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Stem Cells and the Liver: Clinical Applications in Transplantation
Advances in Surgery 43 (2009) 35–51
ADVANCES IN SURGERY Stem Cells and the Liver: Clinical Applications in Transplantation Jayme E. Locke, MD, MPHa, Michael J. Shamblott, PhDb, Andrew M. Cameron, MD, PhDa,* a
Division of Transplantation, Department of Surgery, Johns Hopkins Medical Institutions, 720 Rutland Avenue, Ross Research Building, Room 765, Baltimore, MD 21205, USA b Institute for Cellular Engineering, Johns Hopkins Medical Institutions,720 Rutland Avenue, Ross Research Building, Room 765, Baltimore, MD 21205, USA
T
here currently are more than 5 million people in the United States suffering from end-stage liver disease (ESLD). In adults liver failure is caused primarily by hepatitis C virus (HCV) or alcohol-induced cirrhosis. In children the most common cause is biliary atresia or an inborn metabolic deficiency. Liver failure now is the tenth leading cause of death in the United States. Twenty thousand patients actively await liver transplantation, but only 7000 transplantations are performed annually. Clearly the need for liver replacement far outstrips the current supply of organs. The supply of donor organs is static, and thus it is necessary to evaluate critically alternative approaches to traditional solid-organ transplantation and to devise novel ways to prolong graft survival. Study of humanized animal livers generated via hepatocyte or stem cell transplantation may provide a platform for such alternative approaches and advances in the field. Animal livers that are engineered to be chimeric for human cells will have several important applications in the study of liver disease. First, they will facilitate the study in laboratory animals of human specific diseases such as HCV, which currently has no readily available animal model. HCV infects more than 4 million people in the United States. Development of a novel cell-based animal model of HCV would be of great importance in developing advances for this pandemic disease. A second application of cell transfer to liver is to provide the foundation for cell-based therapy of metabolic diseases currently treated by whole-organ transplantation (eg, alpha-1 antitrypsin deficiency, Wilson’s disease, tyrosinemia, and glycogen storage diseases). Third, humanized animal organs ultimately may prove to be a renewable source of transplantable organs, overcoming the current organ shortage via modified xenotransplantation in Dr. Cameron is a recipient of the American Surgical Association Foundation Fellowship Award.
*Corresponding author. E-mail address: [email protected] (A.M. Cameron). 0065-3411/09/$ – see front matter doi:10.1016/j.yasu.2009.03.002
ª 2009 Elsevier Inc. All rights reserved.
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which animal organs are used as scaffolds for stem cell transfer to engineer organs that ultimately are more human than animal. In a rodent model of liver injury, the authors recently have demonstrated the ability of human embryoid body–derived (EBD) stem cells to migrate to and engraft in animal liver after splenic injection. Their preliminary studies have shown that human EBD stem cells persist long term and ultimately differentiate into mature human hepatocytes. Based on these findings, they speculate that optimization of conditions conducive to cell migration, differentiation, and persistence will reliably generate functional humanized animal livers capable of sustaining HCV infection as well as producing clinically relevant concentrations of hepatic enzymes capable of phenotypic reversal of inborn errors of metabolism. END-STAGE LIVER DISEASE AND LIVER TRANSPLANTATION IN THE UNITED STATES Adult end-stage liver disease and hepatitis C virus More than 5 million Americans suffer from ESRD or cirrhosis. Sixty percent are male, and there are no differences in ethnic or racial groups. Liver disease is responsible for 25,000 deaths per year and has become the tenth most common cause of death in the United States. It is the fourth leading cause of death in the age group from 45 to 54 years, indicating that the numbers will worsen as the population ages. When ESLD in the United States is subdivided by cause, HCV infection accounts for more than 40% of the total. Alcohol use accounts for 8%, and alcohol use complicating HCV infection accounts for another 22%. Thus about three quarters of the cases of ESRD, a large portion of a significant problem, are caused by HCV and/or alcohol use. HCV infection also is the primary risk factor for hepatocellular carcinoma, the incidence of which is steeply rising in the United States. HCV infects 4 million people in the United States and 170 million worldwide [1]. Treatment of the virus remains imperfect, and a cure remains elusive [2]. Host–viral interactions are incompletely understood, and most persons who are infected progress to a chronic condition [3]. Available antiviral agents including interferon and ribavirin have shown improved results in promoting sustained viral clearance in some patients [4]. but most of those infected will not respond or will be unable to tolerate the potentially toxic therapy. As hepatic injury progresses to irreversible cirrhosis and the patient decompensates clinically, liver replacement becomes the only viable option, as illustrated in Fig. 1. Currently 20,000 patients in the United States await liver transplantation [5]. Because of the shortage of appropriate donor organs, only around 7,000 orthotopic liver transplantations (OLTs) are performed each year. HCV cirrhosis has become the most common indication for OLT; in 2005 about 50% of liver transplant recipients carried this diagnosis. Unfortunately, OLT is not a cure for HCV. Graft re-infection is universal after transplantation, and damage to the new liver occurs routinely [6,7]. Twenty percent of recipients develop recurrent cirrhosis within 5 years, and recurrent HCV disease is the most
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Acute Hepatitis C Infection Chronic HCV Infection (75%)
Resolution (25%) Cirrhosis (20% in 20 years)
Liver Failure (5% per year)
HCC (5% per year)
OLT or Death
Fig. 1. The natural history of hepatitis C infection. HCC, hepatocellular carcinoma.
common cause of graft loss and retransplantation [8]. Controlling the virus before and after transplantation is the most important challenge in the field of OLT and is an active area of investigation in the authors’ laboratory. The work described in this article would greatly facilitate this line of investigation. Molecularly characterized in 1989, HCV is a 9-kb single-stranded RNA virus [9,10]. Until recently the lack of simple culture systems in which to infect and propagate the virus hampered progress in understanding the viral life cycle and pathogenesis of infection, including the molecular mechanisms implicated in HCV-induced hepatocellular carcinoma (for review, see Ref. [11]). Development of small animal models of HCV would greatly assist in the study of HCV-associated pathogenesis in the laboratory but has remained largely elusive. Efforts thus far at developing an animal model have involved human cell transplantation and are described later in this article. Pediatric liver transplantation in the United States and inborn errors of metabolism Given its common modes of transmission (contaminated blood products before1992 or sharing of intravenous drug needles), HCV is uncommon in children, although it may be transmitted during childbirth. In most pediatric liver transplant centers, hepatic metabolic defects are the second most common indication for liver transplant, after biliary atresia. The Studies of Pediatric Liver Transplantation database revealed that, of 1187 pediatric liver transplantations performed in the United States between 1995 and 2002, 12% were performed for metabolic defects, most commonly alpha 1-antitrypsin deficiency, urea cycle defects, or tyrosinemia (Table 1). The long-term survival and quality of life of children who undergo OLT for metabolic disease is similar to that of children who have liver failure secondary to other diseases and is likewise limited by organ availability, a problem worsened by the rarity of appropriate-sized pediatric donors. Conceptually, liver transplantation is an
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Table 1 Indications for pediatric liver transplantation Pediatric liver transplantation Cause of liver failure
Number
Percentage
Biliary atresia Fulminant liver failure Metabolic disease Alpha-1 antitrypsin Urea cycle defects Tyrosinemia Cystic fibrosis Wilson’s disease Neonatal hemachromatosis Primary hyperoxaluria Glycogen storage disease Crigler-Najjar syndrome Other
494 145 141 39 22 16 12 10 9 8 7 6 12
41.6 12.2 11.9 27.7 15.6 11.3 8.5 7.1 6.4 5.7 5.0 4.2 8.5
