Cell Therapies for Liver Diseases

Transcription

1 LIVER TRANSPLANTATION 18:9-21, 2012 REVIEW Cell Therapies for Liver Diseases Yue Yu, 1,2 James E. Fisher, 1 Joseph B. Lillegard, 1 Brian Rodysill, 1 Bruce Amiot, 3 and Scott L. Nyberg 1 1 Division of Experimental Surgery, Department of Surgery, Mayo Clinic, Rochester, MN; 2 Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, People s Republic of China; and 3 Brami Biomedical, Incorporated, Minneapolis, MN Cell therapies, which include bioartificial liver support and hepatocyte transplantation, have emerged as potential treatments for a variety of liver diseases. Acute liver failure, acute-on-chronic liver failure, and inherited metabolic liver diseases are examples of liver diseases that have been successfully treated with cell therapies at centers around the world. Cell therapies also have the potential to be widely applied to other liver diseases, including noninherited liver diseases and liver cancer, and to improve the success of liver transplantation. Here we briefly summarize current concepts of cell therapy for liver diseases. Liver Transpl 18:9-21, VC 2011 AASLD. Received June 25, 2011; accepted October 11, Cell therapies can be categorized by the cell organization and the route of therapy, as summarized in Table 1. Extracorporeal or ex vivo therapies such as bioartificial livers are intended for the short-term, intermittent support of patients in liver failure, whereas implantable or in vivo therapies such as cell transplantation can be used for short-term, longterm, or permanent liver replacement. Both ex vivo and in vivo forms of cell therapy can be further classified by the types of cell organization used for therapy, which include individually isolated cells, cellular aggregates, synthetically engineered liver tissue constructs, and naturally occurring liver organs. Synthetic liver tissues, which are also called cellular scaffolds and liver tissue constructs, can be vascularized or can be avascular and supported by diffusion. Livers can be used as whole, intact organs or can be divided along well-defined lobar and segmental planes. In this review, we address each category and subcategory, which are based on features such as cell sources (primary hepatocytes, cell lines, stem cells, other progenitor cells, and other nonparenchymal cell types) and liver diseases that may be appropriate for each cell therapy, as summarized in Table 2. We summarize historical, current, and future forms of these cell therapies and emphasize clinical therapies and emerging therapies currently under evaluation in animal models. NEED FOR LIVER SUPPORT THERAPIES Tens of millions of patients are affected by liver disease worldwide. Many of these patients can be treated or their disease phenotype can be prevented with therapy involving biologically active living cells. Liver transplantation, the ultimate cell therapy, is presently the only proven treatment for many medically refractory liver diseases, including end-stage liver disease and many inherited liver diseases. However, there is a profound shortage of transplantable donor livers. Because of this shortage, approximately 40% of listed patients each year do not receive a liver transplant, and a significant number of these patients either die or become too sick for transplantation according to the United Network for Organ Sharing. Therefore, new Abbreviations: AAV, adeno-associated virus; ALF, acute liver failure; ELAD, extracorporeal liver assist device; FAH, fumaryl acetoacetate hydrolase; HEK293, human embryonic kidney 293; ips, induced pluripotent stem; ITR, inverted terminal repeat; raav, recombinant adeno-associated virus; Treg, regulatory T cell. This research was supported by the Marriott Foundation, the Coulter Foundation, Yecuris Corporation, and the National Institutes of Health (R01-DK56733, R41 DK092105). Address reprint requests to Scott L. Nyberg, M.D., Ph.D., Division of Experimental Surgery, Department of Surgery, Mayo Clinic, 200 First Street Southwest, Rochester, MN. nyberg.scott@mayo.edu DOI /lt View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI /lt. Published on behalf of the American Association for the Study of Liver Diseases VC 2011 American Association for the Study of Liver Diseases.

