Hepatocyte Transplantation for Metabolic Liver Disease
The use of hepatocyte infusions to correct inborn errors of metabolism is logical when a specific metabolic deficiency, with well-studied animal modeling, can be measured. Then, with infusion of donor liver cells natively expressing the gene involved, objective measures of required hepatocyte mass, engraftment percent, and survival advantage can be obtained. Worldwide, 21 humans have been recipients of hepatocyte infusions (Table 1) to correct a metabolic liver disease. In 1976, Matas and the Minnesota transplant group, using the Gunn rat model, a rat corollary of human Crigler-Najjar syndrome type 1 (CN type 1), proved that portal infusion of hepatocytes heterozygous for diphosphate-glucuronosyltransferase could reduce plasma bilirubin in enzyme deficient Gunn rats (27). Familial hypercholesterolemia, an autosomal dominant low-density lipoprotein (LDL) receptor gene defect associated with myocardial infarct in young heterozygote patients and with life-threatening coronary occlusion in homozygotes, was chosen for the first human trial of intraportal hepatocyte infusion to treat an inborn error of hepatocellular metabolism (9, 28). To avoid immunosuppression and allogenic immune variables, an ex vivo LDL receptorwas transplanted with retroviral gene transduction of autologous hepatocytes; when transplanted intraportal into the Watanabe Heritable Hyperlipidemic rabbit (WHHL rabbit) the result was a 40% decrease in serum cholesterol with a measurable LDL receptor for 6 months (29). Importantly, allogenic intraportal hepatocyte transplantation of 2% of liver cell mass, with an expected 0.5% donor hepatocyte survival, resulted in a long-term 40% reduction of serum cholesterol in the Watanabe Heritable Hyperlipidemic rabbit (30). Preclinical work to verify the safety and efficacy of the hepatocellular directed gene therapy, including the safety of infusing intraportal 3–8×107 adult hepatocytes per kg body weight was conducted by Grossman and colleagues in dogs and primates (31, 32). Five patients with homozygous familial hypercholesterolemia underwent left lateral liver segment resection, and isolation of their segment hepatocytes with ex vivo transduced LDL receptor gene, as worked out in the animal models (9, 10). The transduced autologous hepatocytes were infused intraportal, three days postresection, taking advantage of the regenerative stimulus peaking at five to six days with a maximum by two weeks in human regeneration stimulated by resection (33–35); importantly, they differed from the 20–24 hr peak regenerative hepatocyte response to partial hepatectomy noted in animal models (36, 37). Safety from tumor and infection, with over two-year follow up and over 20% reduction in LDL cholesterol, was documented in three of the five patients transplanted, but <5% transgene expression at four months in donor hepatocytes ended further liver directed human gene hepatocyte infusion therapy (38).
Hepatocyte transplantation is more often done as therapy for inborn errors of hepatic metabolism in which a specific absent protein can be measured from transplanted unmodified donor hepatocytes expressing the gene. In the Nagase analbuminemic rat (NAR) model, intraportal and splenic hepatocyte infusion of 1% of the rat liver mass (106 to 107 hepatocytes) increased albumin expression 15 times with stable 16 months correction (39). In this same model, together with cyclosporin immunosuppression and regenerative stimulus of portal vein flow interruption to the recipient target liver, near normal albumin levels were maintained for three months (40). In the Gunn rat model, a surrogate for human Crigler-Najjar Syndrome type 1 defect of bilirubin uridine diphosphate glucuronosyltransferase (bili UDPG transferase), 12% of the whole liver mass normalized bilirubin metabolism (41). KoKudo et al. used normal, untreated, congenic rats (Wistar Shi) as sources of whole liver (for orthotopic liver transplantation [OLT]), or as donors of fetal or adult hepatocytes for transplant into Gunn rats, and studied bilirubin metabolism for 12 months (42). The intrasplenic transplanted fetal and adult congenic hepatocytes (107 cells, representing 2% liver mass) reduced total bilirubin by 30% at two months (compared to nl total bilirubin at two months with whole liver grafts), and at four months the hepatocytes expressed bilirubin glucuronide fractions 28 to 36% of total bilirubin (compared to nl bilirubin glucuronide fractions with whole liver grafts at four months) (42). Colocalization studies of adenosine triphosphate and dipeptidyl peptidase IV (to illustrate gap junctions) of hepatocytes transplanted into the spleens of rats deficient in dipeptidyl peptidase IV, activity found that the transplanted hepatocytes rapidly overcame structural barriers, entered liver plates, and even formed chimeric bile canaliculi (43). Crigler-Najjar Syndrome type 1 in humans, caused by a mutation in the gene UGT1A1, which is