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Am J Transplant. Author manuscript; available in PMC 2016 June 01. Published in final edited form as: Am J Transplant. 2016 June ; 16(6): 1688–1696. doi:10.1111/ajt.13678.
Liver-Regenerative Transplantation: Regrow and Reset A. Collin de l’Hortet1, K. Takeishi1, J. Guzman-Lepe1, K. Handa3, K. Matsubara3, K. Fukumitsu4, K. Dorko5, S. C. Presnell5, H. Yagi3, and A. Soto-Gutierrez1,2,* 1Department
of Pathology, University of Pittsburgh, Pittsburgh, PA
2Thomas
E. Starzl Transplantation Institute and McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA
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3Department
of Surgery, School of Medicine, Keio University, Tokyo, Japan
4Division
of Hepato-Biliary-Pancreatic and Transplant Surgery, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 5Organovo
Holdings Inc., San Diego, CA
Abstract
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Liver transplantation, either a partial liver from a living or deceased donor or a whole liver from a deceased donor, is the only curative therapy for severe end-stage liver disease. Only one-third of those on the liver transplant waiting list will be transplanted, and the demand for livers is projected to increase 23% in the next 20 years. Consequently, organ availability is an absolute constraint on the number of liver transplants that can be performed. Regenerative therapies aim to enhance liver tissue repair and regeneration by any means available (cell repopulation, tissue engineering, biomaterials, proteins, small molecules, and genes). Recent experimental work suggests that liver repopulation and engineered liver tissue are best suited to the task if an unlimited availability of functional induced pluripotent stem (iPS)–derived liver cells can be achieved. The derivation of iPS cells by reprogramming cell fate has opened up new lines of investigation, for instance, the generation of iPS-derived xenogeneic organs or the possibility of simply inducing the liver to reprogram its own hepatocyte function after injury. We reviewed current knowledge about liver repopulation, generation of engineered livers and reprogramming of liver function. We also discussed the numerous barriers that have to be overcome for clinical implementation.
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Introduction Approximately 30 million people in the United States have liver disorders, which are responsible for ≈30 000 deaths annually in the United States and nearly 1 million deaths in developing countries (1–3). Medical therapy can extend life, but the only curative therapy for terminal liver failure (acute or chronic) is allogeneic liver transplantation—either a *
Corresponding author: Alejandro Soto-Gutierrez, ; Email: [email protected]. Disclosure The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. K.D. and S.P. are full-time employees and shareholders of Organovo Holdings, Inc., a publicly held corporation specializing in the production and application of three-dimensional human tissues for in vitro and in vivo applications. The other authors have no conflicts of interest to disclose.
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partial liver from a living or deceased donor or a whole liver from a deceased donor. Organ demand, however, exceeds supply by tens of thousands. In the United States, the annual number of deceased donor livers decreased from 7014 in 2006 to 5710 in 2013 (4). Living donation numbers have also declined, falling from 524 donors in 2001 to 211 in 2013 (4,5). In addition, it is projected that the demand for livers will increase 10% in 10 years and 23% in 20 years (6).
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Exacerbating the organ shortage problem, the donor pool is expected to shrink further because of the obesity epidemic. Liver steatosis is increasingly common in donors and is a significant risk factor in liver transplantation (7). Moreover, the top four causes of chronic liver disease in patients on the U.S. liver transplant waitlist are currently chronic hepatitis C virus (HCV) infection, alcoholic liver disease, nonalcoholic steatohepatitis (NASH), and a combination of chronic HCV infection and alcoholic liver disease; however, these rankings may change because new developments could modify these statistics (e.g. new direct-acting antiviral agents are transforming the treatment of chronic HCV). Meanwhile, NASH incidence has increased 170%, making it the second leading etiology of chronic liver disease among new liver transplant waitlist registrants in 2013 (8). The number of patients with NASH awaiting liver transplantation is anticipated to continue climbing in the near future, whereas availability of donor organs is expected to decline. Together, these factors reveal the changing epidemiology of patients awaiting liver transplantation in the United States.
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Several strategies have been explored to increase the number of livers available for transplantation. These include the use of marginal donors, donors aged >60 years; donors with >30% macrosteatosis; donors with positive serology for the hepatitis B virus (e.g. hepatitis B core positive) or HCV—donor seropositivity for HCV has been considered a contraindication for liver transplantation, but again, the new panorama after the administration of the new generation of anti-HCV therapies might materially change the future in marginal liver donors; donors with a cold ischemia time of >12 h; donation after cardiac death; and grafts from split livers or living donors (9,10) and even resuscitation of marginal-quality donor organs using machine perfusion (11). In addition, several novel cellular therapies to induce tolerance in solid organ transplant patients have entered early phase clinical trials (12). If successful, organ tolerance is expected to have a substantial impact on morbidity and mortality risks for transplant recipients but a limited impact on the organ donor pool. These data point to two solutions: regrow livers for transplantation or “reset” injured livers to fitness.
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New regenerative approaches to investigating liver organogenesis seek to provide novel insights into liver repopulation, organ engineering (recellularization of natural scaffold technologies, three-dimensional [3D] bioprinting of liver tissue or generation of chimeric organs through blastocyst complementation) and reprogramming the injured liver. We have highlighted these promising areas of investigation (Figure 1). Liver repopulation Liver cell therapy has been under intensive investigation for decades (13), and we have learned that the ideal candidates for this kind of therapy are patients with acute liver failure and patients with liver-based inborn errors of metabolism, especially children (13–15). The Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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native architecture of the liver is intact in these diseases; therefore, the transplant procedure involves simple injection of liver cells through the portal vein into the liver, in which the cells integrate into the host liver. In the case of liver-based inborn errors of metabolism, clinical observations have demonstrated the safety of the procedure, and patients who have undergone liver cell therapy showed clinical improvement and/or partial correction of the underlying metabolic defect (16). In the long term, however, transplanted liver cells appear to survive poorly. The main gap that prevents advancement of the field is poor repopulation of the transplanted cells and the inability to detect acute rejection episodes.
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Recent observations have demonstrated that transplanted liver cells subjected to a certain advantageous regenerative stimulus are able to expand over recipient liver cells (15). Consequently, conditions should be established to facilitate the entry of hepatocytes into the parenchyma (cell engraftment) and the enduring survival and expansion of transplanted liver cells. Preparative liver-directed radiation has been shown to facilitate early engraftment of transplanted liver cells by reversibly disrupting the sinusoidal endothelium and enhancing the entry of transplanted liver cells into the liver parenchyma. Preparative radiation also induces postmitotic hepatocyte death, allowing donor liver cells to preferentially proliferate and repopulate the irradiated host liver. This strategy has been used to correct a rodent model of Crigler–Najjar syndrome (17,18). In the case of acute liver failure, it is required that the transplanted liver cells function immediately at their full capacity; however, the lack of a clinically relevant experimental model and limited clinical trials have restricted our ability to advance the field. The number of liver cells, for example, that need to engraft to reverse hepatic failure is essentially unknown, and whether acute hepatic failure is associated with changes in the local microenvironment that might interfere with engraftment and function of transplanted liver cells is also not yet known. Response to these questions requires a reliable supply of high-quality hepatocytes.