2 10 YU ET AL. LIVER TRANSPLANTATION, January 2012 TABLE 1. Status of Cell Therapies for Liver Disease Treatment Option Ex Vivo (Bioartificial Liver) In Vivo (Transplantation) Cells Individual cells Clinically experimental: historical studies Clinically experimental: current studies Aggregates (ie, spheroids) Clinically experimental: current studies Cellular scaffolds Avascular Clinically experimental: historical studies Clinically experimental: historical studies Vascular Partial/split liver Clinically established Whole liver Clinically experimental: historical studies Clinically established therapies are needed to supplement whole organ liver transplantation and to reduce the waiting-list mortality rate. Furthermore, a number of innovative cellbased therapies and animal model studies of human liver disorders highlight the remarkable regenerative capacity of hepatocytes in vivo. These studies indicate the feasibility of cell therapies as a means of replacing lost or diseased hepatic tissue. 1,2 HISTORICAL OVERVIEW The story of ex vivo cell therapy and in vivo cell transplantation for the treatment of liver disease has many parallels. The treatment of liver failure with an ex vivo device composed of living liver tissue was first reported by Eiseman et al. 3 in Matas et al. 4 first performed hepatocyte transplantation in a rodent model in 1976, that is, 9 years after the first human solid organ liver transplant was performed by Starzl et al. 5 Many other forms of liver support therapy and artificial liver support have also been evaluated over the past 50 years. 6 A partial list of these techniques includes hemodialysis, 7 hepatodialysis, 8 extracorporeal heterologous 9 and homologous liver perfusion, 10 cross-circulation, 11 activated charcoal hemoperfusion, 12,13 simple exchange transfusion, 14 and plasmapheresis with plasma exchange. 15 At least 2 positive observations were made from these early clinical trials. First, the neurological status or the extent of hepatic encephalopathy often improved temporarily; however, long-term survival was not significantly affected in comparison with the survival of historical controls. 16 As expected, the underlying liver disease did influence survival, with patients without cirrhosis having improved survival in comparison with patients with cirrhosis in the days before liver transplantation. Second, toxin removal correlated with recovery from TABLE 2. Organizational Considerations for Review Cell sources Primary hepatocytes Cell lines Progenitor cells Hepatocyte-like sources (discarded human livers and liver resections) Human fetal livers Healthy donor animals Tumor-derived lines (C3A and HepG2) Immortalized normal hepatocytes ips cells Embryonic stem cells Fetal stem cells Adult stem cells (oval cells, mesenchymal stem cells, and others) Matured in vitro Matured in vivo Other sources (eg, lymphocytes) Liver abnormalities ALF Inherited metabolic liver disease Hepatitis (alcoholic and viral) Cirrhosis (cholestatic and other) Liver cancer (hepatocellular carcinoma and cholangiocarcinoma) Recurrent disease after transplantation Rejection after transplantation

3 LIVER TRANSPLANTATION, Vol. 18, No. 1, 2012 YU ET AL. 11 hepatic encephalopathy. In fact, many of these early therapies appeared to provide benefits in case reports and small series, but none stood the test of a randomized, prospective trial. 17 Charcoal hemoperfusion is a good example of an artificial support therapy that appeared hopeful in small series but could not stand the test of a randomized, prospective trial. 18 The limitations of early liver support therapies fell into the categories of safety, immune response, reproducibility, functionality, cell dose, and therapy duration. For example, the reproducibility of heterologous and homologous liver perfusion was highly variable because of the inconsistent quality of the organs and the lack of modern preservation techniques. To overcome the safety problems of cross-circulation, in which the patient s blood was directly exchanged with the blood of another human being, a membrane was introduced as a barrier for preventing the transmission of infections and for blocking immune-mediated responses. Lessons learned from these early clinical treatments have helped us to select better and safer membranes for new bioartificial liver devices As a result, modern bioartificial liver devices employ a membrane to prevent both the need for immunosuppression and the harmful immune response due to the direct contact of blood or plasma with the hepatocytes. The term bioartificial liver was first coined by Matsumura et al. 22 in 1987 when they perfused a suspension of porcine hepatocytes in an extracorporeal suspension bioreactor. The treatment duration with the first bioartificial liver device was limited to a few hours because of the death of anchorage-dependent hepatocytes in the suspension culture. The limitations of suspension cultures have been addressed with spherical aggregates of cells (ie, hepatocyte spheroids), which are detailed later in this review. Spheroids allow for greater cell doses and higher functionality. Spheroid cell aggregates also allow for longer periods of therapy and eliminate the need for the frozen storage of isolated cells before extracorporeal use. The first successful clinical hepatocyte transplant was reported by Fox et al years after the report of the first bioartificial liver and involved a child with Crigler-Najjar disease. Since these pioneering studies, many novel clinical applications of both in vivo and ex vivo therapies have been reported for the treatment of acute liver failure (ALF), chronic and end-stage liver diseases, and metabolic liver disorders. Moreover, the variety of current cell sources is vast. Modern cell sources include primary hepatocytes, immortalized cell lines, and an array of stem cells [eg, liver stem cells, bone marrow stem cells, embryonic stem cells, and induced pluripotent stem (ips) cells]. CELL SOURCES FOR LIVER DISEASE THERAPY Primary Hepatocytes Isolated hepatocytes have been used as cell sources for both extracorporeal and injectable cell transplantation procedures. As anchorage-dependent epithelial cells, primary hepatocytes must attach to an extracellular support matrix to avoid programmed cell death, which is termed anoikis. 