responsible for bilirubin conjugation (44), has been treated with allogenic mature hepatocyte intraportal infusions in four children (Table 1). Intraportal human hepatocyte transplantation of 5% of calculated liver mass with fresh and cryopreserved allogenic hepatocytes in multiple infusions has safely and reproducibly reduced hyperbilirubinemia by 30 to 50%, reducing risk of Kernicterus, with sustained bilirubin conjugation for more than three years (7, 8, 11, 12; Allen et al., personal communication). All investigators have used standard calcineurin inhibitor immunosuppression. Pharmacologic induction of donor hepatocytes in vivo (phenobarbital induction of CYP and UGT1A1 gene expression as initially described in liver failure treatment with cellular transplant [20]) has been verified, and human hepatocytes isolated by standardized techniques (45, 46) have been shown to have long-term viability and metabolic function.
The use of mouse models with transplanted donor hepatocytes that produced peripherally measurable transgene products in the circulation of congenic naïve recipients showed that intrasplenic and intraportal transplantation of HbsAg-secreting transgenic mouse hepatocytes resulted in four to five times more HbsAg production than other transplantation sites (47). In similar optimal site cell transplant experiments, α-1 antitrypsin secreting transgenic mouse hepatocytes transplanted into the spleen of congenic naïve mice migrated and functioned indefinitely in the liver, producing the protease inhibitor alpha-1-antitrypsin (48). Human hepatocyte allotransplants for α-1-antitrypsin deficiency have been performed in an 18-week-old infant found to have cirrhosis on explant at liver transplant done on day four postcell infusion (Table 1) (7) and an adult with chronic liver disease (Table 2) who had normal MM phenotype α-1 antitrypsin in the circulation prior to successful liver and kidney transplant at day two postintrasplenic cell infusion (20).
Among 16 human allotransplants done for metabolic disease (Table 1), two brothers with Factor VII deficiency had approximately 5% of liver mass replaced with fresh and previously cryopreserved hepatocytes infused intraportal; they showed 80% reduction in exogenous factor VII replacement up to the time of whole segment transplant at six months (13). Two children with glycogen storage disease IA achieved improved glucose control on a normal diet, and one patient achieved normal glucose 6 phosphatase activity starting at three weeks postintraportal cell infusion and lasting for seven months.(14; Lee KW, personal communication, 2005). In a four-year-old with infantile Refsum’s disease (secondary to absent or diminished hepatic peroxisomes and decreased phytanic acid oxidase activity) treatment with eight intraportal hepatocyte infusions resulted in 0.25% engraftment and measurable pipecholic acid reduction (15). Intraportal hepatocyte transplant had no benefit for two children with progressive familial intrahepatic cholestasis, but the failure was attributed to significant liver fibrosis found unexpectedly at the time of orthotopic liver transplant at 5 and 14 months (8). In a mouse model of progressive intrahepatic cholestasis in which the liver lacks a bile salt export pump, hepatocyte transplant in noncirrhotic recipients led to 70% native liver replacement in nine months (49). Among patients with urea cycle defects who were treated with hepatocyte transplant, three children with ornithine transcarbamylase deficiency (OTC) had NH3 control and documented OTC activity on liver biopsy (16) and normalization of NH3 on a regular protein diet for 11 days to 7 months (17, 18). Finally, with citrullinemia, a 25-month-old child who was infused intraportal with up to 10% of the native liver hepatocyte mass had ammonia control and measurably decreased citrulline levels from two weeks to six months posttransplant (Lee KW, et al., personal communication, 2005). Although inborn errors of liver metabolism in the human seem the most practical model for testing and refining hepatocyte transplant therapy, the 21 human clinical experiences lack the impressive stimulant to regeneration found in an experiment with a transgenic mouse experiment of a urokinase gene with an albumin promoter lethal to the modified recipient. The transplanted 104 normal donor mouse hepatocytes (0.3% of the liver mass) stimulated repopulation of 80% of the recipient’s liver by four weeks (50). Similarly, in a transgenic mouse model with hereditary tyrosinemia, mature normal mouse hepatocytes transplanted into the liver resulted in more than 70 cell doublings (51). Although the cancer risk in human tyrosinemia makes native liver replacement by solid liver allotransplant the therapy of choice (52), the efficacy of whole organ transplant and the uncertainty of cellular therapy have proven to be equally potent barriers to testing regenerative stimuli to improve metabolic liver disease treatment with cell infusion (7, 12).