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Creating an immediately available and inexhaustible supply of functioning liver cells from autologous tissue would allow early intervention in patients with hepatic failure and would allow liver cells to be infused over a longer period of time. Combined with recent advances in genome-editing technology, such liver cells could be used widely to treat devastating liver-based inborn errors of metabolism and to eliminate the need for a life-long regimen of immunosuppressive drugs and their complications. Engineering pluripotency of human somatic cells by the ectopic expression of transcription factors has opened the possibility of generating autologous cells for liver cell replacement therapies. Moreover, induced pluripotent stem (iPS) cells are of extraordinary interest because they could be patient specific, can be propagated indefinitely as undifferentiated cells and can differentiate into practically any cell type (19). Although it is not yet possible to differentiate iPS cells to liver cells with functional and regenerative characteristics identical to those residing in the human liver, it is likely a matter of time before these problems can be overcome (20). The enthusiasm for liver cell therapy has been fueled by recent breakthroughs in stem cell biology and hepatic differentiation, especially since the derivation of iPS cells. Interests in the use of iPS cells in regenerative medicine is growing, and several programs have been created for the development of allogeneic cell banks of iPS cells all over the world. By providing a la carte iPS cells that are ready to use, these banks might accelerate and facilitate this clinical procedure. Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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Although much progress has been made toward enhancing selective repopulation of transplanted liver cells, many fundamental questions surrounding regeneration and acute rejection of transplanted liver cells remain unanswered. For liver repopulation to realize its full potential, continued funding support is necessary to address these challenges, especially if iPS-derived liver cells are to be used clinically in the near future. Regrowing livers
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Although cell therapy for acute liver failure and metabolic liver diseases may benefit greatly by the generation of iPS-derived liver cells, the vast majority of patients in need of lifesaving intervention are those with end-stage cirrhosis and chronic hepatic failure. Because fibrotic or cirrhotic livers have abnormal architecture induced by scarring, liver repopulation for end-stage liver disease is more problematic. Cell transplantation into the portal vein of a cirrhotic liver can generate severe portal hypertension and may cause portal thrombosis. Several experimental studies have suggested that liver cell therapy in extrahepatic sites can improve liver function and prolong survival in end-stage cirrhosis while avoiding the complications of severe portal hypertension and portal thrombosis (21–23). Treatment of chronic liver failure might benefit more from the transplantation of a large mass of healthy functioning liver cells; however, their function is influenced by the architecture of the organ and the microenviroment in which the cells reside. Current efforts are aiming to recapitulate the complex organization of liver tissue by using multiple supporting and functional cell types, such as liver nonparenchymal cells (e.g. fibroblasts, macrophages, cholangiocytes, sinusoidal endothelial cells) for transplantation (24). A synergistic goal is to develop useful tools for drug screening and toxicity testing. Both of these objectives would benefit from a means of replicating the function of whole organs.
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Whole organ assembly—A popular strategy being developed for construction of implantable organs involves the decellularization of whole organs and subsequent reseeding with relevant cell types, followed by maturation of the neo-organ in a physiologically appropriate bioreactor (25–33). Numerous protocols for whole liver engineering, cell seeding and implantation of these constructs in animals have demonstrated the feasibility of this approach (25–33). Regeneration of the tissue is initiated by multiple injections of parenchymal cells throughout the liver scaffold, and endothelial cells are perfused through to reestablish the vascular tree prior to transplanting. Recent advances in liver recellularization technology have demonstrated that human fetal liver progenitor and endothelial cells can be grown in a bioreactor in vitro for >1 month. The fetal liver cells reformed into clusters, and evidence of biliary canaliculi and ducts was seen (27). The scalability of the system has been demonstrated recently in which a whole pig liver scaffold was repopulated with primary porcine hepatocytes (30). Another laboratory demonstrated methods for reestablishing the vascular network within decellularized liver scaffolds by conjugating anti–endothelial cell antibodies to maximize coverage of the vessel walls with endothelial cells and, when implanted, were maintained for up to 24 h (34). In each of these studies, the engineered graft failed to survive or function for more than a few hours after transplantation. These disappointing results have been largely attributed to failure to maintain a durable vascular network and to rebuild the complex liver microarchitecture required for function by
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the assembled liver graft after transplantation. There is also an especially important need to prevent blood clotting and to maintain enough blood circulation for the implanted engineered graft. Several other important challenges remain for the recellularized scaffold approach to succeed. Systems to quantify and maximize the parenchymal and nonparenchymal components of the liver, for instance, will be essential for future scaling-up systems and technologies to boost regeneration of hepatocytes inside the scaffold. Furthermore, the sinusoidal endothelium and the bile duct are highly specialized. In vitro results have used only microvascular endothelial cells, and no attempts have been reported yet to reestablish the intrahepatic portion of the biliary tree.
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Perhaps the greatest potential of human iPS cells lies in autologous derivation. Availability of a liver graft from autologous tissue and cells would require no or little immune suppression. To do so may also require the establishment of protocols for the directed differentiation of human pluripotent stem cells into specialized liver non-parenchymal cells; however, recent advances in differentiation of pluripotent cells into endothelial cells (35,36), hepatocytes (18,20) and cholangiocytes (37,38) may accelerate the progress and feasibility of these studies. Will decellularization provide the basis of the protocol to bioengineer a liver for transplantation? Will it simply be a platform for studying the self-organization of the cells that compose a liver? It remains to be seen.
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Liver organoid generation—This interesting approach has been proposed by exploiting the general capacity of cells to self-organize and form structures with similar histogenic properties, such as those seen in vivo using 3D spatial arrangement and several cell types. Through mechanisms for which molecular bases are largely unknown, cells can self-sort and self-assemble to guide tissue formation. These principles were applied in vitro by culturing human iPS-derived endodermal cells, human mesenchymal stem cells and human umbilical vein endothelial cells (HUVEC). The mixture aggregated and self-organized into 3D spheroid structures termed liver buds, or heterotypic cell collectives. This microenviron ment promoted hepatocyte differentiation of the human iPS-derived endodermal cells, eventually producing human iPS-derived hepatocytes that were still immature but that expressed certain liver functions (albumin and α1-antitrypsin) at higher levels than human iPS-derived hepatocytes generated in two-dimensional (2D) cultures. Surprisingly, only 2 days after implantation in different anatomical sites, the organoids exhibited HUVEC-derived blood vessels that were connected to the host circulation and rapidly formed a complex vascular network. Following transplantation in the mesentery of mice that underwent liver injury, the liver buds were vascularized and integrated with host tissues to increase host survival (39).
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In recent studies, the same authors have investigated the mechanism driving organ bud generation. By analyzing the formation and function of liver buds, Takebe and colleagues concluded that the endothelial cells drive vascularization after transplantation and that extracellular matrix properties drive cell condensate formation via effects on mesenchymal stem cells (40). The main limitations of organoid technology include size restriction, but an alternative approach could utilize natural decellularized liver scaffolds that, once repopulated with organoids, can drive cellular self-organization and recapitulate organ function, thereby facilitating the generation of larger, more sophisticated liver structures that incorporate specialized components (e.g. bile duct, sinusoidal endothelial cells). Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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Bioprinting in the fabrication of 3D liver tissues—Automated engineering platforms, such as ink-jet printers and 3D printers, have been actively adapted in the past 10 years to be compatible with manipulation of living mammalian cells (reviewed by Atala and Yoo) (41). This has enabled bioengineers to design and develop patterned 2D cultures and 3D tissue structures in which multiple distinct cell types can be organized in space relative to each other per user specifications. For many tissues, like liver, 3D configurations that preserve the polarity and foster the development and maintenance of intercellular junctions provide demonstrated benefits in extended lifespan and better preservation of function in in vitro cultures (reviewed by LeCluyse et al and Godoy et al) (42,43). The 3D fabrication of structures comprising liver cells has been approached using hydrogels laden with cell spheroids (44,45) or using bioprinting platforms that enable one component or more of the tissue to be made solely from cells (46–48). For the engineering of solid tissues like the liver, in which the majority of cells are in close approximation to other cells without expansive void spaces or extracellular matrix, high cellularity (≥30%) at the time of production may foster cell–cell interactions and lead to more rapid achievement of tissuelike morphology and functionality.
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Regardless of the fabrication method used, the ultimate architecture of the tissue that is created is governed by both the initial fabrication design and the intercellular interactions that occur over time as the structures mature. Bioprinted liver tissues generated by the NovoGen MMX Bioprinter (Invetech, San Diego, CA), for example, are characterized after fabrication by distinct zones in the x-, y- and z-axes that are enriched for primary hepatocytes, endothelial cells, or hepatic stellate cells. Over time, intercellular interactions drive formation of finer structures, highlighted by the close approximation of hepatic stellate cells and hepatocytes and by the formation of microvascular structures within the tissues. In addition, to extended lifespan and function, multicellular tissue constructs that recapitulate some elements of native tissue architecture also provide a means to assess not only the biochemical response to various forms of injury but also the tissue response. Phenomenon such as necrosis, lipid accumulation and fibrosis can be detected. Although it is not yet the case that bioprinted tissues perfectly reproduce native tissue architecture and function, they provide better approximation than simple 2D or monocellular cultures and offer the added benefit of tissue-level organization for interpretation of outcomes. Bioprinted liver tissues were first commercialized in 2014 (Organovo, San Diego, CA) and are being used today for long-term and tissue-level assessment of toxicity, liver biology and disease modeling.