24 Suspension-based bioartificial liver systems were the first to be tested because of their simplicity and convenience, but these therapies were limited in duration because of the short viability of hepatocytes in suspensions even under ideal conditions. In contrast, the transplantation of individual hepatocytes allows the rapid attachment of transplanted cells to the existing extracellular matrix in vivo. Hepatocyte transplantation is a promising alternative to liver transplantation for the treatment of some liver diseases. Primary hepatocytes can be isolated from nontransplantable human livers, human liver resections, human fetal livers, and healthy donor animals such as pigs. Nontransplantable whole human livers are the most common sources of primary hepatocytes for cell therapy. However, the quality and metabolic/functional activity of isolated primary hepatocytes are variable. Bhogal et al. 25 reported that the time delay between hepatectomy and liver perfusion is the most important factor in determining the likelihood of the procedure s success, and the shortest possible digestion time is desirable. Their overall success rate for isolating human hepatocytes from different liver sources was 54%. Their data further highlight the fact that tissue from patients with biliary cirrhosis provides high hepatocyte yields (success rate ¼ 71%), so these cirrhotic livers could also be valuable sources of human hepatocytes for experimental use. Isolated hepatocytes can also be cryopreserved and stored in hepatocyte banks, and this allows scheduled or emergency transplantation. However, hepatocytes are highly susceptible to the freezethaw process, and their functionality after frozen cryopreservation is significantly reduced in comparison with the functionality of freshly isolated hepatocytes. It has been suggested that cryopreservation is a further stimulus for the apoptosis of freshly isolated hepatocytes. 26 Several cryopreservation methods for hepatocytes have been reported, but the functional activity of hepatocytes after thawing is still unsatisfactory, and further research is required for an optimal cryopreservation technique. Tumor Cell Lines A critical barrier to the success of hepatocyte transplantation and bioartificial livers is the limited supply of high-quality human hepatocytes. Conventional methods for obtaining hepatocytes cannot meet the clinical demand because of the shortage of livers from which high-quality hepatocytes can be isolated, and hepatocytes are not easily maintained in cultures for extended periods of time. The demand for human hepatocytes, therefore, heavily outweighs their availability. Hence, some investigators have made immortalized human hepatocytes via spontaneous transformation, the introduction of telomerase constructs, or retroviral transfection. To date, the C3A line, a subclone of the HepG2 hepatoblastoma cell line, is the

4 12 YU ET AL. LIVER TRANSPLANTATION, January 2012 only human-based cell line that has been tested clinically in a bioartificial liver device [the extracorporeal liver assist device (ELAD) system] These clinical studies have suggested safety because no evidence of C3A cell transmission has ever been reported. There is some concern because C3A cells demonstrate reduced levels of ammonia detoxification and urea cycle activity, P450 activity, and amino acid metabolism in comparison with adult porcine hepatocytes. 34 The reduced removal of ammonia by immortalized C3A hepatocytes appears to be due to the reduced expression of the urea cycle genes. 35 To enhance their ammonia detoxification activity, the glutamine synthetase gene was transfected into C3A cells, and the transfected cells showed higher ammonia removal activity in a bioreactor. 36 Because of the potential for tumor transmission, the clinical use of immortalized cell lines such as C3A has been limited to extracorporeal devices that possess a membrane for blocking the spread of cells to the patient. Notably, a pivotal randomized trial of the ELADsystemwasongoingatthetimeofthiswriting. Immortalized Hepatocyte Lines From Normal Human Hepatocytes In an attempt to bypass the limitations associated with tumor cells derived from immortalized human hepatocytes, researchers have tried to immortalize hepatocytes from non tumor-derived hepatocytes. Chen et al. 37 transplanted immortalized human fetal hepatocytes (ie, HepCL cells) into 90% hepatectomized mice to prevent ALF. They demonstrated that HepCL cells functioned similarly to primary human fetal hepatocytes and showed no tumorigenicity; this suggested the superiority of HepCL cells over HepG2 cells with respect to metabolic support during ALF. Immortalized human hepatocytes from non tumor-derived hepatocytes may also be useful as potential sources of liver support in bioartificial liver systems. Xenotransplantation With Primary Pig Hepatocytes Because of the shortage of human hepatocytes for cell therapies, xenogeneic cells have also been considered as potential cell sources for bioartificial liver systems and for the treatment of liver disease. Primary porcine hepatocytes have been used in several liver support devices undergoing preclinical and clinical evaluations. Unlike immortalized cell lines, porcine hepatocytes maintain hepatocyte-specific metabolic functions, including ammonia detoxification. 38 However, concerns about the use of porcine hepatocytes include the risks of humoral and cellular immunological responses 39 and potential functional mismatches between porcine hepatocyte released proteins and their human counterparts. The transmission of a porcine endogenous retrovirus from pig cells to human patients is a potential risk of pig cell therapy. However, no such transmission was identified in an extensive examination of human tissues subjected to pig cell therapies. 40 Figure 1. Possible sources of cells in the liver. A variety of cell types can be induced to form the parenchymal cells in the liver. Hepatocytes can divide to produce daughter hepatocytes; bone marrow cells can add genetic material by fusing with hepatocytes. Oval cells are resident precursor cells for cholangiocytes and hepatocytes. Stem cells can produce all cells in the liver. Fetal liver cells are an experimental source for hepatocytes and cholangiocytes. Reprinted with permission from American Journal of Transplantation. 41 Copyright 2009, John Wiley & Sons, Inc. Stem Cells Recent advances in cell biology have led to the concept of regenerative medicine, which is based on the therapeutic potential of stem cells. Stem cells, which are distinguished by their ability to self-renew and differentiate into a wide variety of cell types, have been proposed as an ideal cell source for generating unlimited numbers of hepatocytes, as illustrated in Fig. 1. Different types of stem cells are theoretically eligible for liver cell replacement. Liver Stem Cells A large number of studies have used liver-derived stem cells, including fetal liver stem cells and adult liver stem cells, to generate primary hepatocytes. 42,43 Fetal liver stem cells, which are also called hepatoblasts, appear with the differentiation of the hepatic endoderm and the growth of the liver bud. Hepatoblasts are bipotent and thus able to give rise to both hepatocytes and bile duct cells. Murine hepatoblast cell lines have been established by various research groups, and their capacity to repopulate the liver upon transplantation has been well studied in animal