Hepatocyte Transplantation for Chronic Liver Disease
Table 2 summarizes the data on 20 human chronic liver disease patients transplanted with unfractionated, genetically unmodified human hepatocytes. With chronic liver failure, the spleen is the most successful site to transplant hepatocytes. In the portal-caval-shunted rat model, hepatic encephalopathy and behavioral disorders, along with ultrastructural changes in the brain’s corpus striatum, were reversed with splenic transplanted hepatocytes (53). Mito and coworkers confirmed long-term function and hepatization of spleens in small animal models of splenic hepatocyte transplantation; in a portal-caval shunt monkey model of chronic liver failure, these results were confirmed by the correction of encephalopathy along with normal chord structure nidation of transplanted splenic hepatocytes out to eight months (54). Splenic hepatocyte transplantation in rats administered phenobarbital and carbon tetrachloride (CCL4), which produce clinical features of cirrhosis resembling human disease, extended survival from 15 days with one transplant to 128 days with a second transplant. Cholestasis and hyperammonemia were also significantly improved in treated rats, compared to controls (55).
The first human hepatocyte transplants for chronic liver disease were 10 direct splenic inoculation autotransplants done by Mito and coworkers in Child-Turcotte-Pugh A through C cirrhotic patients, using the recipient’s cirrhotic left lateral segment as the donor hepatocyte source. Transplanted hepatocytes were detected in the spleens with technetium Tc 99m labeling at one to six months, but were not considered responsible for encephalopathy resolution in these patients (21, 56). The remaining 10 chronic liver disease patients treated underwent hepatocyte allotransplants from noncirrhotic liver donors (Table 2). In three children, single hepatocyte administrations by transfemoral intra-arterial splenic infusion or transhepatic and transjugular portal venous infusion led to clinically significant ammonia and encephalopathy control for up to 6 weeks, and to successful bridging to liver transplant and full recovery (FR) in two of the three (19, 20). In a cirrhotic adult patient, the spleen volume can be 800 to 1200 g, as measured by indirect radiologic means: this differs from 100 to 150 g spleens in acute liver failure humans, and allows safe accommodation of 6×108 to 6×109 infused hepatocytes (22). Among seven adult cirrhotic patients with medically advanced uncontrolled encephalopathy who underwent single intrasplenic hepatocyte allotransplant, one patient with α-1-antitrypsin deficiency has been previously discussed, and one patient with hepatitis C virus (HCV) cirrhosis had histologic evidence of transplanted hepatocytes forming cord like structures with normal tight cellular junctions in the spleen by day two postinfusion (20). The five alcoholic cirrhotic patients treated with splenic hepatocyte transplantation using single-cell infusion either showed no benefit or showed significant ammonia and encephalopathy reduction up to day 33 of the study. (4, 22). Obvious questions arising from these cellular treated chronic liver disease patients are: 1) will multiple infusions improve efficacy, and 2) will unfractionated donor hepatocytes isolated from cirrhotic liver behave in vivo as they do in culture, forming nodular structures instead of confluent single cell monolayers, (5) negatively impacting nidation and function and thus eliminating cirrhotic explants as a source of human hepatocyte transplant supply? In the CCL4 cirrhotic rat model, hepatocytes transplanted in the spleen were fully integrated by one week in the liver and proliferated and survived in the diseased liver for more than a year (57). Transplantation of normal mature hepatocytes can decrease the progression of fibrosis in alkaloid poisoning (58); adenoviral telomerase RNA delivery into the livers of telomerase deficient mice corrects cirrhosis progression (59); and hepatic radiotherapy in rats induces a selective growth advantage for the transplanted syngeneic hepatocytes (60). The number of human patients with hepatocellular carcinoma (HCC) is rapidly growing; over 70% of these patients are not solid organ transplant candidates, but they gain significant life advantage by HCC ablation and ultimately die of their cancer or liver failure (61). Utilizing beta-emitting radiation (Thera Sphere) to ablate HCC (62); together with an immunosuppression regimen, identical to that used in the 21 human patients treated with hepatocytes (5, 22) there has been no increase in HCC recurrence posttransplant (63). A prospective, randomized splenic hepatocyte transplant study of radiation ablated cirrhotic HCC patients will test for the beneficial results of hepatocyte infusion seen in the animal studies cited above (57–60).
Cell Transplantation for Acute Liver Failure
The earliest studies of cell transplantation after lethal 90% hepatectomy in rats, as well as chemically induced acute liver failure (D-galactosamine poisoning), which includes necrosis and cell loss such as occur in acute human liver toxin injury, documented disease reversal with splenic, portal, and peritoneal hepatocyte transplantation of 1 to 2% of the animal’s liver mass (64–67). Even with necrosis and inflammation in the D-galactosamine liver failure model, transplanted hepatocytes infiltrated the liver plates within 48 hr (68). Time-sensitive efficacy of hepatocyte transplantation has been documented in animal models of acute liver failure (67, 69); repeatedly a small number of hepatocytes, even conditionally immortalized hepatocytes, have reversed encephalopathy and prevented death (70). The findings that cryopreserved human hepatocytes as well as fresh cultured hepatocytes nidate in the spleen, travel to the liver, and organize liver structure and function in acute human liver failure (5, 19, 20, 22) parallel the findings in animal studies (71–74). Table 2 summarizes the experiences of 37 acute liver failure patients allotransplanted with unfractionated, genetically unmodified human hepatocytes, categorized by patient age and etiology of disease. Ten children, ages 3.5 months to 16 years, have been treated with intraportal and intraperitoneal (one patient) hepatocyte infusions, under conventional calcineurin inhibitor immunosuppression, for drug, idiopathic or viral acute liver failure; two patients experienced full recovery without solid organ transplant, and three children were successfully bridged to OLT with full recovery (4, 19, 22, 23; Fisher et al. unpublished observation; Table 3). Despite multiple-organ failure in addition to severe liver injury, eight of the 10 cell-treated children had significant ammonia reduction, with half of the patients demonstrating measurable improvement in encephalopathy just as in adults with acute liver failure who were treated with hepatocyte bridging to transplant (20, 22). Soriano and colleagues have not only verified the amount of donor human hepatocyte engraftment with a real-time polymerase chain reaction (PCR) (75), but in biopsy studies of acute liver failure, patients have also shown donor hepatocytes infiltrating the liver plate and bile ductules (76). These findings support similar findings in animal studies of hepatocyte transplantation for acute and metabolic liver diseases (43, 68). Twenty-seven human adults with acute liver failure and multisystem organ failure (MSOF) have been treated with human hepatocyte transplantation (4, 5, 22–24, 26; Fisher et al., unpublished observation). Of 15 patients with drug induced liver and multiple organ failure, 10 showed clinically measurable improvement in encephalopathy, with ammonia reduction. Splenic hepatocyte transplant successfully bridged two adults to OLT at days 2 to10 postcell transplant; one adult experienced complete recovery with intraportal hepatocyte transplant alone and two patients had complete recovery with intraperitoneal fetal hepatocyte transplant (4, 5, 23, 24, Fisher et al., unpublished observation). In idiopathic acute liver failure, splenic hepatocyte transplantation successfully bridged one patient to OLT at day five, with death due to sepsis and MSOF at day 