inefficient means by which to perform the single-gene replacement needed in these congenital deficiencies; with much-needed advances in cell transplant therapy, it is hoped that treatment of these diseases with OLT will become obsolete in the future. CURRENT ANIMAL MODELS OF HEPATITIS C VIRUS AND HEPATIC METABOLIC DEFECTS: GENERATED BY AND TARGETS FOR CELL TRANSPLANTATION Like other human hepatitis viruses, HCV infection requires fully functional human hepatocytes for its development. Because of the poorly understood but strict tropism of HCV, only humans and chimpanzees, among the higher primates, are receptive to HCV infection. Besides the chimpanzee, the only non-human primate susceptible to HCV infection is the marmoset. Members of the Tupaia (Tree shrew) genus also can be infected by HCV (Table 2) (for review, see Ref. [15]). A mouse or rat model of HCV infection appropriate for use in the laboratory is much needed, although three such experimental animals have been described in the literature. All employ xeno-transplantation of adult human liver tissue or human hepatoma cells into the rodent [12–14]. These models represent forward steps in facilitating HCV study, but each has significant shortcomings (Table 3). Two of the models generate only low-level viremia, and the model with higher viral levels depends on fresh hepatocytes obtained from a human source. Such cell preparations are likely to be heterogeneous, because each is from a different donor, and their availability is limited. Furthermore, the model with high viremia is generated in a mouse that is frail, and thus mortality rates are high. A model that uses a more robust host and a cell source that is
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Table 2 Animals permissive to HCV infection Animal
Comment
Reference
Chimpanzee
Few uninfected animals available for research; cost per animal is high: $50,000 Only non-human primate besides chimpanzee that carries HCV Member of the tree shrew genus Scandentia
Alter and colleagues, 1978 [12]
Marmoset
Tupai
Feinstone and colleagues, 1981 [13] Xie and colleagues, 1994 [14]
homogenous, amenable to manipulation, and in unlimited supply would be preferable. A chimeric animal based on such a cell source is described in the authors’ specific aims and would represent an advance in the study of liver disease and hepatic transplantation and a useful application of emerging stem cell biology (for review, see Ref. [16]). Animal models of hepatic metabolic errors Rodent models for several of the metabolic human liver diseases have been developed and serve as a platform for testing novel therapies (Table 4). Success with cell-based or other therapies in these animals will allow the development of treatments to be used in a clinical setting. As mentioned earlier, some success already has been demonstrated experimentally and in the clinic with hepatocyte transplantation for these conditions. Using stem cells for this application would permit further manipulation of cells before transplantation (ie, gene therapy) and would improve the homogeneity and availability of the cells to be transplanted. Alternatively, epithelial cell adhesion molecule–positive (EpCAMþ) hepatic progenitors could be purified from resected specimens from affected children, stably transfected with the missing gene of interest, and auto-transplanted back into the patient. Two well-described animal models are discussed briefly here. Wilson’s disease as model for cell therapy Wilson’s disease is an autosomal recessive condition in which pathologic copper deposition occurs in the liver, brain, and eyes. It occurs in 1 in 100,000 live births and manifests with liver function abnormalities in early adolescence. Liver lesions begin as fatty change and progress to a chronic hepatitis with eventual cirrhosis and resultant portal hypertension, jaundice, and liver failure. In patients who have Wilson’s disease, copper absorption and transport to the liver are normal, but biliary copper excretion is abnormal, and serum ceruloplasmin is low. Copper accumulates in the liver and causes toxic hepatic injury. Excess copper becomes bound to serum albumin, is deposited in
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Table 3 Animal models of HCV infection Model
Hepatocyte Source
Level of viremia (copies/ml) 6
Timing of viremia
Human hepatocytes prepared from cancer resections
High: 110
1–9 months
Trimera mouse
Human liver implant
Low: 1104
18 days to 1 month
Rat
Human hepatoma cell line
Low: 1104
3–4 months
Reference
Heterogeneous and limited hepatocyte supply Immunodeficient host High death rate Low viremia Heterogeneous and limited liver tissue supply Immunodeficient host Low viremia Use of hepatoma Immunocompetent host
Mercer and colleagues, 2001 [16]
Iian and colleagues, 2002 [17]
Wu and colleagues, 2005 [18]
LOCKE, SHAMBLOTT, & CAMERON
uPA/SCID mouse
Comments
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Table 4 Rodent models of hepatic diseases that may be targets of cell-based therapy Human disease
Rodent model
Genetic defect
Reference
Tyrosinemia
FAH/ mouse
Crigler-Najjar I
Gunn rat
Wilson’s disease
Long-Evans cinnamon rat Mdr2/ mouse
Fumaryl-acetoacetate hydrolase UDP-glucuronyl transferase ATP7B
Overturf and colleagues, 1997 [19] Matas and colleagues, 1976 [20] Yoshida and colleagues, 1996 [21] De Vree and colleagues, 1998 [22] Michel and colleagues, 1993 [23]
Intrahepatic cholestasis Ornithine transcarbamylase deficiency
Spf-ash mouse
Mdr2 Ornithine transcarbamylase
peripheral tissues, and is excreted in the urine. The molecular defect responsible for Wilson’s disease has been identified as the copper transporter P-type ATPase gene (ATP7B) located on chromosome 13, and a variety of mutations within this gene have been described in patients who have Wilson’s disease. The Long-Evans Cinnamon (LEC) rat is an accepted animal model of Wilson’s disease with many biochemical and phenotypic features (eg, excess hepatic copper levels and hepatitis development) similar to the human disease. The gene responsible in rat is homologous to the defect in human. Yoshida and colleagues [21] have shown that transplantation of wild-type rat hepatocytes from Long-Evans Agouti rats generates liver chimerism in around 20% of LEC rats and reduces the mortality seen in LEC rats from around 50% to 10%. Transplantation of hepatocytes to humans who have Wilson’s disease has not been reported, nor have investigators attempted treatment in the LEC model with either human hepatocytes or human progenitor cells as described by the author. Intrahepatic cholestasis as model for cell therapy Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal recessive liver disorders that progress to cirrhosis and liver failure before adulthood. One group of patients who have PFIC is characterized by high serum levels of gamma-glutamyl transferase and characteristic early hepatitis seen on liver histology. Genetic analyses have shown these patients carry deletional or nonsense mutations in the multi-drug resistance type 3 (MDR3) gene. MDR3, or P-glycoprotein, mediates the translocation of phosphatidylcholine across the canulicular membrane in hepatocytes. Mice with a mutation in a homologous MDR2 transporter completely lack biliary phospholipid excretion and develop progressive liver disease characterized by hepatitis similar to that seen in humans who have PFIC. De Vree and colleagues [22] have reported transfer of mdr3-expressing mouse hepatocytes to mice with PFIC with
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resultant 20% chimerism and improvement in cholestasis. No attempts to use human cell therapy to reverse the mdr2/ PFIC mouse phenotype have been reported, although anecdotal reports of small series of hepatocyte transplantation in children who have PFIC suggest proof of principle.
CELL SOURCES FOR TRANSPLANTATION Transplantation of healthy hepatocytes or hepatic progenitors to support the failing or deficient liver is an innovative proposal with several important applications. Compared with whole-organ transplantation, cell transfer is a technically simple procedure with numerous advantages. It is nonsurgical, with relatively low morbidity and mortality. One donor liver may provide therapy for multiple patients, and livers deemed unfit for whole-organ transplantation may be a usable source to generate hepatocytes for transplantation. Hepatocytes can be cryopreserved, unlike whole grafts. Advances in hepatocyte isolation, handling, culture, and transplantation techniques have made clinical application realistic, and recent clinical studies suggest promise in replacing liver function in the short term. Both mature hepatocytes and hepatic progenitor cells can be considered for these applications. Although the technical aspects of cell delivery are largely resolved, the optimal cell source and the optimization for each application remain important questions to be addressed in animal models. The following sections present several of the important options. Mature hepatocytes The large number of controlled animal experiments during the last 3 decades has enabled implementation of hepatocyte transplantation trials in humans. The first report came from Japan in 1993 in which intrasplenic injection of human hepatocytes into patients who had cirrhosis or chronic hepatitis yielded no benefit. A year later, seven patients in India who had fulminant hepatic failure were injected with hepatocytes, and a significant increase in survival and improvement in laboratory tests was seen as compared with controls. In the United States 19 patients who had fulminant liver failure have undergone hepatocyte transplantation. Results have been inconclusive, and success is only anecdotal. As described earlier, a small group of patients who had inherited diseases have undergone hepatocyte transplantation, including efforts to improve familial hypercholesterolemia, ornithine transcarbamylase deficiency, alpha 1-antitrypsin deficiency, glycogen storage disease 1a, and the Crigler-Najjar syndrome. Varying degrees of success have been documented in each instance. Hepatocyte availability for transplantation ultimately would suffer from the same shortage that currently plagues whole-organ transplantation. Hepatocytes generated from a renewable source such as stem cells, as proposed herein, would provide an alternative and unlimited resource for this application and could be of donor origin after in vitro manipulation.