5 LIVER TRANSPLANTATION, Vol. 18, No. 1, 2012 YU ET AL. 13 models. 42 Weber et al. 44 reported that human fetal liver cells can be isolated and cultured. These cells can also engraft and differentiate into mature hepatocytes in situ after transplantation into immunodeficient mice. The adult liver has particularly extensive regenerative potential in response to injury and parenchymal loss, and this is mainly granted by mature hepatocytes. However, whenever the replication ability of hepatocytes is experimentally inhibited or impaired by advanced chronic injury, liver regeneration can still be accomplished by the activation, expansion, and differentiation of so-called hepatic progenitor/stem cells. Oval cells represent the majority of these hepatic progenitor cells. Like hepatoblasts, oval cells are also bipotent. 43 Although there have been great advances in liver stem cell biology, these cells are rare within liver tissue because hepatoblasts compose only approximately 0.1% of the fetal liver mass, and oval cells compose 0.3% to 0.7% of the adult liver mass. This makes the isolation of both types difficult and renders expansion unfavorable for large-scale applications. 45,46 Hepatocyte-Like Cells From Bone Marrow Derived Stem Cells In the last decade, studies have suggested that cells derived from bone marrow can give rise to hepatocytes. At least 4 different bone marrow derived cell populations have been described: hematopoietic stem cells, mesenchymal stem cells, multipotent adult progenitor cells, and very small embryonic-like cells. The transplantation of nonliver stem cells (eg, bone marrow derived stem cells) has also demonstrated the feasibility of generating functioning hepatocytes. Li et al. 47 demonstrated that adipose tissue derived mesenchymal stem cells can be transduced by recombinant adeno-associated virus (raav) vectors, be engrafted into recipient livers, contribute to liver regeneration, and serve as a platform for transgene expression without eliciting an immune response. Mesenchymal stem cells are rare multipotent residents of bone marrow and other tissues (eg, adipose tissue) that can rapidly expand in culture and are able to develop into several tissue types. Mesenchymal stem cells might become a more suitable source for stem cell based therapies than hematopoietic stem cells because of their favorable immunological properties, ease of access, and potential for transdifferentiation. Hepatocyte-Like Cells From Annex Stem Cells Another promising source of human stem cells is human placental tissue, which contains cells with higher proliferation and differentiation potential in comparison with adult stem cells and does not seem to form teratomas or teratocarcinomas in humans. Several studies have indicated that the umbilical cord and umbilical cord blood, the placenta, and the amniotic fluid are easily accessible sources of pluripotent stem cells, which may be readily available for transplantation or for further expansion and manipulation before cell therapy. Using flow cytometry, histology, immunohistochemistry, and reverse-transcriptase polymerase chain reaction for human hepatic markers to monitor the engraftment of human cells into animals, Piscaglia et al. 48,49 demonstrated that human umbilical cord blood stem cells could colonize the liver and differentiate into hepatocytes after acute toxic liver damage in nonobese diabetic/severe combined immunodeficient mice and in immunocompetent rats. Hepatocyte-Like Cells From Embryonic Stem Cells In other studies, embryonic stem cells have been induced to differentiate into hepatocyte-like cells. So far, many promising studies have shown the therapeutic potential of differentiated derivatives of embryonic stem cells for ameliorating a range of diseases in animal models. These derivatives are able to colonize the injured liver and function as mature hepatocytes. 42 Hepatocyte-Like Cells From ips Cells Human ips cells could prove to be an unlimited source of hepatocytes. For clinical applications, the all-autologous setting seems to be the most promising approach. Because ips cells can bypass the ethical concerns of embryo destruction related to the derivation of embryonic stem cells as well as potential issues of allogeneic rejection, ips cells may be a more ideal source for producing patient- and diseasespecific adult cells for future clinical applications. Hepatocyte-like cells generated from ips cells have been shown to secrete human albumin, synthesize urea, and express human cytochrome P450 enzymes, and they could represent a very promising cell population for future therapeutic transplantation. 50 Differentiated cell types produced from a patient s ips cells have many potential therapeutic applications, including tissue replacement and gene therapy. Investigators have recently reported that ips cells can be used to treat type 1 diabetes and several inherited liver diseases. 51 The transplantation of hepatocytes derived from human ips cells could be an alternative to liver transplantation for patients with ALF or for the correction of genetic disorders that result in metabolically deficient states. Hepatocyte-Like Cells From Other Sources Several efforts have been made to generate hepatocyte-like cells from other sources (eg, monocytederived hepatocyte-like cells). Ehnert et al. 52 demonstrated that by bridging the time to whole organ transplantation or supporting the regeneration of the liver, the transplantation of hepatocyte-like cells derived from peripheral blood monocytes may represent a possible therapy for patients suffering from acute or acute-on-chronic liver disease.