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It is important to consider some of the inherent limitations of automated 3D fabrication platforms as they relate to the production of structures containing living cells. The size and biomechanical forces imposed on cells subjected to bioprinting are a key consideration. Hepatocytes, for example, are highly sensitive to shear force and would be unlikely to survive high-shear deposition methods such as the traditional ink-jet approaches. Unlike simple culture systems wherein dead or dying cells are simply removed with the next media change, shear-damaged hepatocytes may become trapped within the thick bioprinted tissues and create areas of necrosis that disrupt the architecture and further damage neighboring cells. Consequently, there are limits of resolution and deposition speed imposed by the
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tolerances of the cell types used in fabrication, and the parameters must be defined for each cell type and ultimately limited by the most fragile component. Although it is attractive, conceptually, to envision placing individual cells relative to each other and building up a 3D structure cell by cell, there are challenges with respect to the time it takes to make a tissue of appreciable size that meets the metabolic needs of the living cells during the production window. Speed and resolution, the limits of which are dictated by the tolerance of the living cells being used, must be balanced to preserve cell health and phenotype while producing tissue at a scale that is useful for downstream applications. As 3D fabrication technologies evolve to address the challenges of living cells, it may be possible to reproduce the intricate architecture of the liver acinus at a scale that enables tissue-level outcomes to be assessed and that incorporates fluid paths to model circulation and biliary excretion.
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Disease modeling will likely be a primary focus of future liver organogenesis (49). These diseases can include developmental disorders, cancer, infectious disease and liver degenerative diseases (50). In this context, patient-derived or engineered iPS cells will be useful tools in future disease modeling. Laboratory-grown miniature human livers, for instance, also have the potential to be used for testing efficacy and toxicity of drug compounds. This approach could be applied to model degenerative conditions—for example, liver fibrosis or NASH—for which one could study specific pathways related to human pathology or screen for effective treatments. The human liver often metabolizes drugs in a manner distinct from animal metabolism. If successful, the ability to practically engineer liver grafts would go far beyond modeling liver steatosis or fibrosis and could be a tool to accurately predict the metabolism or toxicity of a compound in healthy and diseased human liver grafts in vitro prior to exposure to the whole body. This approach potentially translates into reduced costs and time in drug development and less harmful patient exposure in clinical trials.
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Blastocyst complementation for organ generation—Generation of iPS cells has opened the possibilities to practically producing any cell lineage from any particular person. The differentiation approaches in the laboratory have attempted to mimic normal organ development in a dish, producing cells that do not necessarily reflect the full spectrum of functions of normal hepatocytes (14). In a recent important work, Kobayashi et al showed that is possible to grow a rat pancreas from iPS cells in chimera mice using blastocyst complementation principles (51). This elegant study demonstrated that knocking out the host’s blastocyst master regulator of pancreas development (the Pdx1 gene) and injecting with donor iPS-derived cells, and thus “complementing” this deficiency, generated a newly formed pancreas composed entirely of injected cells (although vessels, nerves and interstitial cells were composed of both host and donor cells, complementing iPS cells contributed to nearly 95% of all pancreatic tissues, including pancreatic islets, exocrine tissues and duct epithelia examined by imaging techniques). These complemented embryos survived to adulthood without abnormalities, indicating that the iPS-derived pancreas was functional. Recently, the same group was able to successfully generate iPS-derived kidney (52). Consistent with this concept of developmental compensation, they were able to induce this pancreatogenesis in large animals (cloned pigs) (53). Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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The exciting possibility of growing patient-specific transplantable organs in chimeric large animals is far in the future. The generation of an entire interspecific liver has not been reported and seems currently challenging with this nascent technology but is clearly possible. To create a whole liver from patient-specific iPS cells that includes the parenchymal and nonparenchymal components (e.g. vasculature, bile duct, gallbladder, nerves), embryos lacking several developmental pathways would have to be created. If chimeric livers were generated containing an animal host’s cells, those cells could pose a threat for immunological rejection once the organs are transplanted into patients. Because the field of liver xenotransplantation has been limited and has had limited survival (54), one can speculate that the expected immune responses could be problematic. Nevertheless, further studies are indispensable to analyze the immunogenicity of such chimeric organs. Resetting liver function
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The vast majority of patients in need of liver transplantation are those with end-stage cirrhosis and chronic hepatic failure. Late-stage liver cirrhosis is characterized by portal hypertension and hepatic encephalopathy, terminal processes that result from fibrosis and vascular remodeling of the cirrhotic liver (55,56). These extrahepatic pathologies are superimposed on liver failure, namely, the inability of hepatocytes to adequately synthesize coagulation factors, conjugate and secrete bilirubin, and regulate metabolism. Hepatocyte decompensation in advanced liver disease is difficult to explain because the liver is capable of full function with less than half its normal hepatocyte content (57,58). In addition, hepatic failure persists even after amelioration of the source of liver injury, indicating that hepatocytes undergo dedifferentiation (epithelial-to-mesenchymal-transition).
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The concept of cellular reprogramming introduced by Yamanaka et al in 2007 (19) has revolutionized cell biology and increased excitement for regenerative medicine. The strategy behind successful lineage reprogramming has its fundamentals in developmental biology and is based on the knowledge of transcriptional networks underlying the establishment and maintenance of cellular identity. In other words, rewiring the transcriptional circuitry by relevant transcription factors can force a new molecular program and thus a new cellular identity. The past decade has seen enormous progress in reprogramming pluripotency and even differentiated hepatocyte-like cells in vitro (59). Recent pioneering work indicates that in vivo lineage reprogramming is possible and has been demonstrated in a handful of organs (60–62).
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To understand this process better in terms of chronic liver failure, Nishikawa et al have studied how hepatocyte metabolic adaptations in response to the microenvironment might lead to cellular breakdown (63,64). Nishikawa et al found that the state of the host microenvironment is critical to the regenerative and functional potential of hepatocytes (63). In other words, hepatocytes can be reprogrammed to functional states by the microenviroment in which they reside. Nishikawa et al demonstrated that fatal chronic liver injury exhibits a disruption of the critical balance of transcription factors and that diseased or dedifferentiated cells can be returned to normal function by reexpression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent (65). In these studies, Nishikawa et al demonstrated that a
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network of hepatocyte-enriched transcription factors (hepatocyte nuclear factor 4α [HNF4A], forkhead box A2, CCAAT/enhancer binding protein α and hepatocyte nuclear factor 1α) regulates the mature hepatocyte phenotype by controlling expression of proteins needed for coagulation, biliary and lipid metabolism. Conversely, the forced reexpression of one factor, HNF4A, improved cellular function and reversed liver failure. This work indicates that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest that reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement. It is possible that such disruption of the hepatic transcription factor network in liver injury is caused the microenviroment; rigorous testing of this hypothesis remains to be performed.
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Currently, it is tantalizing to consider how rewiring of the hepatic transcription factor network might resolve liver failure and fibrosis by resetting hepatocyte function in humans. New therapies centered on hepatic reprogramming mechanisms may become viable strategies in the future by simply inducing the liver to reset the hepatic function of its own cells after injury.
Conclusions
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The worldwide shortage of livers available for transplants has led scientists to develop other promising therapies and to shape the future of regenerative medicine (66). In our opinion, two regenerative strategies can prevail: designing and fabricating entirely brand new livers from fresh materials and/or resetting sick livers back to their hepatic “default settings.” The first approach has the potential to generate an unlimited source of grafts, to provide patients with better timing for the procedure and to improve patient quality of life after surgery. The second approach has the advantage of avoiding any invasive surgery, eliminating long waiting times and eventually decreasing the overall need for liver transplantations. Moreover, even if full liver replacement cannot be achieved with these therapies, they could be used in the near future as a temporary bridge in clinical transplantation (e.g. auxiliary partial orthotopic or heterotopic transplantation of the engineered liver graft) until a liver becomes available.