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Bone marrow–derived cells Several researchers have described the contribution of bone marrow–derived cells to liver regeneration. Utilizing sex-mismatched bone marrow and liver transplantation in the mouse, rat, and human, it is possible to observe Y-chromosome–positive hepatocytes as single cells or clusters in female recipients [24,25]. Lagasse and colleagues [26] have shown rescue from lethal tyrosinemia with hematopoietic stem cells. In humans, bone marrow–derived cells have been used as clinical therapy for chronic liver disease in several noncontrolled phase I clinical trials. Autologous CD133þ bone marrow cells have been used to improve regeneration of the residual liver after portal vein embolization of tumor-bearing liver segments with a increase in size greater than that seen in control patients [27,28]. Whether cell fusion, actual trans-differentiation, or even secretion of regenerative factors by transplanted cells underlies the mechanism is unknown. Embryonic stem cells Embryonic stem cells (ESCs) form all somatic cell types of the body, including endoderm-derived cells and hepatic progenitors. Nondirected differentiation can be achieved through the formation of embryoid bodies that contain cells representing lineages from the three germ layers. In the presence of growth factors such as hepatocyte growth factor, fibroblast growth factor, and dexamethasone, differentiation into hepatocyte-like cells occurs. Numerous similar protocols recently have described success in generating hepatocytes from ESCs. [29–31]. Transplantation of ESCs carries the risk of teratoma formation, even in the case of prior in vitro differentiation, via contaminating pluripotent ESCs (although cell-sorting techniques can be used to minimize this danger). The use of EBD cells minimizes this risk as well. Transplantation with hepatocytes derived from human ESCs has been reported in fumarylacetoacetate hydrolase (FAH)-deficient mice, indicating the potential use of these cells as therapy, but engraftment rates have been lower than seen with transplantation of adult hepatocytes [32–34]. Human hepatic stem cells from fetal/postnatal donors A recent report described the isolation of human hepatic stem cells (hHpSCs) from organs discarded as inappropriate for transplantation [4]. hHpSCs as characterized in these experiments are pluripotent precursors of hepatoblasts and thence of hepatocytes and biliary epithelia. These cells were isolated readily by immunoselection for EpCAM-positive cells. hHpSCs constitute around 1% to 2% of liver parenchyma, regardless of donor age, and self-renew with phenotypic stability in culture. When transplanted into carbon tetrachloride (CCl4)-injured mice with severe combined immune deficiency (SCID), these progenitor cells differentiated into mature liver tissue expressing human-specific proteins. These cells would be particularly attractive for cell therapy applications, because they could be purified from portions of liver resected from persons needing transplantation. Such cells could be expanded and
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modified in vitro and retransplanted, avoiding the need for exogenous immunosuppression. Embryoid body–derived cells from embryonic germ cell lines Pluripotent stem cells (PSCs) have been derived from the inner cell mass of blastocysts and from primordial germ cells colonizing the developing gonadal ridge and are thus referred to as ‘‘embryonic stem cells’’ or as ‘‘embryonic germ cells,’’ respectively. When PSCs differentiate in vitro, they form complex three-dimensional cell aggregates termed ‘‘embryoid bodies,’’ as described earlier in this article. Some early developmental processes are recapitulated within the environment of an embryoid body, resulting in a haphazard collection of precursor and more fully differentiated cells from a wide variety of lineages. Human embryonic germ cells also have been shown to form embryoid bodies when they differentiate; these embryoid bodies are composed of endodermal, ectodermal, and mesodermal derivatives. At the Johns Hopkins Medical Institutions, the Gearhart laboratory has isolated progenitor cells from embryoid bodies derived from embryonic germ cells that show desirable proliferation and expression characteristics [35]. These cells are not considered ESCs, because they are not totipotent, but they are capable of long-term and robust proliferation in culture [36]. As shown in Fig. 1, they express a wide array of mRNA and protein markers that normally are associated with distinct developmental lineages, suggesting that they may be a useful resource in the study of human cell differentiation and for cellular transplantation therapies. Last, an independent group has shown the potential of these cells to differentiate into hepatocytes in vivo upon injection into fetal sheep liver [37]. The authors’ EBD stem cells proliferate over the long term with a normal karyotype and have the ability to be cryopreserved, cloned, and genetically manipulated [35]. Therefore they represent an excellent starting material for the authors’ experiments with generation of chimeric animal livers.
THE NONOBESE DIABETIC/SEVERE COMBINED IMMUNODEFICIENT ANIMAL MODEL The authors are using a variety of host animals for cell transplantation applications to test the optimal suitability for generation of chimeric animal liver. Others at the Johns Hopkins Medical Institutions previously have evaluated a number of immunodeficient mouse strains (including nonobese diabetic/ severe combined immunodeficient [NOD/SCID], Rag2null/ccnull, NOD/SCID/ ccnull, and others) to define the strain that most efficiently accepts hHpSCs for stable and sustained engraftment. Among these strains, the NOD/SCID mouse line harboring a complete null mutation of the common cytokine receptor gamma chain (NOD/SCID/ccnull or NOG/SCID), which recently was shown to support efficient development of functional human hematolymphopoiesis, was significantly superior to all other strains tested [38].
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Human embryoid body–derived stem cells EBD stem cells express a wide array of mRNA and protein markers that normally are associated with distinct developmental lineages (Figs. 1 and 2). The authors’ human EBD stem cells (LVEC P4) proliferate over the long term with a normal karyotype and can be cryopreserved, cloned, and genetically manipulated. Cell migration and engraftment Dark Agouti rat immunocompetent model Initial studies were aimed at determining the conditions necessary for human EBD stem cell migration, engraftment, and persistence in animal liver. To follow and characterize EBD stem cell migration, cells were labeled with Vybrant DiI (Molecular Probes) (5 ul of DiI per 1 10^6 cells/ml) before transplantation. Vybrant DiI is a lipophilic dye that inserts into the cell membrane and is observed as red fluorescence (570 nm). Each animal received an intrasplenic injection of 1 10^6 DiI-labeled cells (Fig. 3A). Preliminary data indicate that liver injury via CCl4 (1 ml/kg) is necessary for stem cell migration and
Fig. 2. Baseline expression of hepatocyte differentiation markers in the human EBD stem cell line (original magnification 40). (A) HuH 7.0 cells: positive control for human alpha-fetoprotein (aFP) production as evidenced by green cytoplasmic staining on immunocytochemistry. (B) Human EBD stem cell line negative for human aFP. (C) HuH 7.0 cell positive control for human albumin production as evidenced by green cytoplasmic staining on immunocytochemistry. (D) Human EBD stem cell line negative for human albumin.
Fig. 3. Conditions necessary for human EBD stem cell migration and engraftment in animal liver (original magnification 20). (A) Human EBD stem cells labeled with DiI. (B) At 2 days with no CCl4 injury and no immunosuppression. (C) At 2 days with Cci4 injury and no immunosuppression. (D) At 7 days with CCl4 injury and no immunosuppression. (E) At 2 days with CCl4 injury and with immunosuppression. (F) At 7 days with CCl4 injury and with immunosuppression. (G) At 30 days with CCl4 injury and 30 days of immunosuppression. (H) At 30 days with CCl4 injury and with immunosuppression for only 10 days.