6 14 YU ET AL. LIVER TRANSPLANTATION, January 2012 EXPANSION OPTIONS FOR PRIMARY HEPATOCYTES Although other cell types have shown promise as alternatives to primary hepatocytes, primary hepatocytes continue to be the dominant source of cells for liver cell therapy. Unfortunately, isolated primary hepatocytes show little proliferation capacity ex vivo no matter which architectural configuration is employed, and the most significant problem with cultured hepatocytes is that they rapidly lose their differentiated structures and liver-specific functions after isolation. Therefore, research into the most effective culture method for primary hepatocytes continues. Small Scale: In Vitro Primary Hepatocyte Culture Techniques The most common primary hepatocyte culture technique is the seeding of the cells as a single layer onto collagen gel coated dishes in a conditioned medium. When primary hepatocytes are cultured on a single collagen layer, they produce albumin and urea and show cytochrome P450 activity, but their liver-specific functions steadily decline within the first week of the culture. To mimic the matrix surrounding the hepatocytes in the sinusoid, a second layer of collagen is added on top of the cultured hepatocytes; this is called a collagen sandwich configuration. This scheme maintains hepatocyte function and polarity and induces distinct apical and lateral membrane formation. 53 In an effort to re-create the interactions between parenchymal and nonparenchymal cells, several types of liver nonparenchymal cells, such as fibroblasts, stellate cells, Kupffer cells, and endothelial cells, have been cocultured with hepatocytes, and they have shown remarkably liverlike structure and function. 54 Michalopoulos et al. 55 showed that parenchymal and nonparenchymal cells self-organize in roller bottles to form simple epithelial structures consisting of an outer layer of biliary epithelial cells, a middle layer of hepatocytes and connective tissue, and an inner layer of endothelial cells. Large Scale: Bioreactor Options for Cell Cultures The generation of a system for culturing large numbers of hepatocytes may be required for clinical applications such as bioartificial livers and hepatic tissue engineering. A bioartificial liver is designed for use either before liver transplantation (until the recovery of the native liver) or as a chronic supportive therapy. Similarly to hemodialysis for the treatment of kidney failure, a bioartificial liver functions outside the patient s body, but it is unique in that it contains metabolically active liver cells (ie, hepatocytes), which provide liver functionality to the patient. Hepatocytes are the functional components of a bioartificial liver. The design configurations of bioartificial livers include flat-plate membranes, hollow-fiber cartridges, encapsulation technology, and cell aggregates (eg, spheroids) that are either attached to a support or placed in a suspension culture. 56 Flat-Membrane Culture Systems The use of a flat-membrane bioreactor allows for the control of the internal flow distribution and the perfusion of all hepatocytes under a stable oxygen and hormone gradient in vitro. Hepatocytes cultured with this technique have shown specific in vivo zonal differentiation characteristics, such as the expression of phosphoenolpyruvate carboxykinase in the upstream oxygen-rich region and cytochrome P450 2B in the downstream oxygen-poor region. Recently, this system was applied to study the effects of acetaminophen toxicity on metabolically zonated hepatocytes. 57 Hollow-Fiber Systems Most bioartificial liver devices that have been tested clinically employ hollow-fiber cartridges containing either porcine or human hepatocytes. The hollow-fiber cartridges provide a large surface area for the mass transfer of waste molecules from the patient to hepatocytes in the bioartificial liver. The hollow fibers also serve as a semipermeable barrier that separates cells in these devices from cytotoxic proteins and cells in the patient s blood. Most cell-based ex vivo devices require an oxygenator membrane in the flow circuit to meet the large demand of the cells for oxygen. Nyberg et al. 58 used a device in which hepatocytes in a supporting matrix are seeded into the intrafiber space of hollow fibers; this allows oxygenated plasma to flow over the outer surface of the fibers. This technology is being used to culture other cell types, including mesenchymal stem cells, embryonic stem cells, and stem cells in various stages of hepatic differentiation. 59 Encapsulation Technology Hepatocyte microencapsulation techniques using synthetic semipermeable membranes have been developed to physically separate and protect xenogeneic cells from a recipient s immune system within a support system. In theory, the biomaterial excludes high-molecular-weight components of the immune system but allows the free passage of low-molecularweight nutrients and oxygen across the semipermeable membranes. 60,61 Spherical Aggregate Culture Systems Hepatocyte spheroids, which are spherical, multicellular aggregates of hepatocytes greater than 50 lm in diameter, provide a useful 3-dimensional tissue construct for cell transplantation and bioartificial livers. Several methods, such as the culturing of hepatocytes on nonadherent plastic surfaces for self-assembly 62 and rotational culturing via spinner flasks, 63 have been employed for the formation of spheroids from