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We are living in exciting times: Technology and scientific accomplishments in past decades have placed these new approaches within reach and should be reflected in patient care in coming years. Nevertheless, we need to prioritize carefully and not let science be driven by fashion. The irony is that for all of these new technologies, old questions remain to be solved. Even if we are successful, for example, in creating iPS-derived hepatocytes of high standards for liver cell transplantation, we still need to solve the old challenges of engraftment and repopulation. Similarly, we might generate organs through blastocyst complementation, but primordial questions of xenorejection will persist. In other words, working on creating new, elegant tools will never be enough if we do not envision the future needs and uses of technology properly. A combined research effort and appropriate funding in different areas of investigation are crucial and, if done efficiently, have the potential to achieve great clinical success.
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Acknowledgments Funding from the US National Institutes of Health (DK099257 to A.S.-G. and UH3TR000503 to D Lansing Taylor and A.S.-G. as collaborator), and the Competitive Medical Research Fund Program from UPMC Health System to A.S.-G. and Japan Agency for Medical Research and Development, Research Center Network for Realization of Regenerative Medicine, Projects for Technological Development to H.Y. also supported this work.
Abbreviations
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2D
two-dimensional
3D
three-dimensional
HCV
hepatitis C virus
HNF4A
hepatocyte nuclear factor 4a
HUVEC
human umbilical vein endothelial cell
iPS
induced pluripotent stem
NASH
nonalcoholic steatohepatitis
References
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1. Hoyert DL, Xu J. Deaths: Preliminary data for 2011. Natl Vital Stat Rep. 2012; 61(6):1–51. [PubMed: 24984457] 2. Alqahtani SA. Update in liver transplantation. Curr Opin Gastroenterol. 2012; 28(3):230–238. [PubMed: 22450898] 3. Mokdad AA, Lopez AD, Shahraz S, et al. Liver cirrhosis mortality in 187 countries between 1980 and 2010: A systematic analysis. BMC Med. 2014; 12:145. [PubMed: 25242656] 4. Kim WR, Lake JR, Smith JM, et al. OPTN/SRTR 2013 Annual Data Report: Liver. Am J Transplant. 2015; 15(Suppl 2):1–28. [PubMed: 25626341] 5. U.S. Department of Health & Human Services HRaSA. Organ Procurement and Transplantation Network (OPTN). [cited 2015 Nov 3]. Available from: http://optn.transplant.hrsa.gov/ 6. Habka D, Mann D, Landes R, Soto-Gutierrez A. Future economics of liver transplantation: A 20year cost modeling forecast and the prospect of bioengineering autologous liver grafts. PLoS ONE. 2015; 10(7):e0131764. [PubMed: 26177505] 7. Busuttil RW, Tanaka K. The utility of marginal donors in liver transplantation. Liver Transpl. 2003; 9(7):651–663. [PubMed: 12827549] 8. Wong RJ, Aguilar M, Cheung R, et al. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology. 2015; 148(3):547–555. [PubMed: 25461851] 9. Jimenez-Romero C, Caso Maestro O, Cambra Molero F, et al. Using old liver grafts for liver transplantation: Where are the limits? World J Gastroenterol. 2014; 20(31):10691–10702. [PubMed: 25152573] 10. Zarrinpar A, Busuttil RW. Liver transplantation: Past, present and future. Nat Rev Gastroenterol Hepatol. 2013; 10(7):434–440. [PubMed: 23752825] 11. Graham JA, Guarrera JV. “Resuscitation” of marginal liver allografts for transplantation with machine perfusion technology. J Hepatol. 2014; 61(2):418–431. [PubMed: 24768755] 12. Geissler EK, Hutchinson JA. Cell therapy as a strategy to minimize maintenance immunosuppression in solid organ transplant recipients. Curr Opin Organ Transplant. 2013; 18(4): 408–415. [PubMed: 23838645] 13. Puppi J, Strom SC, Hughes RD, et al. Improving the techniques for human hepatocyte transplantation: Report from a consensus meeting in London. Cell Transplant. 2012; 21(1):1–10. [PubMed: 21457616] Am J Transplant. Author manuscript; available in PMC 2016 June 01.
de l’Hortet et al.
Page 11
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
14. Fox IJ, Daley GQ, Goldman SA, Huard J, Kamp TJ, Trucco M. Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease. Science. 2014; 345(6199):1247391. [PubMed: 25146295] 15. Soltys KA, Soto-Gutierrez A, Nagaya M, et al. Barriers to the successful treatment of liver disease by hepatocyte transplantation. J Hepatol. 2010; 53(4):769–774. [PubMed: 20667616] 16. Fox IJ, Chowdhury JR, Kaufman SS, et al. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med. 1998; 338(20):1422–1426. [PubMed: 9580649] 17. Zhou H, Dong X, Kabarriti R, et al. Single liver lobe repopulation with wildtype hepatocytes using regional hepatic irradiation cures jaundice in Gunn rats. PLoS ONE. 2012; 7(10):e46775. [PubMed: 23091601] 18. Chen Y, Li Y, Wang X, et al. Amelioration of hyperbilirubinemia in gunn rats after transplantation of human induced pluripotent stem cell-derived hepatocytes. Stem Cell Reports. 2015; 5(1):22–30. [PubMed: 26074313] 19. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861–872. [PubMed: 18035408] 20. Zhu S, Rezvani M, Harbell J, et al. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature. 2014; 508(7494):93–97. [PubMed: 24572354] 21. Kobayashi N, Ito M, Nakamura J, et al. Hepatocyte transplantation in rats with decompensated cirrhosis. Hepatology. 2000; 31(4):851–857. [PubMed: 10733539] 22. Navarro-Alvarez N, Soto-Gutierrez A, Chen Y, et al. Intramuscular transplantation of engineered hepatic tissue constructs corrects acute and chronic liver failure in mice. J Hepatol. 2010; 52(2): 211–219. [PubMed: 20022655] 23. Hoppo T, Komori J, Manohar R, Stolz DB, Lagasse E. Rescue of lethal hepatic failure by hepatized lymph nodes in mice. Gastroenterology. 2011; 140(2):656–666.e2. [PubMed: 21070777] 24. Soto-Gutierrez A, Wertheim JA, Ott HC, Gilbert TW. Perspectives on whole-organ assembly: Moving toward transplantation on demand. J Clin Invest. 2012; 122(11):3817–3823. [PubMed: 23114604] 25. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartificial heart. Nat Med. 2008; 14(2):213–221. [PubMed: 18193059] 26. Soto-Gutierrez A, Zhang L, Medberry C, et al. A whole-organ regenerative medicine approach for liver replacement. Tissue Eng Part C Methods. 2011; 17(6):677–686. [PubMed: 21375407] 27. Baptista PM, Siddiqui MM, Lozier G, Rodriguez SR, Atala A, Soker S. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology. 2011; 53(2):604– 617. [PubMed: 21274881] 28. Zhou P, Lessa N, Estrada DC, et al. Decellularized liver matrix as a carrier for the transplantation of human fetal and primary hepatocytes in mice. Liver Transpl. 2011; 17(4):418–427. [PubMed: 21445925] 29. Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, Ott HC. Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat Med. 2013; 19(5):646–651. [PubMed: 23584091] 30. Yagi H, Fukumitsu K, Fukuda K, et al. Human-scale whole-organ bioengineering for liver transplantation: A regenerative medicine approach. Cell Transplant. 2013; 22(2):231–242. [PubMed: 22943797] 31. Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med. 2010; 16(7): 814–820. [PubMed: 20543851] 32. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science. 2010; 329(5991):538–541. [PubMed: 20576850] 33. Ott HC, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med. 2010; 16(8):927–933. [PubMed: 20628374] 34. Ko IK, Peng L, Peloso A, et al. Bioengineered transplantable porcine livers with re-endothelialized vasculature. Biomaterials. 2015; 40:72–79. [PubMed: 25433603] 35. Yoder MC. Differentiation of pluripotent stem cells into endothelial cells. Curr Opin Hematol. 2015; 22(3):252–257. [PubMed: 25767955] Am J Transplant. Author manuscript; available in PMC 2016 June 01.
de l’Hortet et al.