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Fig. 4. In vivo migration of human EBD stem cells to rodent liver 7 days after liver injury and intrasplenic human EBD stem cell transplantation (original magnification 20). (A) Rodent liver 7 days after liver injury and intrasplenic saline injection. (B) Rodent liver 7 days after liver injury and intrasplenic human EBD stem cell transplantation demonstrating evidence of human EBD stem cells in rodent liver (black arrows).
engraftment to animal liver (Fig. 3B and C) and that in a xenogeneic immunocompetent animal model immunosuppression is required for human EBD stem cells to persist long term (cyclosporine 10 mg/kg/d) (Fig. 3C–F). Furthermore, the authors were able to titrate the duration of immunosuppression and determined that a maximum of 10 days of cyclosporine-based immunosuppression was required (Fig. 3G and H). Although human EBD cells were present at day 2, cell engraftment was most robust at day 7. Labeled human EBD stem cells were seen consistently in clusters around central veins, and chimerism was approximately 10% to 20% (Fig. 4), in contrast to observations in animals that were treated with CCl4 alone (Fig. 5).
Fig. 5. Rodent livers treated with CCl4 alone (original magnification 20). (A) Rodent liver 2 days after injury with CCl4. (B) Higher powered view of the same showing periportal injury.
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CELL DIFFERENTIATION Dark Agouti female rats treated with 10 days of cyclosporine-based immunosuppression that underwent hepatic injury and human EBD stem cell transplantation showed evidence of in vivo differentiation of human EBD cells into functional human hepatocytes. At 7 days after human EBD stem cell transplantation, rat livers stained positive for human alpha-fetoprotein (Fig. 6E), and
Fig. 6. In vivo differentiation of human EBD stem cells in a Dark Agouti rat model (original magnification 40). (A) Human liver positive for human alpha-fetoprotein (aFP) as evidenced by green cytoplasmic straining. (B) Normal rat liver negative for human aFP. (C) Human liver positive for human albumin as evidenced by green cytoplasmic staining. (D) Normal rat liver negative for human albumin. (E) Rodent liver 7 days after human EBD stem cell transplantation positive for human aFP. (F) Rodent liver positive for human albumin 30 days after human EBD stem cell transplantation.
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Fig. 7. RT-PCR confirmation of human albumin production 30 days after cell transplantation. (A) Both the positive control human liver tissue (Hu) and the rat liver 30 days after human EBD stem cell transplantation (RH) are positive for human-specific albumin production (100-bp product). Rat liver 30 days after syngeneic rat hepatocyte transplant (RR) is negative for human-specific albumin production. (B) Both Hu and RH are positive for human-specific b-actin (140-bp product), whereas RR is negative. (C) Hu, RH, and RR are all positive for GAPDH (340-bp product), a liver enzyme that is 100% homologous across species.
at 30 days after human EBD stem cell transplantation rat livers showed staining for human albumin (Fig. 6F). Human-specific albumin production in the chimeric animal was confirmed with RT-PCR, and the PCR product was confirmed further with sequencing (Fig. 7). In addition, the authors have demonstrated similar evidence of durable engraftment and differentiation after human EBD stem cell transplantation in NOG-SCID mice, as evidenced by the production of human albumin (Fig. 8A). Finally, the authors also have shown that mature human hepatocytes can be transplanted in this model and maintain the ability to produce human albumin (Fig. 8B). SUMMARY ESLD affects millions of Americans, and HCV is a worldwide pandemic. Unfortunately, the ability to study liver disease and novel therapeutics
Fig. 8. Human albumin production in animal livers 30 days after human EBD stem cell transplantation (original magnification 40). (A) SCID mouse liver 30 days after human EBD stem cell transplantation with evidence of human albumin production. (B) SCID mouse liver 7 days after human mature hepatocyte transplantation with evidence of human albumin production.
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experimentally in the laboratory is limited by an ongoing lack of small animal models. The development of rodents with livers chimeric for human hepatocytes may improve this situation. The authors’ efforts currently use an immunodeficient or exogenously immunosuppressed animal with subsequent liver injury provided by chemical or surgical means. Cell transplantation with either human hepatocytes or human stem cells results in engraftment and subsequent ‘‘humanization’’ of an animal liver. Study of these animal models may lead to innovative approaches to the management of ESLD in both children and adults. References [1] Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276(5309):60–6. [2] Yovchev MI, Grozdanov PN, Joseph B, et al. Novel hepatic progenitor cell surface markers in the adult rat liver. Hepatology 2007;45(1):139–49. [3] Gleiberman AS, Encinas JM, Mignone JL, et al. Expression of nestin-green fluorescent protein transgene marks oval cells in the adult liver. Dev Dyn 2005;234(2):413–21. [4] Schmelzer E, Zhang L, Bruce A, et al. Human hepatic stem cells from fetal and postnatal donors. J Exp Med 2007;204(8):1973–87. [5] Alison MR, Lovell MJ. Liver cancer: the role of stem cells. Cell Prolif 2005;38(6):407–21. [6] Piscaglia AC, Shupe TD, Petersen BE, et al. Stem cells, cancer, liver, and liver cancer stem cells: finding a way out of the labyrinth. Curr Cancer Drug Targets 2007;7(6):582–90. [7] Theise ND, Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000;31(1):235–40. [8] Tanimizu N, Miyajima A. Molecular mechanism of liver development and regeneration. Int Rev Cytol 2007;259:1–48. [9] Rice CM. Flaviviridae: the viruses and their replication. 3rd edition. Philadelphia: Lippencott-Raven Publishers; 1996. [10] Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244(4902):359–62. [11] Tellinghuisen TL, Evans MJ, von Hahn T, et al. Studying hepatitis C virus: making the best of a bad virus. J Virol 2007;81(17):8853–67. [12] Alter HJ, Purcell RH, Holland PV, et al. Transmissible agent in non-A, non-B hepatitis. Lancet 1978;1(8062):459–63. [13] Feinstone SM, Alter HJ, Dienes HP, et al. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis 1981;144(6):588–98. [14] Xie ZC, Riezu-Boj JI, Lasarte JJ, et al. Transmission of hepatitis C virus infection to tree shrews. Virology 1998;244(2):513–20. [15] Kremsdorf D, Brezillon N. New animal models for hepatitis C viral infection and pathogenesis studies. World J Gastroenterol 2007;13(17):2427–35. [16] Mercer DF, Schiller DE, Elliott JF, et al. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7(8):927–33. [17] Ilan E, Arazi J, Nussbaum O, et al. The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV. J Infect Dis 2002;185(2):153–61. [18] Wu GY, Konishi M, Walton CM, et al. A novel immunocompetent rat model of HCV infection and hepatitis. Gastroenterology 2005;128(5):1416–23. [19] Overturf K, al-Dhalimy M, Ou CN, et al. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151(5):1273–80. [20] Matas AJ, Sutherland DE, Steffes MW, et al. Hepatocellular transplantation for metabolic deficiencies: decrease of plasma bilirubin in Gunn rats. Science 1976;192(4242):892–4. [21] Yoshida Y, Tokusashi Y, Lee GH, et al. Intrahepatic transplantation of normal hepatocytes prevents Wilson’s disease in Long-Evans cinnamon rats. Gastroenterology 1996;111(6): 1654–60.