7 LIVER TRANSPLANTATION, Vol. 18, No. 1, 2012 YU ET AL. 15 mammalian hepatocytes. More recently, Nyberg et al. 64 reported the preliminary observation that hepatocytes form spheroids spontaneously when they are rocked in a suspension culture. Hepatic spheroids and encapsulated hepatocyte aggregates can be maintained in a suspension culture at a high cell density under oxygenated bioreactor conditions. Suspension culture systems can be easily scaled up to cell mass levels appropriate for sustaining a patient s life. In Vivo Incubators: Humanized Animal Livers As stated earlier, a large demand exists for an abundant, routinely available, high-quality source of human hepatocytes for therapeutic and diagnostic applications. To meet this demand, in vivo (ie, animal) models were proposed for expanding primary human hepatocytes. However, the engraftment of human hepatocytes after their transplantation into animals was very low and corresponded to only approximately 0.5% of the recipient s liver mass under normal conditions. Various approaches have been employed to improve the engraftment of human hepatocytes in animal models. Repeated hepatocyte transplantation has been shown to increase the level of engrafted cells beyond 5%, and this is sufficient to correct some metabolic defects. Primary adult hepatocytes lose their functionality and viability after isolation and culture, and they have limited proliferation potential in vitro. However, hepatocytes have a remarkable regenerative capacity in vivo. On the basis of the in vivo properties of hepatocytes, animal models have been developed specifically for the selective expansion of transplanted human hepatocytes. One such model is the immunodeficient urokinase-type plasminogen activator transgenic mouse, 65 in which an albumin promoter directs the high-level toxic expression of urokinase-type plasminogen activator. The hepatotoxicity in this model creates a permissive environment for the expansion of transplanted hepatocytes, which undergo more than 12 cell divisions on average. Azuma et al. 66 introduced another method by which primary human hepatocytes can be efficiently expanded to nearly complete (>90%) hepatocyte replacement in the livers of mice triply mutant for fumaryl acetoacetate hydrolase (Fah), recombination activation gene 2, and the common c-chain of the interleukin receptor. In the absence of the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, the native mouse hepatocytes die, whereas the transplanted human hepatocytes expand. This model s unique capacity for the expansion of primary human hepatocytes is based on 2 essential features: extensive and continuous liver injury and a strong selective advantage for the transplanted cells to survive in comparison with the host cells. However, a limitation of the repopulated liver of the FAH-deficient mouse is related to the absolute number of primary human hepatocytes that can be obtained. Thus, an alternative to mice for the large-scale expansion of primary human hepatocytes would be an FAH-deficient pig. Pigs offer a 1000-fold scale-up of the potential for the expansion of primary human hepatocytes. The strategy that we have used for the cloning and herd development of heterozygote and homozygote Fah-null pigs is illustrated in Fig Perfusion of Decellularized Liver Matrix and Hepatocytes Recent progress in hepatic tissue engineering has been hampered by low initial levels of hepatocyte engraftment and an insufficient blood supply in vivo. Uygun et al. 68 demonstrated a novel approach to the generation of transplantable liver grafts with decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network and allows the efficient recellularization of the liver matrix with adult hepatocytes. The recellularized graft supports liverspecific functions, including albumin secretion, urea synthesis, and cytochrome P450 expression, at levels comparable to those of normal livers in vitro. Recellularized liver grafts have been perfused with minimal ischemic damage after transplantation into rats. 69 Bao et al. 69 developed an intact 3-dimensional scaffold of an extracellular matrix derived from a decellularized liver lobe, repopulated it with hepatocytes, and successfully implanted this tissue construct into the portal system. The tissue-engineered liver provided sufficient volume for the transplantation of cells representing up to 10% of whole liver equivalents. The treatment of rats that underwent extended hepatectomy with tissueengineered livers improved their liver function and prolonged their survival. These results provide a proof of principle for the generation of transplantable liver grafts as a potential treatment for liver disease. CELL THERAPIES FOR LIVER FAILURE The potential indications for cell-based therapies are ALF, acute-on-chronic liver failure, and acute decompensation after liver resection. Acute-on-chronic liver failureisdefinedastheacutedeteriorationofachronic liver disease. Pareja et al. 70 demonstrated that hepatocyte transplantation may decrease the mortality rate for patients with end-stage liver disease who are awaiting liver transplantation and possibly even prevent death for patients with ALF. So far, more than 30 cases of hepatocyte transplantation in children and adults have been reported. In addition to hepatocyte transplantation for ALF treatment, Jin et al. 71 suggested that bone marrow mononuclear cell transplantation in combination with hepatocyte growth factor administration also has a synergistic beneficial effect by improving both functional and histological liver recovery in a mouse model of ALF. Moreover, stem cells, including mesenchymal stem cells, show promise for the attenuation of ALF. Parekkadan et al. 72 provided the first experimental evidence for the medicinal use of mesenchymal stem cell derived molecules in the treatment of an inflammatory condition, and their findings support the role of chemokines and altered