Page 12
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
36. Prasain N, Lee MR, Vemula S, et al. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells. Nat Biotechnol. 2014; 32(11):1151–1157. [PubMed: 25306246] 37. Sampaziotis F, Cardoso de Brito M, Madrigal P, et al. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nat Biotechnol. 2015; 33(8):845– 852. [PubMed: 26167629] 38. Ogawa M, Ogawa S, Bear CE, et al. Directed differentiation of cholangiocytes from human pluripotent stem cells. Nat Biotechnol. 2015; 33(8):853–861. [PubMed: 26167630] 39. Takebe T, Sekine K, Enomura M, et al. Vascularized and functional human liver from an iPSCderived organ bud transplant. Nature. 2013; 499(7459):481–484. [PubMed: 23823721] 40. Takebe T, Enomura M, Yoshizawa E, et al. Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell. 2015; 16(5):556–565. [PubMed: 25891906] 41. Atala, A.; Yoo, JJ. Essentials of 3D biofabrication and translation. 1st. Atlanta, GA: Elsevier; 2015. 42. LeCluyse EL, Witek RP, Andersen ME, Powers MJ. Organotypic liver culture models: Meeting current challenges in toxicity testing. Crit Rev Toxicol. 2012; 42(6):501–548. [PubMed: 22582993] 43. Godoy P, Hewitt NJ, Albrecht U, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013; 87(8):1315–1530. [PubMed: 23974980] 44. Chang R, Emami K, Wu H, Sun W. Biofabrication of a three-dimensional liver micro-organ as an in vitro drug metabolism model. Biofabrication. 2010; 2(4):045004. [PubMed: 21079286] 45. Skardal A, Devarasetty M, Kang HW, et al. A hydrogel bioink toolkit for mimicking native tissue biochemical and mechanical properties in bioprinted tissue constructs. Acta Biomater. 2015; 25:24–34. [PubMed: 26210285] 46. Jakab K, Norotte C, Marga F, Murphy K, Vunjak-Novakovic G, Forgacs G. Tissue engineering by self-assembly and bio-printing of living cells. Biofabrication. 2010; 2(2):022001. [PubMed: 20811127] 47. Norotte C, Marga FS, Niklason LE, Forgacs G. Scaffold-free vascular tissue engineering using bioprinting. Biomaterials. 2009; 30(30):5910–5917. [PubMed: 19664819] 48. Robbins JB, Gorgen V, Min P, Sheperd B, Presnell S. A novel in vitro three-dimensional bioprinted liver tissue system for drug development. FASEB J. 2013; 27(872):812. 49. Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol. 2006; 7(3):211–224. [PubMed: 16496023] 50. Lancaster MA, Knoblich JA. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science. 2014; 345(6194):1247125. [PubMed: 25035496] 51. Kobayashi T, Yamaguchi T, Hamanaka S, et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell. 2010; 142(5):787–799. [PubMed: 20813264] 52. Usui J, Kobayashi T, Yamaguchi T, Knisely AS, Nishinakamura R, Nakauchi H. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am J Pathol. 2012; 180(6): 2417–2426. [PubMed: 22507837] 53. Matsunari H, Nagashima H, Watanabe M, et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc Natl Acad Sci USA. 2013; 110(12):4557–4562. [PubMed: 23431169] 54. Ekser B, Ezzelarab M, Hara H, et al. Clinical xenotransplantation: The next medical revolution? Lancet. 2012; 379(9816):672–683. [PubMed: 22019026] 55. Gines P, Cardenas A, Arroyo V, Rodes J. Management of cirrhosis and ascites. N Engl J Med. 2004; 350(16):1646–1654. [PubMed: 15084697] 56. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet. 2008; 371(9615):838–851. [PubMed: 18328931] 57. Lin TY, Lee CS, Chen CC, Liau KY, Lin WS. Regeneration of human liver after hepatic lobectomy studied by repeated liver scanning and repeated needle biopsy. Ann Surg. 1979; 190(1):48–53. [PubMed: 464678] Am J Transplant. Author manuscript; available in PMC 2016 June 01.
de l’Hortet et al.
Page 13
Author Manuscript Author Manuscript
58. Pack GT, Islami AH, Hubbard JC, Brasfield RD. Regeneration of human liver after major hepatectomy. Surgery. 1962; 52:617–623. [PubMed: 14483060] 59. Huang P, Zhang L, Gao Y, et al. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell. 2014; 14(3):370–384. [PubMed: 24582927] 60. Song K, Nam YJ, Luo X, et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 2012; 485(7400):599–604. [PubMed: 22660318] 61. Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature. 2008; 455(7213):627–632. [PubMed: 18754011] 62. Muraoka N, Ieda M. Direct reprogramming of fibroblasts into myocytes to reverse fibrosis. Annu Rev Physiol. 2014; 76:21–37. [PubMed: 24079415] 63. Liu L, Yannam GR, Nishikawa T, et al. The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis. Hepatology. 2012; 55(5):1529–1539. [PubMed: 22109844] 64. Nishikawa T, Bellance N, Damm A, et al. A switch in the source of ATP production and a loss in capacity to perform glycolysis are hallmarks of hepatocyte failure in advance liver disease. J Hepatol. 2014; 60(6):1203–1211. [PubMed: 24583248] 65. Nishikawa T, Bell A, Brooks JM, et al. Resetting the transcription factor network reverses terminal chronic hepatic failure. J Clin Investig. 2015; 125(4):1533–1544. [PubMed: 25774505] 66. Lechler RI, Sykes M, Thomson AW, Turka LA. Organ transplantation–how much of the promise has been realized? Nat Med. 2005; 11(6):605–613. [PubMed: 15937473]
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Figure 1. Schematic representation of different approaches under investigation in regenerative medicine for liver replacement and regeneration
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Cell source for liver repopulation (A) and liver engineering (B) should be facilitated by an unlimited supply of induced pluripotent stem–derived hepatocytes, but the underlying tumor potential would remain to be elucidated. Successful liver repopulation will require optimizing functional hepatic cells to achieve high engraftment and repopulation efficiency. “Resetting” liver functions (C) to normal will require improvement of in vivo genetic delivery systems and better understanding of complex molecular networks associated with liver diseases. Important health and ethical issues associated with blastocyst complementation (D) will need to be addressed. The liver may contain host cells and pathogens that could pose a threat of immunological rejection and transmission of interspecies diseases. Ethical concerns could be solved once the genetic toolbox makes it possible to restrict differentiation toward particular organs (brain and gonads). 3D, threedimensional.
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Am J Transplant. Author manuscript; available in PMC 2016 June 01. Published in final edited form as: Am J Transplant. 2016 June ; 16(6): 1688–1696. doi:10.1111/ajt.13678.
Liver-Regenerative Transplantation: Regrow and Reset A. Collin de l’Hortet1, K. Takeishi1, J. Guzman-Lepe1, K. Handa3, K. Matsubara3, K. Fukumitsu4, K. Dorko5, S. C. Presnell5, H. Yagi3, and A. Soto-Gutierrez1,2,* 1Department
of Pathology, University of Pittsburgh, Pittsburgh, PA
2Thomas
E. Starzl Transplantation Institute and McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA
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3Department
of Surgery, School of Medicine, Keio University, Tokyo, Japan
4Division
of Hepato-Biliary-Pancreatic and Transplant Surgery, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 5Organovo
Holdings Inc., San Diego, CA
Abstract
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Liver transplantation, either a partial liver from a living or deceased donor or a whole liver from a deceased donor, is the only curative therapy for severe end-stage liver disease. Only one-third of those on the liver transplant waiting list will be transplanted, and the demand for livers is projected to increase 23% in the next 20 years. Consequently, organ availability is an absolute constraint on the number of liver transplants that can be performed. Regenerative therapies aim to enhance liver tissue repair and regeneration by any means available (cell repopulation, tissue engineering, biomaterials, proteins, small molecules, and genes). Recent experimental work suggests that liver repopulation and engineered liver tissue are best suited to the task if an unlimited availability of functional induced pluripotent stem (iPS)–derived liver cells can be achieved. The derivation of iPS cells by reprogramming cell fate has opened up new lines of investigation, for instance, the generation of iPS-derived xenogeneic organs or the possibility of simply inducing the liver to reprogram its own hepatocyte function after injury. We reviewed current knowledge about liver repopulation, generation of engineered livers and reprogramming of liver function. We also discussed the numerous barriers that have to be overcome for clinical implementation.