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[22] de Vree JM, Jacquemin E, Sturm E, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci U S A 1998;95(1):282–7. [23] Michel JL, Rabier D, Rambaud C, et al. [Intrasplenic transplantation of hepatocytes in spf-ash mice with congenital ornithine transcarbamylase deficiency]. Chirurgie 1993;119(10): 666–71 [in French]. [24] Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284(5417):1168–70. [25] Theise ND, Nimmakayalu M, Gardner R, et al. Liver from bone marrow in humans. Hepatology 2000;32(1):11–6. [26] Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6(11):1229–34. [27] am Esch JS 2nd, Knoefel WT, Klein M, et al. Portal application of autologous CD133þ bone marrow cells to the liver: a novel concept to support hepatic regeneration. Stem Cells 2005;23(4):463–70. [28] Furst G, Schulte am Esch J, Poll LW, et al. Portal vein embolization and autologous CD133þ bone marrow stem cells for liver regeneration: initial experience. Radiology 2007;243(1): 171–9. [29] Cai J, Zhao Y, Liu Y, et al. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 2007;45(5):1229–39. [30] Duan Y, Catana A, Meng Y, et al. Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 2007;25(12):3058–68. [31] Hamazaki T, Iiboshi Y, Oka M, et al. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett 2001;497(1):15–9. [32] Azuma H, Paulk N, Ranade A, et al. Robust expansion of human hepatocytes in Fah-/-/ Rag2-/-/Il2rg-/- mice. Nat Biotechnol 2007;25(8):903–10. [33] Gouon-Evans V, Boussemart L, Gadue P, et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol 2006;24(11): 1402–11. [34] Heo J, Factor VM, Uren T, et al. Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver. Hepatology 2006;44(6):1478–86. [35] Shamblott MJ, Axelman J, Littlefield JW, et al. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A 2001;98(1):113–8. [36] Kerr CL, Shamblott MJ, Gearhart JD. Pluripotent stem cells from germ cells. Meth Enzymol 2006;419:400–26. [37] Chen B, Shi J, Zheng J, et al. Differentiation of liver cells from human primordial germ cellderived progenitors. Differentiation 2007;75(5):350–9. [38] Ishikawa F, Yasukawa M, Lyons B, et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood 2005;106(5):1565–73.
ADVANCES IN SURGERY Stem Cells and the Liver: Clinical Applications in Transplantation Jayme E. Locke, MD, MPHa, Michael J. Shamblott, PhDb, Andrew M. Cameron, MD, PhDa,* a
Division of Transplantation, Department of Surgery, Johns Hopkins Medical Institutions, 720 Rutland Avenue, Ross Research Building, Room 765, Baltimore, MD 21205, USA b Institute for Cellular Engineering, Johns Hopkins Medical Institutions,720 Rutland Avenue, Ross Research Building, Room 765, Baltimore, MD 21205, USA
T
here currently are more than 5 million people in the United States suffering from end-stage liver disease (ESLD). In adults liver failure is caused primarily by hepatitis C virus (HCV) or alcohol-induced cirrhosis. In children the most common cause is biliary atresia or an inborn metabolic deficiency. Liver failure now is the tenth leading cause of death in the United States. Twenty thousand patients actively await liver transplantation, but only 7000 transplantations are performed annually. Clearly the need for liver replacement far outstrips the current supply of organs. The supply of donor organs is static, and thus it is necessary to evaluate critically alternative approaches to traditional solid-organ transplantation and to devise novel ways to prolong graft survival. Study of humanized animal livers generated via hepatocyte or stem cell transplantation may provide a platform for such alternative approaches and advances in the field. Animal livers that are engineered to be chimeric for human cells will have several important applications in the study of liver disease. First, they will facilitate the study in laboratory animals of human specific diseases such as HCV, which currently has no readily available animal model. HCV infects more than 4 million people in the United States. Development of a novel cell-based animal model of HCV would be of great importance in developing advances for this pandemic disease. A second application of cell transfer to liver is to provide the foundation for cell-based therapy of metabolic diseases currently treated by whole-organ transplantation (eg, alpha-1 antitrypsin deficiency, Wilson’s disease, tyrosinemia, and glycogen storage diseases). Third, humanized animal organs ultimately may prove to be a renewable source of transplantable organs, overcoming the current organ shortage via modified xenotransplantation in Dr. Cameron is a recipient of the American Surgical Association Foundation Fellowship Award.
*Corresponding author. E-mail address: [email protected] (A.M. Cameron). 0065-3411/09/$ – see front matter doi:10.1016/j.yasu.2009.03.002
ª 2009 Elsevier Inc. All rights reserved.
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which animal organs are used as scaffolds for stem cell transfer to engineer organs that ultimately are more human than animal. In a rodent model of liver injury, the authors recently have demonstrated the ability of human embryoid body–derived (EBD) stem cells to migrate to and engraft in animal liver after splenic injection. Their preliminary studies have shown that human EBD stem cells persist long term and ultimately differentiate into mature human hepatocytes. Based on these findings, they speculate that optimization of conditions conducive to cell migration, differentiation, and persistence will reliably generate functional humanized animal livers capable of sustaining HCV infection as well as producing clinically relevant concentrations of hepatic enzymes capable of phenotypic reversal of inborn errors of metabolism. END-STAGE LIVER DISEASE AND LIVER TRANSPLANTATION IN THE UNITED STATES Adult end-stage liver disease and hepatitis C virus More than 5 million Americans suffer from ESRD or cirrhosis. Sixty percent are male, and there are no differences in ethnic or racial groups. Liver disease is responsible for 25,000 deaths per year and has become the tenth most common cause of death in the United States. It is the fourth leading cause of death in the age group from 45 to 54 years, indicating that the numbers will worsen as the population ages. When ESLD in the United States is subdivided by cause, HCV infection accounts for more than 40% of the total. Alcohol use accounts for 8%, and alcohol use complicating HCV infection accounts for another 22%. Thus about three quarters of the cases of ESRD, a large portion of a significant problem, are caused by HCV and/or alcohol use. HCV infection also is the primary risk factor for hepatocellular carcinoma, the incidence of which is steeply rising in the United States. HCV infects 4 million people in the United States and 170 million worldwide [1]. Treatment of the virus remains imperfect, and a cure remains elusive [2]. Host–viral interactions are incompletely understood, and most persons who are infected progress to a chronic condition [3]. Available antiviral agents including interferon and ribavirin have shown improved results in promoting sustained viral clearance in some patients [4]. but most of those infected will not respond or will be unable to tolerate the potentially toxic therapy. As hepatic injury progresses to irreversible cirrhosis and the patient decompensates clinically, liver replacement becomes the only viable option, as illustrated in Fig. 1. Currently 20,000 patients in the United States await liver transplantation [5]. Because of the shortage of appropriate donor organs, only around 7,000 orthotopic liver transplantations (OLTs) are performed each year. HCV cirrhosis has become the most common indication for OLT; in 2005 about 50% of liver transplant recipients carried this diagnosis. Unfortunately, OLT is not a cure for HCV. Graft re-infection is universal after transplantation, and damage to the new liver occurs routinely [6,7]. Twenty percent of recipients develop recurrent cirrhosis within 5 years, and recurrent HCV disease is the most
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Acute Hepatitis C Infection Chronic HCV Infection (75%)
Resolution (25%) Cirrhosis (20% in 20 years)
Liver Failure (5% per year)
HCC (5% per year)
OLT or Death
Fig. 1. The natural history of hepatitis C infection. HCC, hepatocellular carcinoma.