8 16 YU ET AL. LIVER TRANSPLANTATION, January 2012 Figure 2. Schematic of AAVdirected gene targeting and somatic cell nuclear transfer for the production of heterozygote Fah-null pigs. 67 The cloning of genetic knockout pigs involves 3 steps. First, create the targeting construct with the disruption cassette with production in HEK293 cells (yellow). Second, select clones of porcine fetal fibroblasts that have been successfully transfected with the targeting construct (blue). Third, produce genetically engineered piglets by somatic cell nuclear transfer, that is, the fusion of a targeted porcine fetal fibroblast with an enucleated pig embryo (green). leukocyte migration as a novel therapeutic modality for ALF. Shi et al. 73 demonstrated that the encapsulation of hepatocytes and mesenchymal stem cells can improve hepatocyte-specific functions in vitro and in vivo and that the transplantation of these cells may be a promising strategy for cell-based therapy for acute liver diseases. Another cell-based therapy for ALF is the bioartificial liver. A bioartificial liver system removes toxins by filtration or adsorption (an artificial liver) and performs biotransformation and synthetic functions of biochemically active hepatocytes. Several bioartificial liver modalities have been tested in the clinical arena, and other improved configurations are under development. 77,78 CELL THERAPIES FOR INHERITED LIVER DISEASES Major indications for hepatocyte transplantation include inherited metabolic liver diseases in children. Hepatocyte transplantation has been used to treat inherent liver diseases either as a bridge to whole organ transplantation or as a long-term correction of the underlying metabolic deficiency. 23 Some genetic diseases that have been treated with hepatocyte transplantation include familial hypercholesterolemia, Crigler-Najjar syndrome type I, glycogen storage disease type 1a, urea cycle defects, and a congenital deficiency of coagulation factor VII. Dhawan et al. 79

9 LIVER TRANSPLANTATION, Vol. 18, No. 1, 2012 YU ET AL. 17 Figure 3. Schematic summarizing the approaches to hepatocyte transplantation for the treatment of inherited metabolic disease. The standard procedure under clinical evaluation uses cryopreserved human hepatocytes from a human donor liver (green). Alternative procedures for producing transplantable hepatocyte-like cells from human stem cells are under development. Also under development are individualized approaches to the treatment of inherited metabolic disease via ex vivo gene therapy and in vivo cell transplantation. An individualized approach would involve the production of hepatocytes from the patient s own cells and thereby avoid the need for immunosuppression. Patient-derived hepatocytes may be produced in vitro or expanded in vivo in genetically engineered animals such as the FAH-deficient pig. Patient-derived hepatocytes could also be used in an extracorporeal bioartificial liver. observed that the most encouraging outcomes of hepatocyte transplantation occurred in patients with inborn errors of metabolism. Research on hepatocyte transplantation for inherited liver diseases is actively being pursued. In a thorough review, Flohr et al. 80 summarized animal experimentation with hepatocyte transplantation for the treatment of inherited liver diseases. The development of ips cells from adult somatic tissues may provide a unique approach to the creation of patient- and disease-specific treatments for inherited liver diseases. ips-based cell therapies have been applied to several animal models of liver-based metabolic disorders, and the results have been encouraging. 81 An example of a method for potentially individualizing the treatment of inherited liver disease with ips cell technologies is illustrated in Fig. 3. CELL THERAPIES FOR NONINHERITED LIVER DISEASES Cell Therapy for Hepatitis Fernandez-Ruiz et al. 82 reported that the genetic engineering of endothelial progenitor cells for the overexpression of cytokine cardiotrophin 1 enhances the hepatoprotective properties of endothelial progenitor cells and constitutes a therapy that deserves consideration for fulminant hepatitis. Longhi et al. 83 used autoantigen-specific regulatory T cells (Tregs) and engineered designer T cells as potential tools for immune tolerance reconstitution in patients with type 2 autoimmune hepatitis and chronic hepatitis B, respectively. Farag et al. 84 demonstrated that vaccination with ex vivo activated dendritic cells may be a promising tool for therapeutic or prophylactic approaches to hepatitis B virus.