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Introduction Approximately 30 million people in the United States have liver disorders, which are responsible for ≈30 000 deaths annually in the United States and nearly 1 million deaths in developing countries (1–3). Medical therapy can extend life, but the only curative therapy for terminal liver failure (acute or chronic) is allogeneic liver transplantation—either a *
Corresponding author: Alejandro Soto-Gutierrez, ; Email: [email protected]. Disclosure The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. K.D. and S.P. are full-time employees and shareholders of Organovo Holdings, Inc., a publicly held corporation specializing in the production and application of three-dimensional human tissues for in vitro and in vivo applications. The other authors have no conflicts of interest to disclose.
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partial liver from a living or deceased donor or a whole liver from a deceased donor. Organ demand, however, exceeds supply by tens of thousands. In the United States, the annual number of deceased donor livers decreased from 7014 in 2006 to 5710 in 2013 (4). Living donation numbers have also declined, falling from 524 donors in 2001 to 211 in 2013 (4,5). In addition, it is projected that the demand for livers will increase 10% in 10 years and 23% in 20 years (6).
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Exacerbating the organ shortage problem, the donor pool is expected to shrink further because of the obesity epidemic. Liver steatosis is increasingly common in donors and is a significant risk factor in liver transplantation (7). Moreover, the top four causes of chronic liver disease in patients on the U.S. liver transplant waitlist are currently chronic hepatitis C virus (HCV) infection, alcoholic liver disease, nonalcoholic steatohepatitis (NASH), and a combination of chronic HCV infection and alcoholic liver disease; however, these rankings may change because new developments could modify these statistics (e.g. new direct-acting antiviral agents are transforming the treatment of chronic HCV). Meanwhile, NASH incidence has increased 170%, making it the second leading etiology of chronic liver disease among new liver transplant waitlist registrants in 2013 (8). The number of patients with NASH awaiting liver transplantation is anticipated to continue climbing in the near future, whereas availability of donor organs is expected to decline. Together, these factors reveal the changing epidemiology of patients awaiting liver transplantation in the United States.
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Several strategies have been explored to increase the number of livers available for transplantation. These include the use of marginal donors, donors aged >60 years; donors with >30% macrosteatosis; donors with positive serology for the hepatitis B virus (e.g. hepatitis B core positive) or HCV—donor seropositivity for HCV has been considered a contraindication for liver transplantation, but again, the new panorama after the administration of the new generation of anti-HCV therapies might materially change the future in marginal liver donors; donors with a cold ischemia time of >12 h; donation after cardiac death; and grafts from split livers or living donors (9,10) and even resuscitation of marginal-quality donor organs using machine perfusion (11). In addition, several novel cellular therapies to induce tolerance in solid organ transplant patients have entered early phase clinical trials (12). If successful, organ tolerance is expected to have a substantial impact on morbidity and mortality risks for transplant recipients but a limited impact on the organ donor pool. These data point to two solutions: regrow livers for transplantation or “reset” injured livers to fitness.
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New regenerative approaches to investigating liver organogenesis seek to provide novel insights into liver repopulation, organ engineering (recellularization of natural scaffold technologies, three-dimensional [3D] bioprinting of liver tissue or generation of chimeric organs through blastocyst complementation) and reprogramming the injured liver. We have highlighted these promising areas of investigation (Figure 1). Liver repopulation Liver cell therapy has been under intensive investigation for decades (13), and we have learned that the ideal candidates for this kind of therapy are patients with acute liver failure and patients with liver-based inborn errors of metabolism, especially children (13–15). The Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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native architecture of the liver is intact in these diseases; therefore, the transplant procedure involves simple injection of liver cells through the portal vein into the liver, in which the cells integrate into the host liver. In the case of liver-based inborn errors of metabolism, clinical observations have demonstrated the safety of the procedure, and patients who have undergone liver cell therapy showed clinical improvement and/or partial correction of the underlying metabolic defect (16). In the long term, however, transplanted liver cells appear to survive poorly. The main gap that prevents advancement of the field is poor repopulation of the transplanted cells and the inability to detect acute rejection episodes.
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Recent observations have demonstrated that transplanted liver cells subjected to a certain advantageous regenerative stimulus are able to expand over recipient liver cells (15). Consequently, conditions should be established to facilitate the entry of hepatocytes into the parenchyma (cell engraftment) and the enduring survival and expansion of transplanted liver cells. Preparative liver-directed radiation has been shown to facilitate early engraftment of transplanted liver cells by reversibly disrupting the sinusoidal endothelium and enhancing the entry of transplanted liver cells into the liver parenchyma. Preparative radiation also induces postmitotic hepatocyte death, allowing donor liver cells to preferentially proliferate and repopulate the irradiated host liver. This strategy has been used to correct a rodent model of Crigler–Najjar syndrome (17,18). In the case of acute liver failure, it is required that the transplanted liver cells function immediately at their full capacity; however, the lack of a clinically relevant experimental model and limited clinical trials have restricted our ability to advance the field. The number of liver cells, for example, that need to engraft to reverse hepatic failure is essentially unknown, and whether acute hepatic failure is associated with changes in the local microenvironment that might interfere with engraftment and function of transplanted liver cells is also not yet known. Response to these questions requires a reliable supply of high-quality hepatocytes.
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Creating an immediately available and inexhaustible supply of functioning liver cells from autologous tissue would allow early intervention in patients with hepatic failure and would allow liver cells to be infused over a longer period of time. Combined with recent advances in genome-editing technology, such liver cells could be used widely to treat devastating liver-based inborn errors of metabolism and to eliminate the need for a life-long regimen of immunosuppressive drugs and their complications. Engineering pluripotency of human somatic cells by the ectopic expression of transcription factors has opened the possibility of generating autologous cells for liver cell replacement therapies. Moreover, induced pluripotent stem (iPS) cells are of extraordinary interest because they could be patient specific, can be propagated indefinitely as undifferentiated cells and can differentiate into practically any cell type (19). Although it is not yet possible to differentiate iPS cells to liver cells with functional and regenerative characteristics identical to those residing in the human liver, it is likely a matter of time before these problems can be overcome (20). The enthusiasm for liver cell therapy has been fueled by recent breakthroughs in stem cell biology and hepatic differentiation, especially since the derivation of iPS cells. Interests in the use of iPS cells in regenerative medicine is growing, and several programs have been created for the development of allogeneic cell banks of iPS cells all over the world. By providing a la carte iPS cells that are ready to use, these banks might accelerate and facilitate this clinical procedure. Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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Although much progress has been made toward enhancing selective repopulation of transplanted liver cells, many fundamental questions surrounding regeneration and acute rejection of transplanted liver cells remain unanswered. For liver repopulation to realize its full potential, continued funding support is necessary to address these challenges, especially if iPS-derived liver cells are to be used clinically in the near future. Regrowing livers
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Although cell therapy for acute liver failure and metabolic liver diseases may benefit greatly by the generation of iPS-derived liver cells, the vast majority of patients in need of lifesaving intervention are those with end-stage cirrhosis and chronic hepatic failure. Because fibrotic or cirrhotic livers have abnormal architecture induced by scarring, liver repopulation for end-stage liver disease is more problematic. Cell transplantation into the portal vein of a cirrhotic liver can generate severe portal hypertension and may cause portal thrombosis. Several experimental studies have suggested that liver cell therapy in extrahepatic sites can improve liver function and prolong survival in end-stage cirrhosis while avoiding the complications of severe portal hypertension and portal thrombosis (21–23). Treatment of chronic liver failure might benefit more from the transplantation of a large mass of healthy functioning liver cells; however, their function is influenced by the architecture of the organ and the microenviroment in which the cells reside. Current efforts are aiming to recapitulate the complex organization of liver tissue by using multiple supporting and functional cell types, such as liver nonparenchymal cells (e.g. fibroblasts, macrophages, cholangiocytes, sinusoidal endothelial cells) for transplantation (24). A synergistic goal is to develop useful tools for drug screening and toxicity testing. Both of these objectives would benefit from a means of replicating the function of whole organs.