common cause of graft loss and retransplantation [8]. Controlling the virus before and after transplantation is the most important challenge in the field of OLT and is an active area of investigation in the authors’ laboratory. The work described in this article would greatly facilitate this line of investigation. Molecularly characterized in 1989, HCV is a 9-kb single-stranded RNA virus [9,10]. Until recently the lack of simple culture systems in which to infect and propagate the virus hampered progress in understanding the viral life cycle and pathogenesis of infection, including the molecular mechanisms implicated in HCV-induced hepatocellular carcinoma (for review, see Ref. [11]). Development of small animal models of HCV would greatly assist in the study of HCV-associated pathogenesis in the laboratory but has remained largely elusive. Efforts thus far at developing an animal model have involved human cell transplantation and are described later in this article. Pediatric liver transplantation in the United States and inborn errors of metabolism Given its common modes of transmission (contaminated blood products before1992 or sharing of intravenous drug needles), HCV is uncommon in children, although it may be transmitted during childbirth. In most pediatric liver transplant centers, hepatic metabolic defects are the second most common indication for liver transplant, after biliary atresia. The Studies of Pediatric Liver Transplantation database revealed that, of 1187 pediatric liver transplantations performed in the United States between 1995 and 2002, 12% were performed for metabolic defects, most commonly alpha 1-antitrypsin deficiency, urea cycle defects, or tyrosinemia (Table 1). The long-term survival and quality of life of children who undergo OLT for metabolic disease is similar to that of children who have liver failure secondary to other diseases and is likewise limited by organ availability, a problem worsened by the rarity of appropriate-sized pediatric donors. Conceptually, liver transplantation is an
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Table 1 Indications for pediatric liver transplantation Pediatric liver transplantation Cause of liver failure
Number
Percentage
Biliary atresia Fulminant liver failure Metabolic disease Alpha-1 antitrypsin Urea cycle defects Tyrosinemia Cystic fibrosis Wilson’s disease Neonatal hemachromatosis Primary hyperoxaluria Glycogen storage disease Crigler-Najjar syndrome Other
494 145 141 39 22 16 12 10 9 8 7 6 12
41.6 12.2 11.9 27.7 15.6 11.3 8.5 7.1 6.4 5.7 5.0 4.2 8.5
inefficient means by which to perform the single-gene replacement needed in these congenital deficiencies; with much-needed advances in cell transplant therapy, it is hoped that treatment of these diseases with OLT will become obsolete in the future. CURRENT ANIMAL MODELS OF HEPATITIS C VIRUS AND HEPATIC METABOLIC DEFECTS: GENERATED BY AND TARGETS FOR CELL TRANSPLANTATION Like other human hepatitis viruses, HCV infection requires fully functional human hepatocytes for its development. Because of the poorly understood but strict tropism of HCV, only humans and chimpanzees, among the higher primates, are receptive to HCV infection. Besides the chimpanzee, the only non-human primate susceptible to HCV infection is the marmoset. Members of the Tupaia (Tree shrew) genus also can be infected by HCV (Table 2) (for review, see Ref. [15]). A mouse or rat model of HCV infection appropriate for use in the laboratory is much needed, although three such experimental animals have been described in the literature. All employ xeno-transplantation of adult human liver tissue or human hepatoma cells into the rodent [12–14]. These models represent forward steps in facilitating HCV study, but each has significant shortcomings (Table 3). Two of the models generate only low-level viremia, and the model with higher viral levels depends on fresh hepatocytes obtained from a human source. Such cell preparations are likely to be heterogeneous, because each is from a different donor, and their availability is limited. Furthermore, the model with high viremia is generated in a mouse that is frail, and thus mortality rates are high. A model that uses a more robust host and a cell source that is
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Table 2 Animals permissive to HCV infection Animal
Comment
Reference
Chimpanzee
Few uninfected animals available for research; cost per animal is high: $50,000 Only non-human primate besides chimpanzee that carries HCV Member of the tree shrew genus Scandentia
Alter and colleagues, 1978 [12]
Marmoset
Tupai
Feinstone and colleagues, 1981 [13] Xie and colleagues, 1994 [14]
homogenous, amenable to manipulation, and in unlimited supply would be preferable. A chimeric animal based on such a cell source is described in the authors’ specific aims and would represent an advance in the study of liver disease and hepatic transplantation and a useful application of emerging stem cell biology (for review, see Ref. [16]). Animal models of hepatic metabolic errors Rodent models for several of the metabolic human liver diseases have been developed and serve as a platform for testing novel therapies (Table 4). Success with cell-based or other therapies in these animals will allow the development of treatments to be used in a clinical setting. As mentioned earlier, some success already has been demonstrated experimentally and in the clinic with hepatocyte transplantation for these conditions. Using stem cells for this application would permit further manipulation of cells before transplantation (ie, gene therapy) and would improve the homogeneity and availability of the cells to be transplanted. Alternatively, epithelial cell adhesion molecule–positive (EpCAMþ) hepatic progenitors could be purified from resected specimens from affected children, stably transfected with the missing gene of interest, and auto-transplanted back into the patient. Two well-described animal models are discussed briefly here. Wilson’s disease as model for cell therapy Wilson’s disease is an autosomal recessive condition in which pathologic copper deposition occurs in the liver, brain, and eyes. It occurs in 1 in 100,000 live births and manifests with liver function abnormalities in early adolescence. Liver lesions begin as fatty change and progress to a chronic hepatitis with eventual cirrhosis and resultant portal hypertension, jaundice, and liver failure. In patients who have Wilson’s disease, copper absorption and transport to the liver are normal, but biliary copper excretion is abnormal, and serum ceruloplasmin is low. Copper accumulates in the liver and causes toxic hepatic injury. Excess copper becomes bound to serum albumin, is deposited in
40
Table 3 Animal models of HCV infection Model
Hepatocyte Source
Level of viremia (copies/ml) 6
Timing of viremia
Human hepatocytes prepared from cancer resections
High: 110
1–9 months
Trimera mouse
Human liver implant
Low: 1104
18 days to 1 month
Rat
Human hepatoma cell line
Low: 1104
3–4 months
Reference
Heterogeneous and limited hepatocyte supply Immunodeficient host High death rate Low viremia Heterogeneous and limited liver tissue supply Immunodeficient host Low viremia Use of hepatoma Immunocompetent host
Mercer and colleagues, 2001 [16]
Iian and colleagues, 2002 [17]
Wu and colleagues, 2005 [18]
LOCKE, SHAMBLOTT, & CAMERON
uPA/SCID mouse
Comments
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Table 4 Rodent models of hepatic diseases that may be targets of cell-based therapy Human disease
Rodent model
Genetic defect
Reference
Tyrosinemia
FAH/ mouse
Crigler-Najjar I
Gunn rat
Wilson’s disease
Long-Evans cinnamon rat Mdr2/ mouse
Fumaryl-acetoacetate hydrolase UDP-glucuronyl transferase ATP7B
Overturf and colleagues, 1997 [19] Matas and colleagues, 1976 [20] Yoshida and colleagues, 1996 [21] De Vree and colleagues, 1998 [22] Michel and colleagues, 1993 [23]
Intrahepatic cholestasis Ornithine transcarbamylase deficiency
Spf-ash mouse
Mdr2 Ornithine transcarbamylase
peripheral tissues, and is excreted in the urine. The molecular defect responsible for Wilson’s disease has been identified as the copper transporter P-type ATPase gene (ATP7B) located on chromosome 13, and a variety of mutations within this gene have been described in patients who have Wilson’s disease. The Long-Evans Cinnamon (LEC) rat is an accepted animal model of Wilson’s disease with many biochemical and phenotypic features (eg, excess hepatic copper levels and hepatitis development) similar to the human disease. The gene responsible in rat is homologous to the defect in human. Yoshida and colleagues [21] have shown that transplantation of wild-type rat hepatocytes from Long-Evans Agouti rats generates liver chimerism in around 20% of LEC rats and reduces the mortality seen in LEC rats from around 50% to 10%. Transplantation of hepatocytes to humans who have Wilson’s disease has not been reported, nor have investigators attempted treatment in the LEC model with either human hepatocytes or human progenitor cells as described by the author. Intrahepatic cholestasis as model for cell therapy Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal recessive liver disorders that progress to cirrhosis and liver failure before adulthood. One group of patients who have PFIC is characterized by high serum levels of gamma-glutamyl transferase and characteristic early hepatitis seen on liver histology. Genetic analyses have shown these patients carry deletional or nonsense mutations in the multi-drug resistance type 3 (MDR3) gene. MDR3, or P-glycoprotein, mediates the translocation of phosphatidylcholine across the canulicular membrane in hepatocytes. Mice with a mutation in a homologous MDR2 transporter completely lack biliary phospholipid excretion and develop progressive liver disease characterized by hepatitis similar to that seen in humans who have PFIC. De Vree and colleagues [22] have reported transfer of mdr3-expressing mouse hepatocytes to mice with PFIC with
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resultant 20% chimerism and improvement in cholestasis. No attempts to use human cell therapy to reverse the mdr2/ PFIC mouse phenotype have been reported, although anecdotal reports of small series of hepatocyte transplantation in children who have PFIC suggest proof of principle.