10 18 YU ET AL. LIVER TRANSPLANTATION, January 2012 Cell Therapy for Liver Cirrhosis Chronic liver disease is usually accompanied by progressive fibrosis. Paradoxically, the intravenous injection of bone marrow cells and particularly mesenchymal stem cells appears to be therapeutically useful for animals with ongoing hepatic damage. Phase 1 trials involving the injection of autologous bone marrow cells into patients with cirrhosis have reported modest improvements in clinical scores. 85 Terai et al. 86 reported improvements in the Child-Pugh scores and albumin levels of 9 patients with cirrhosis who received a portal vein infusion of unsorted autologous mesenchymal stem cells. Also, a significant increase in liver function after liver resection was documented in patients with cirrhosis who were pretreated with autologous mesenchymal stem cell transplantation. 87 Transplanted mesenchymal stem cells have been shown to improve insulin resistance and thereby contribute to glucose homeostasis and the amelioration of liver cirrhosis in rodent models of carbon tetrachloride induced liver disease. 88 Autologous peripheral bone marrow cell transplantation has also been shown to be a novel and potentially beneficial treatment for patients with decompensated liver cirrhosis. 89 Cell Therapy for Liver Cancer The possible therapeutic benefits of bone marrow stem cells for patients with liver cancer were first investigated in 2005 when autologous CD133 þ bone marrow stem cells were transplanted via the portal vein into patients with liver cancer before they underwent portal venous embolization. After embolization, these patients underwent extensive liver resection, which caused a subsequent degree of clinical improvement. 90 Fürst et al. 91 concluded that a combination of portal vein embolization and CD133 þ bone marrow stem cell administration increased the degree of hepatic regenerationincomparisonwithembolizationaloneinpatients with malignant liver lesions. Similarly, patients with hepatocellular carcinoma who first received autologous bone marrow stem cells experienced a significant increase in liver function after liver resection. 87 Allogeneic hematopoietic stem cell transplantation produces a graft-versus-tumor effect in patients with solid tumors. The most optimal graft-versus-tumor effect has been demonstrated in patients with advanced primary liver cancer who previously underwent liver transplantation. The suppression of the tumor and its progression may be undertaken before and after stem cell transplantation in combination with adjuvant cell therapies because donor-derived immune cells allow the allogeneic graft-versus-tumor effect. 92 There are currently no survival data. More clinical trials are needed to evaluate such issues in the future. Cell Therapy for Inducing Immunity Tolerance After Liver Transplantation Liver transplantation has evolved over the past 4 decades into the most effective method for treating endstage liver failure. Despite this evolution, liver transplantation has numerous limitations, including rejection. Although immunosuppressive drugs are highly effective in protecting allografts from acute rejection, current immunosuppression targets all arms of the immune system. In addition to its high cost, immunosuppression has side effects that can worsen patient morbidity and mortality rates. Therefore, it is important to induce tolerance that is specific to the donor antigen so that immunosuppression can be avoided. A couple of alternatives to pharmacological immunosuppression are described in the following paragraphs. Tregs Compelling evidence from animal transplant models and clinical data show that manipulating the balance between Tregs and responder T cells is a potentially effective strategy for controlling immunoresponsiveness after transplantation because Tregs play a critical role in promoting immunological unresponsiveness to allogeneic organ transplants. CD4 þ CD25 þ Tregs represent a subset of T cells without specific antigen stimulation that can suppress immune responses through a mechanism of cell-to-cell contact. Thus, CD4 þ CD25 þ Tregs may have effects on both the induction and maintenance of tolerance. Such studies could help us to determine whether the enhancement of Tregs also correlates with the immunological features associated with clinical transplant tolerance. 93 Mesenchymal Stem Cells Mesenchymal stem cells exert strong immunosuppressive effects, including the suppression of B, T, and natural killer cell activity, the complement pathway, and the differentiation and maturation of dendritic cells (the most important antigen-processing cells). Mesenchymal stem cells also exert a profound inhibitory effect on T cell proliferation in vitro and in vivo. Moreover, a recent major breakthrough was the discovery that mesenchymal stem cells induce the generation of Tregs both in vivo and in vitro Cell Therapy for Liver Regeneration Living donor liver transplantation has been developed to increase the number of donor livers. However, living donor liver transplantation makes use of small-for-size liver transplantation. In small-for-size liver transplantation, a size-mismatched graft is used that may not be large enough for the recipient s required level of liver function. The size of the graft in small-for-size liver transplantation has an inverse relationship with the degree of nonfunction. Poorly functioning grafts show delayed and impaired regeneration, which frequently leads to liver failure. Therefore, immediate regeneration of the mismatched graft is required in patients undergoing living donor liver transplantation. Current work has established the feasibility of using ips cells generated in a clinically acceptable fashion for rapid and stable liver

11 LIVER TRANSPLANTATION, Vol. 18, No. 1, 2012 YU ET AL. 19 regeneration. 97 Similarly, mesenchymal stem cell based therapies may provide a novel approach to hepatic regeneration and hepatocyte differentiation and thereby support hepatic function in diseased individuals. 98 CONCLUSION Despite the notable achievements of hepatocyte transplantation and bioartificial liver therapy in treating various liver diseases by acting as bridges to transplantation or allowing recovery without transplantation, the limited supply of primary human hepatocytes remains a major barrier to the successful expansion of liver cell therapies. Stem cells are promising tools for the treatment of many liver diseases and the service of regenerative medicine. However, the in vivo functionality of stem cell derived hepatocytelike cells is largely unknown. The normal differentiation of ips cells into hepatocyte-like cells and thus mature hepatocytes has yet to be reported. It is likely that the normal and complete differentiation of liver progenitor cells into mature hepatocytes requires an in vivo milieu. Moreover, critical issues, including the long-term safety, tolerability, and effectiveness of the engraftment of primary hepatocytes and stem cells in diseased human livers, need to be addressed. The efficacy of novel cell-based treatments must be established in clinical practice. Clinical trials are needed to evaluate unresolved issues of liver cell therapies. These hurdles are likely to be overcome, and this will be associated with the wider application of cell-based therapies to the treatment of liver diseases. Meanwhile, parallel strategies for increasing organ donation are also required in conjunction with advances in cell therapies to combat liver disease; these strategies include living donor and split liver transplantation and tissue-engineered transplantable livers. REFERENCES 1. 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