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Whole organ assembly—A popular strategy being developed for construction of implantable organs involves the decellularization of whole organs and subsequent reseeding with relevant cell types, followed by maturation of the neo-organ in a physiologically appropriate bioreactor (25–33). Numerous protocols for whole liver engineering, cell seeding and implantation of these constructs in animals have demonstrated the feasibility of this approach (25–33). Regeneration of the tissue is initiated by multiple injections of parenchymal cells throughout the liver scaffold, and endothelial cells are perfused through to reestablish the vascular tree prior to transplanting. Recent advances in liver recellularization technology have demonstrated that human fetal liver progenitor and endothelial cells can be grown in a bioreactor in vitro for >1 month. The fetal liver cells reformed into clusters, and evidence of biliary canaliculi and ducts was seen (27). The scalability of the system has been demonstrated recently in which a whole pig liver scaffold was repopulated with primary porcine hepatocytes (30). Another laboratory demonstrated methods for reestablishing the vascular network within decellularized liver scaffolds by conjugating anti–endothelial cell antibodies to maximize coverage of the vessel walls with endothelial cells and, when implanted, were maintained for up to 24 h (34). In each of these studies, the engineered graft failed to survive or function for more than a few hours after transplantation. These disappointing results have been largely attributed to failure to maintain a durable vascular network and to rebuild the complex liver microarchitecture required for function by
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the assembled liver graft after transplantation. There is also an especially important need to prevent blood clotting and to maintain enough blood circulation for the implanted engineered graft. Several other important challenges remain for the recellularized scaffold approach to succeed. Systems to quantify and maximize the parenchymal and nonparenchymal components of the liver, for instance, will be essential for future scaling-up systems and technologies to boost regeneration of hepatocytes inside the scaffold. Furthermore, the sinusoidal endothelium and the bile duct are highly specialized. In vitro results have used only microvascular endothelial cells, and no attempts have been reported yet to reestablish the intrahepatic portion of the biliary tree.
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Perhaps the greatest potential of human iPS cells lies in autologous derivation. Availability of a liver graft from autologous tissue and cells would require no or little immune suppression. To do so may also require the establishment of protocols for the directed differentiation of human pluripotent stem cells into specialized liver non-parenchymal cells; however, recent advances in differentiation of pluripotent cells into endothelial cells (35,36), hepatocytes (18,20) and cholangiocytes (37,38) may accelerate the progress and feasibility of these studies. Will decellularization provide the basis of the protocol to bioengineer a liver for transplantation? Will it simply be a platform for studying the self-organization of the cells that compose a liver? It remains to be seen.
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Liver organoid generation—This interesting approach has been proposed by exploiting the general capacity of cells to self-organize and form structures with similar histogenic properties, such as those seen in vivo using 3D spatial arrangement and several cell types. Through mechanisms for which molecular bases are largely unknown, cells can self-sort and self-assemble to guide tissue formation. These principles were applied in vitro by culturing human iPS-derived endodermal cells, human mesenchymal stem cells and human umbilical vein endothelial cells (HUVEC). The mixture aggregated and self-organized into 3D spheroid structures termed liver buds, or heterotypic cell collectives. This microenviron ment promoted hepatocyte differentiation of the human iPS-derived endodermal cells, eventually producing human iPS-derived hepatocytes that were still immature but that expressed certain liver functions (albumin and α1-antitrypsin) at higher levels than human iPS-derived hepatocytes generated in two-dimensional (2D) cultures. Surprisingly, only 2 days after implantation in different anatomical sites, the organoids exhibited HUVEC-derived blood vessels that were connected to the host circulation and rapidly formed a complex vascular network. Following transplantation in the mesentery of mice that underwent liver injury, the liver buds were vascularized and integrated with host tissues to increase host survival (39).
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In recent studies, the same authors have investigated the mechanism driving organ bud generation. By analyzing the formation and function of liver buds, Takebe and colleagues concluded that the endothelial cells drive vascularization after transplantation and that extracellular matrix properties drive cell condensate formation via effects on mesenchymal stem cells (40). The main limitations of organoid technology include size restriction, but an alternative approach could utilize natural decellularized liver scaffolds that, once repopulated with organoids, can drive cellular self-organization and recapitulate organ function, thereby facilitating the generation of larger, more sophisticated liver structures that incorporate specialized components (e.g. bile duct, sinusoidal endothelial cells). Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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Bioprinting in the fabrication of 3D liver tissues—Automated engineering platforms, such as ink-jet printers and 3D printers, have been actively adapted in the past 10 years to be compatible with manipulation of living mammalian cells (reviewed by Atala and Yoo) (41). This has enabled bioengineers to design and develop patterned 2D cultures and 3D tissue structures in which multiple distinct cell types can be organized in space relative to each other per user specifications. For many tissues, like liver, 3D configurations that preserve the polarity and foster the development and maintenance of intercellular junctions provide demonstrated benefits in extended lifespan and better preservation of function in in vitro cultures (reviewed by LeCluyse et al and Godoy et al) (42,43). The 3D fabrication of structures comprising liver cells has been approached using hydrogels laden with cell spheroids (44,45) or using bioprinting platforms that enable one component or more of the tissue to be made solely from cells (46–48). For the engineering of solid tissues like the liver, in which the majority of cells are in close approximation to other cells without expansive void spaces or extracellular matrix, high cellularity (≥30%) at the time of production may foster cell–cell interactions and lead to more rapid achievement of tissuelike morphology and functionality.
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Regardless of the fabrication method used, the ultimate architecture of the tissue that is created is governed by both the initial fabrication design and the intercellular interactions that occur over time as the structures mature. Bioprinted liver tissues generated by the NovoGen MMX Bioprinter (Invetech, San Diego, CA), for example, are characterized after fabrication by distinct zones in the x-, y- and z-axes that are enriched for primary hepatocytes, endothelial cells, or hepatic stellate cells. Over time, intercellular interactions drive formation of finer structures, highlighted by the close approximation of hepatic stellate cells and hepatocytes and by the formation of microvascular structures within the tissues. In addition, to extended lifespan and function, multicellular tissue constructs that recapitulate some elements of native tissue architecture also provide a means to assess not only the biochemical response to various forms of injury but also the tissue response. Phenomenon such as necrosis, lipid accumulation and fibrosis can be detected. Although it is not yet the case that bioprinted tissues perfectly reproduce native tissue architecture and function, they provide better approximation than simple 2D or monocellular cultures and offer the added benefit of tissue-level organization for interpretation of outcomes. Bioprinted liver tissues were first commercialized in 2014 (Organovo, San Diego, CA) and are being used today for long-term and tissue-level assessment of toxicity, liver biology and disease modeling.
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It is important to consider some of the inherent limitations of automated 3D fabrication platforms as they relate to the production of structures containing living cells. The size and biomechanical forces imposed on cells subjected to bioprinting are a key consideration. Hepatocytes, for example, are highly sensitive to shear force and would be unlikely to survive high-shear deposition methods such as the traditional ink-jet approaches. Unlike simple culture systems wherein dead or dying cells are simply removed with the next media change, shear-damaged hepatocytes may become trapped within the thick bioprinted tissues and create areas of necrosis that disrupt the architecture and further damage neighboring cells. Consequently, there are limits of resolution and deposition speed imposed by the
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tolerances of the cell types used in fabrication, and the parameters must be defined for each cell type and ultimately limited by the most fragile component. Although it is attractive, conceptually, to envision placing individual cells relative to each other and building up a 3D structure cell by cell, there are challenges with respect to the time it takes to make a tissue of appreciable size that meets the metabolic needs of the living cells during the production window. Speed and resolution, the limits of which are dictated by the tolerance of the living cells being used, must be balanced to preserve cell health and phenotype while producing tissue at a scale that is useful for downstream applications. As 3D fabrication technologies evolve to address the challenges of living cells, it may be possible to reproduce the intricate architecture of the liver acinus at a scale that enables tissue-level outcomes to be assessed and that incorporates fluid paths to model circulation and biliary excretion.