CELL SOURCES FOR TRANSPLANTATION Transplantation of healthy hepatocytes or hepatic progenitors to support the failing or deficient liver is an innovative proposal with several important applications. Compared with whole-organ transplantation, cell transfer is a technically simple procedure with numerous advantages. It is nonsurgical, with relatively low morbidity and mortality. One donor liver may provide therapy for multiple patients, and livers deemed unfit for whole-organ transplantation may be a usable source to generate hepatocytes for transplantation. Hepatocytes can be cryopreserved, unlike whole grafts. Advances in hepatocyte isolation, handling, culture, and transplantation techniques have made clinical application realistic, and recent clinical studies suggest promise in replacing liver function in the short term. Both mature hepatocytes and hepatic progenitor cells can be considered for these applications. Although the technical aspects of cell delivery are largely resolved, the optimal cell source and the optimization for each application remain important questions to be addressed in animal models. The following sections present several of the important options. Mature hepatocytes The large number of controlled animal experiments during the last 3 decades has enabled implementation of hepatocyte transplantation trials in humans. The first report came from Japan in 1993 in which intrasplenic injection of human hepatocytes into patients who had cirrhosis or chronic hepatitis yielded no benefit. A year later, seven patients in India who had fulminant hepatic failure were injected with hepatocytes, and a significant increase in survival and improvement in laboratory tests was seen as compared with controls. In the United States 19 patients who had fulminant liver failure have undergone hepatocyte transplantation. Results have been inconclusive, and success is only anecdotal. As described earlier, a small group of patients who had inherited diseases have undergone hepatocyte transplantation, including efforts to improve familial hypercholesterolemia, ornithine transcarbamylase deficiency, alpha 1-antitrypsin deficiency, glycogen storage disease 1a, and the Crigler-Najjar syndrome. Varying degrees of success have been documented in each instance. Hepatocyte availability for transplantation ultimately would suffer from the same shortage that currently plagues whole-organ transplantation. Hepatocytes generated from a renewable source such as stem cells, as proposed herein, would provide an alternative and unlimited resource for this application and could be of donor origin after in vitro manipulation.
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Bone marrow–derived cells Several researchers have described the contribution of bone marrow–derived cells to liver regeneration. Utilizing sex-mismatched bone marrow and liver transplantation in the mouse, rat, and human, it is possible to observe Y-chromosome–positive hepatocytes as single cells or clusters in female recipients [24,25]. Lagasse and colleagues [26] have shown rescue from lethal tyrosinemia with hematopoietic stem cells. In humans, bone marrow–derived cells have been used as clinical therapy for chronic liver disease in several noncontrolled phase I clinical trials. Autologous CD133þ bone marrow cells have been used to improve regeneration of the residual liver after portal vein embolization of tumor-bearing liver segments with a increase in size greater than that seen in control patients [27,28]. Whether cell fusion, actual trans-differentiation, or even secretion of regenerative factors by transplanted cells underlies the mechanism is unknown. Embryonic stem cells Embryonic stem cells (ESCs) form all somatic cell types of the body, including endoderm-derived cells and hepatic progenitors. Nondirected differentiation can be achieved through the formation of embryoid bodies that contain cells representing lineages from the three germ layers. In the presence of growth factors such as hepatocyte growth factor, fibroblast growth factor, and dexamethasone, differentiation into hepatocyte-like cells occurs. Numerous similar protocols recently have described success in generating hepatocytes from ESCs. [29–31]. Transplantation of ESCs carries the risk of teratoma formation, even in the case of prior in vitro differentiation, via contaminating pluripotent ESCs (although cell-sorting techniques can be used to minimize this danger). The use of EBD cells minimizes this risk as well. Transplantation with hepatocytes derived from human ESCs has been reported in fumarylacetoacetate hydrolase (FAH)-deficient mice, indicating the potential use of these cells as therapy, but engraftment rates have been lower than seen with transplantation of adult hepatocytes [32–34]. Human hepatic stem cells from fetal/postnatal donors A recent report described the isolation of human hepatic stem cells (hHpSCs) from organs discarded as inappropriate for transplantation [4]. hHpSCs as characterized in these experiments are pluripotent precursors of hepatoblasts and thence of hepatocytes and biliary epithelia. These cells were isolated readily by immunoselection for EpCAM-positive cells. hHpSCs constitute around 1% to 2% of liver parenchyma, regardless of donor age, and self-renew with phenotypic stability in culture. When transplanted into carbon tetrachloride (CCl4)-injured mice with severe combined immune deficiency (SCID), these progenitor cells differentiated into mature liver tissue expressing human-specific proteins. These cells would be particularly attractive for cell therapy applications, because they could be purified from portions of liver resected from persons needing transplantation. Such cells could be expanded and
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modified in vitro and retransplanted, avoiding the need for exogenous immunosuppression. Embryoid body–derived cells from embryonic germ cell lines Pluripotent stem cells (PSCs) have been derived from the inner cell mass of blastocysts and from primordial germ cells colonizing the developing gonadal ridge and are thus referred to as ‘‘embryonic stem cells’’ or as ‘‘embryonic germ cells,’’ respectively. When PSCs differentiate in vitro, they form complex three-dimensional cell aggregates termed ‘‘embryoid bodies,’’ as described earlier in this article. Some early developmental processes are recapitulated within the environment of an embryoid body, resulting in a haphazard collection of precursor and more fully differentiated cells from a wide variety of lineages. Human embryonic germ cells also have been shown to form embryoid bodies when they differentiate; these embryoid bodies are composed of endodermal, ectodermal, and mesodermal derivatives. At the Johns Hopkins Medical Institutions, the Gearhart laboratory has isolated progenitor cells from embryoid bodies derived from embryonic germ cells that show desirable proliferation and expression characteristics [35]. These cells are not considered ESCs, because they are not totipotent, but they are capable of long-term and robust proliferation in culture [36]. As shown in Fig. 1, they express a wide array of mRNA and protein markers that normally are associated with distinct developmental lineages, suggesting that they may be a useful resource in the study of human cell differentiation and for cellular transplantation therapies. Last, an independent group has shown the potential of these cells to differentiate into hepatocytes in vivo upon injection into fetal sheep liver [37]. The authors’ EBD stem cells proliferate over the long term with a normal karyotype and have the ability to be cryopreserved, cloned, and genetically manipulated [35]. Therefore they represent an excellent starting material for the authors’ experiments with generation of chimeric animal livers.
THE NONOBESE DIABETIC/SEVERE COMBINED IMMUNODEFICIENT ANIMAL MODEL The authors are using a variety of host animals for cell transplantation applications to test the optimal suitability for generation of chimeric animal liver. Others at the Johns Hopkins Medical Institutions previously have evaluated a number of immunodeficient mouse strains (including nonobese diabetic/ severe combined immunodeficient [NOD/SCID], Rag2null/ccnull, NOD/SCID/ ccnull, and others) to define the strain that most efficiently accepts hHpSCs for stable and sustained engraftment. Among these strains, the NOD/SCID mouse line harboring a complete null mutation of the common cytokine receptor gamma chain (NOD/SCID/ccnull or NOG/SCID), which recently was shown to support efficient development of functional human hematolymphopoiesis, was significantly superior to all other strains tested [38].
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Human embryoid body–derived stem cells EBD stem cells express a wide array of mRNA and protein markers that normally are associated with distinct developmental lineages (Figs. 1 and 2). The authors’ human EBD stem cells (LVEC P4) proliferate over the long term with a normal karyotype and can be cryopreserved, cloned, and genetically manipulated. Cell migration and engraftment Dark Agouti rat immunocompetent model Initial studies were aimed at determining the conditions necessary for human EBD stem cell migration, engraftment, and persistence in animal liver. To follow and characterize EBD stem cell migration, cells were labeled with Vybrant DiI (Molecular Probes) (5 ul of DiI per 1 10^6 cells/ml) before transplantation. Vybrant DiI is a lipophilic dye that inserts into the cell membrane and is observed as red fluorescence (570 nm). Each animal received an intrasplenic injection of 1 10^6 DiI-labeled cells (Fig. 3A). Preliminary data indicate that liver injury via CCl4 (1 ml/kg) is necessary for stem cell migration and
Fig. 2. Baseline expression of hepatocyte differentiation markers in the human EBD stem cell line (original magnification 40). (A) HuH 7.0 cells: positive control for human alpha-fetoprotein (aFP) production as evidenced by green cytoplasmic staining on immunocytochemistry. (B) Human EBD stem cell line negative for human aFP. (C) HuH 7.0 cell positive control for human albumin production as evidenced by green cytoplasmic staining on immunocytochemistry. (D) Human EBD stem cell line negative for human albumin.