13; an intraportal hepatocyte transplant reduced hyperammonemia but was “too little, too late” to reverse brain edema in an adult Reyes syndrome patient (22; Fisher et al., unpublished observation). A woman with lethal mushroom poisoning who was treated with intraportal hepatocyte infusion recovered completely with discontinuation of immunosuppression at 12 weeks (25). An adult with postresection liver failure received no benefit but had no procedural complications from splenic hepatocyte cell infusion (4). In eight patients with acute viral liver and multiple organ failure treated with intraportal or intrasplenic infusion (or both), or with intraperitoneal hepatocyte infusion, two adults were successfully bridged to OLT and one patient completely recovered after cell transplant alone (4, 22–24, 26; Fisher et al., unpublished observations) Among these critically ill adults, two cases of hepatocyte lung emboli (24), one non-lethal splenic vein thrombosis (22), and one lethal mesenteric vein thrombosis were documented (Fisher, unpublished observations). A male with polysubstance abuse, liver, renal and pulmonary failure (Table 3, Ba9pt) fully recovered after intraportal hepatocyte infusion of 3% of his liver mass with weaning of and complete cessation of immunosuppression by 105 days postcell transplant. Donor engraftment was quantified with liver specific transcript expression (Fig. 1) using serial transjugular liver biopsy and a well-described, reproducible forensic pathologic short tandem repeats PCR technique (77). This case confirms a previous report of a liver failure patient bridged with hepatocyte transplant to native liver regeneration, with a three- to six-month period of normal parenchymal liver architecture recuperation (26), as predicted by animal studies (78).
;)
FIGURE 1.:
Study of engraftment and gene expression after hepatocyte transplantation. (A) AmpFLSTR Profiler Plus PCR Amplification Kit (Applied Biosystems, Foster City, CA) was used for the study of engraftment. Engraftment studies were performed in liver biopsies at days 0, 7, 15, and 32 posthepatocyte transplantation. Although the pretransplantation biopsy showed markers corresponding to the recipient genotype, a mix of markers from donor and recipient was observed at day 7 with lower engraftment percentages at days 15 and 32. (B) Histological evaluation of the liver biopsies performed at the indicated biopsy times showed gradually improving architecture. (C) Quantitation of gene expression of the liver specific transcripts albumin and P450IIB1 was performed using real-time PCR. mRNA levels of both transcripts were increased after transplantation when compared with pretransplantation values.
Cell Source and Banking
Hepatocyte isolation, culture, preservation, and banking from human liver tissue are beyond the scope of this overview and they have been beautifully reviewed elsewhere (3, 5, 46, 79). The limited supply of human liver for hepatocyte isolation has many causes, and we and others have characterized these (5, 7); other sources such as immortalized cell lines, fetal liver, amniotic epithelial cells, stem-cell–derived hepatocytes, to name but a few, continue to be intensively studied (80). Finally, studies of the safety and function of human hepatocytes isolated from adult and fetal donors (79, 81) and studies of condition optimization for clinical human hepatocyte infusion (82), will provide future human studies reproducible standards to work with.
Years of elegant in vitro and animal modeling combined with the lessons learned from 72 brave human souls have provided multiple proofs of the concept of “back to the basic” hepatocyte treatment for human liver disease. The safety of the human hepatocytes transplanted to date is documented by surviving patients free of malignant and infectious morbidity not for just months, but for years (22, 78). The age-old problem of supply and demand and human experimental design difficulties will be solved by the principle that led to this quest, the need to alleviate a devastating human condition.
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