolic liver defect, i.e., conjugation of bilirubin.

Intraperitoneal transplantation of microcarrier-attached enterocytes in rats.

The purpose of this study was to determine whether enterocyte transplantation could be used to correct a specific genetic metabolic defect. Bilirubin uridine diphosphoglucuronyl transferase (UDPGT) activity has been demonstrated in normal rat intestinal mucosa. We hypothesized that normal rat enterocyte transplantation may restore the ability of Gunn rats, which lack bilirubin UDPGT, to conjugate bilirubin. Small intestine was resected from normal Wistar rat donors. Enterocytes were harvested and suspended in Dulbecco's modified Eagle's medium with 10 percent fetal calf serum. Isolated enterocytes were attached to collagen-coated dextran microcarriers and transplanted into congeneic Gunn rat recipients. Recipient rats underwent bile duct cannulation after transplantation, and bile was analyzed for bilirubin glucuronides by high-pressure liquid chromatography. Fifty percent of the transplanted rats showed a significant increase in the concentration of bilirubin monoglucuronide and diglucuronide in their bile 4 and 7 days posttransplantation. Recipient rats had well vascularized microcarrier-enterocyte aggregates in the peritoneal cavity. Our method for intraperitoneal transplantation of isolated enterocytes in rats could potentially be used to correct specific enzymatic and metabolic defects.