Fig. 3. Conditions necessary for human EBD stem cell migration and engraftment in animal liver (original magnification 20). (A) Human EBD stem cells labeled with DiI. (B) At 2 days with no CCl4 injury and no immunosuppression. (C) At 2 days with Cci4 injury and no immunosuppression. (D) At 7 days with CCl4 injury and no immunosuppression. (E) At 2 days with CCl4 injury and with immunosuppression. (F) At 7 days with CCl4 injury and with immunosuppression. (G) At 30 days with CCl4 injury and 30 days of immunosuppression. (H) At 30 days with CCl4 injury and with immunosuppression for only 10 days.
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Fig. 4. In vivo migration of human EBD stem cells to rodent liver 7 days after liver injury and intrasplenic human EBD stem cell transplantation (original magnification 20). (A) Rodent liver 7 days after liver injury and intrasplenic saline injection. (B) Rodent liver 7 days after liver injury and intrasplenic human EBD stem cell transplantation demonstrating evidence of human EBD stem cells in rodent liver (black arrows).
engraftment to animal liver (Fig. 3B and C) and that in a xenogeneic immunocompetent animal model immunosuppression is required for human EBD stem cells to persist long term (cyclosporine 10 mg/kg/d) (Fig. 3C–F). Furthermore, the authors were able to titrate the duration of immunosuppression and determined that a maximum of 10 days of cyclosporine-based immunosuppression was required (Fig. 3G and H). Although human EBD cells were present at day 2, cell engraftment was most robust at day 7. Labeled human EBD stem cells were seen consistently in clusters around central veins, and chimerism was approximately 10% to 20% (Fig. 4), in contrast to observations in animals that were treated with CCl4 alone (Fig. 5).
Fig. 5. Rodent livers treated with CCl4 alone (original magnification 20). (A) Rodent liver 2 days after injury with CCl4. (B) Higher powered view of the same showing periportal injury.
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CELL DIFFERENTIATION Dark Agouti female rats treated with 10 days of cyclosporine-based immunosuppression that underwent hepatic injury and human EBD stem cell transplantation showed evidence of in vivo differentiation of human EBD cells into functional human hepatocytes. At 7 days after human EBD stem cell transplantation, rat livers stained positive for human alpha-fetoprotein (Fig. 6E), and
Fig. 6. In vivo differentiation of human EBD stem cells in a Dark Agouti rat model (original magnification 40). (A) Human liver positive for human alpha-fetoprotein (aFP) as evidenced by green cytoplasmic straining. (B) Normal rat liver negative for human aFP. (C) Human liver positive for human albumin as evidenced by green cytoplasmic staining. (D) Normal rat liver negative for human albumin. (E) Rodent liver 7 days after human EBD stem cell transplantation positive for human aFP. (F) Rodent liver positive for human albumin 30 days after human EBD stem cell transplantation.
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Fig. 7. RT-PCR confirmation of human albumin production 30 days after cell transplantation. (A) Both the positive control human liver tissue (Hu) and the rat liver 30 days after human EBD stem cell transplantation (RH) are positive for human-specific albumin production (100-bp product). Rat liver 30 days after syngeneic rat hepatocyte transplant (RR) is negative for human-specific albumin production. (B) Both Hu and RH are positive for human-specific b-actin (140-bp product), whereas RR is negative. (C) Hu, RH, and RR are all positive for GAPDH (340-bp product), a liver enzyme that is 100% homologous across species.
at 30 days after human EBD stem cell transplantation rat livers showed staining for human albumin (Fig. 6F). Human-specific albumin production in the chimeric animal was confirmed with RT-PCR, and the PCR product was confirmed further with sequencing (Fig. 7). In addition, the authors have demonstrated similar evidence of durable engraftment and differentiation after human EBD stem cell transplantation in NOG-SCID mice, as evidenced by the production of human albumin (Fig. 8A). Finally, the authors also have shown that mature human hepatocytes can be transplanted in this model and maintain the ability to produce human albumin (Fig. 8B). SUMMARY ESLD affects millions of Americans, and HCV is a worldwide pandemic. Unfortunately, the ability to study liver disease and novel therapeutics
Fig. 8. Human albumin production in animal livers 30 days after human EBD stem cell transplantation (original magnification 40). (A) SCID mouse liver 30 days after human EBD stem cell transplantation with evidence of human albumin production. (B) SCID mouse liver 7 days after human mature hepatocyte transplantation with evidence of human albumin production.
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experimentally in the laboratory is limited by an ongoing lack of small animal models. The development of rodents with livers chimeric for human hepatocytes may improve this situation. The authors’ efforts currently use an immunodeficient or exogenously immunosuppressed animal with subsequent liver injury provided by chemical or surgical means. Cell transplantation with either human hepatocytes or human stem cells results in engraftment and subsequent ‘‘humanization’’ of an animal liver. Study of these animal models may lead to innovative approaches to the management of ESLD in both children and adults. References [1] Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276(5309):60–6. [2] Yovchev MI, Grozdanov PN, Joseph B, et al. Novel hepatic progenitor cell surface markers in the adult rat liver. Hepatology 2007;45(1):139–49. [3] Gleiberman AS, Encinas JM, Mignone JL, et al. Expression of nestin-green fluorescent protein transgene marks oval cells in the adult liver. Dev Dyn 2005;234(2):413–21. [4] Schmelzer E, Zhang L, Bruce A, et al. Human hepatic stem cells from fetal and postnatal donors. J Exp Med 2007;204(8):1973–87. [5] Alison MR, Lovell MJ. Liver cancer: the role of stem cells. Cell Prolif 2005;38(6):407–21. [6] Piscaglia AC, Shupe TD, Petersen BE, et al. Stem cells, cancer, liver, and liver cancer stem cells: finding a way out of the labyrinth. Curr Cancer Drug Targets 2007;7(6):582–90. [7] Theise ND, Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000;31(1):235–40. [8] Tanimizu N, Miyajima A. Molecular mechanism of liver development and regeneration. Int Rev Cytol 2007;259:1–48. [9] Rice CM. Flaviviridae: the viruses and their replication. 3rd edition. Philadelphia: Lippencott-Raven Publishers; 1996. [10] Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244(4902):359–62. [11] Tellinghuisen TL, Evans MJ, von Hahn T, et al. Studying hepatitis C virus: making the best of a bad virus. J Virol 2007;81(17):8853–67. [12] Alter HJ, Purcell RH, Holland PV, et al. Transmissible agent in non-A, non-B hepatitis. Lancet 1978;1(8062):459–63. [13] Feinstone SM, Alter HJ, Dienes HP, et al. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis 1981;144(6):588–98. [14] Xie ZC, Riezu-Boj JI, Lasarte JJ, et al. Transmission of hepatitis C virus infection to tree shrews. Virology 1998;244(2):513–20. [15] Kremsdorf D, Brezillon N. New animal models for hepatitis C viral infection and pathogenesis studies. World J Gastroenterol 2007;13(17):2427–35. [16] Mercer DF, Schiller DE, Elliott JF, et al. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7(8):927–33. [17] Ilan E, Arazi J, Nussbaum O, et al. The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV. J Infect Dis 2002;185(2):153–61. [18] Wu GY, Konishi M, Walton CM, et al. A novel immunocompetent rat model of HCV infection and hepatitis. Gastroenterology 2005;128(5):1416–23. [19] Overturf K, al-Dhalimy M, Ou CN, et al. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151(5):1273–80. [20] Matas AJ, Sutherland DE, Steffes MW, et al. Hepatocellular transplantation for metabolic deficiencies: decrease of plasma bilirubin in Gunn rats. Science 1976;192(4242):892–4. [21] Yoshida Y, Tokusashi Y, Lee GH, et al. Intrahepatic transplantation of normal hepatocytes prevents Wilson’s disease in Long-Evans cinnamon rats. Gastroenterology 1996;111(6): 1654–60.
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