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Disease modeling will likely be a primary focus of future liver organogenesis (49). These diseases can include developmental disorders, cancer, infectious disease and liver degenerative diseases (50). In this context, patient-derived or engineered iPS cells will be useful tools in future disease modeling. Laboratory-grown miniature human livers, for instance, also have the potential to be used for testing efficacy and toxicity of drug compounds. This approach could be applied to model degenerative conditions—for example, liver fibrosis or NASH—for which one could study specific pathways related to human pathology or screen for effective treatments. The human liver often metabolizes drugs in a manner distinct from animal metabolism. If successful, the ability to practically engineer liver grafts would go far beyond modeling liver steatosis or fibrosis and could be a tool to accurately predict the metabolism or toxicity of a compound in healthy and diseased human liver grafts in vitro prior to exposure to the whole body. This approach potentially translates into reduced costs and time in drug development and less harmful patient exposure in clinical trials.
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Blastocyst complementation for organ generation—Generation of iPS cells has opened the possibilities to practically producing any cell lineage from any particular person. The differentiation approaches in the laboratory have attempted to mimic normal organ development in a dish, producing cells that do not necessarily reflect the full spectrum of functions of normal hepatocytes (14). In a recent important work, Kobayashi et al showed that is possible to grow a rat pancreas from iPS cells in chimera mice using blastocyst complementation principles (51). This elegant study demonstrated that knocking out the host’s blastocyst master regulator of pancreas development (the Pdx1 gene) and injecting with donor iPS-derived cells, and thus “complementing” this deficiency, generated a newly formed pancreas composed entirely of injected cells (although vessels, nerves and interstitial cells were composed of both host and donor cells, complementing iPS cells contributed to nearly 95% of all pancreatic tissues, including pancreatic islets, exocrine tissues and duct epithelia examined by imaging techniques). These complemented embryos survived to adulthood without abnormalities, indicating that the iPS-derived pancreas was functional. Recently, the same group was able to successfully generate iPS-derived kidney (52). Consistent with this concept of developmental compensation, they were able to induce this pancreatogenesis in large animals (cloned pigs) (53). Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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The exciting possibility of growing patient-specific transplantable organs in chimeric large animals is far in the future. The generation of an entire interspecific liver has not been reported and seems currently challenging with this nascent technology but is clearly possible. To create a whole liver from patient-specific iPS cells that includes the parenchymal and nonparenchymal components (e.g. vasculature, bile duct, gallbladder, nerves), embryos lacking several developmental pathways would have to be created. If chimeric livers were generated containing an animal host’s cells, those cells could pose a threat for immunological rejection once the organs are transplanted into patients. Because the field of liver xenotransplantation has been limited and has had limited survival (54), one can speculate that the expected immune responses could be problematic. Nevertheless, further studies are indispensable to analyze the immunogenicity of such chimeric organs. Resetting liver function
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The vast majority of patients in need of liver transplantation are those with end-stage cirrhosis and chronic hepatic failure. Late-stage liver cirrhosis is characterized by portal hypertension and hepatic encephalopathy, terminal processes that result from fibrosis and vascular remodeling of the cirrhotic liver (55,56). These extrahepatic pathologies are superimposed on liver failure, namely, the inability of hepatocytes to adequately synthesize coagulation factors, conjugate and secrete bilirubin, and regulate metabolism. Hepatocyte decompensation in advanced liver disease is difficult to explain because the liver is capable of full function with less than half its normal hepatocyte content (57,58). In addition, hepatic failure persists even after amelioration of the source of liver injury, indicating that hepatocytes undergo dedifferentiation (epithelial-to-mesenchymal-transition).
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The concept of cellular reprogramming introduced by Yamanaka et al in 2007 (19) has revolutionized cell biology and increased excitement for regenerative medicine. The strategy behind successful lineage reprogramming has its fundamentals in developmental biology and is based on the knowledge of transcriptional networks underlying the establishment and maintenance of cellular identity. In other words, rewiring the transcriptional circuitry by relevant transcription factors can force a new molecular program and thus a new cellular identity. The past decade has seen enormous progress in reprogramming pluripotency and even differentiated hepatocyte-like cells in vitro (59). Recent pioneering work indicates that in vivo lineage reprogramming is possible and has been demonstrated in a handful of organs (60–62).
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To understand this process better in terms of chronic liver failure, Nishikawa et al have studied how hepatocyte metabolic adaptations in response to the microenvironment might lead to cellular breakdown (63,64). Nishikawa et al found that the state of the host microenvironment is critical to the regenerative and functional potential of hepatocytes (63). In other words, hepatocytes can be reprogrammed to functional states by the microenviroment in which they reside. Nishikawa et al demonstrated that fatal chronic liver injury exhibits a disruption of the critical balance of transcription factors and that diseased or dedifferentiated cells can be returned to normal function by reexpression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent (65). In these studies, Nishikawa et al demonstrated that a
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network of hepatocyte-enriched transcription factors (hepatocyte nuclear factor 4α [HNF4A], forkhead box A2, CCAAT/enhancer binding protein α and hepatocyte nuclear factor 1α) regulates the mature hepatocyte phenotype by controlling expression of proteins needed for coagulation, biliary and lipid metabolism. Conversely, the forced reexpression of one factor, HNF4A, improved cellular function and reversed liver failure. This work indicates that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest that reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement. It is possible that such disruption of the hepatic transcription factor network in liver injury is caused the microenviroment; rigorous testing of this hypothesis remains to be performed.
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Currently, it is tantalizing to consider how rewiring of the hepatic transcription factor network might resolve liver failure and fibrosis by resetting hepatocyte function in humans. New therapies centered on hepatic reprogramming mechanisms may become viable strategies in the future by simply inducing the liver to reset the hepatic function of its own cells after injury.
Conclusions
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The worldwide shortage of livers available for transplants has led scientists to develop other promising therapies and to shape the future of regenerative medicine (66). In our opinion, two regenerative strategies can prevail: designing and fabricating entirely brand new livers from fresh materials and/or resetting sick livers back to their hepatic “default settings.” The first approach has the potential to generate an unlimited source of grafts, to provide patients with better timing for the procedure and to improve patient quality of life after surgery. The second approach has the advantage of avoiding any invasive surgery, eliminating long waiting times and eventually decreasing the overall need for liver transplantations. Moreover, even if full liver replacement cannot be achieved with these therapies, they could be used in the near future as a temporary bridge in clinical transplantation (e.g. auxiliary partial orthotopic or heterotopic transplantation of the engineered liver graft) until a liver becomes available.
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We are living in exciting times: Technology and scientific accomplishments in past decades have placed these new approaches within reach and should be reflected in patient care in coming years. Nevertheless, we need to prioritize carefully and not let science be driven by fashion. The irony is that for all of these new technologies, old questions remain to be solved. Even if we are successful, for example, in creating iPS-derived hepatocytes of high standards for liver cell transplantation, we still need to solve the old challenges of engraftment and repopulation. Similarly, we might generate organs through blastocyst complementation, but primordial questions of xenorejection will persist. In other words, working on creating new, elegant tools will never be enough if we do not envision the future needs and uses of technology properly. A combined research effort and appropriate funding in different areas of investigation are crucial and, if done efficiently, have the potential to achieve great clinical success.
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Acknowledgments Funding from the US National Institutes of Health (DK099257 to A.S.-G. and UH3TR000503 to D Lansing Taylor and A.S.-G. as collaborator), and the Competitive Medical Research Fund Program from UPMC Health System to A.S.-G. and Japan Agency for Medical Research and Development, Research Center Network for Realization of Regenerative Medicine, Projects for Technological Development to H.Y. also supported this work.
Abbreviations
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2D
two-dimensional
3D
three-dimensional
HCV
hepatitis C virus
HNF4A
hepatocyte nuclear factor 4a
HUVEC
human umbilical vein endothelial cell
iPS
induced pluripotent stem
NASH
nonalcoholic steatohepatitis
References
Author Manuscript Author Manuscript
1. Hoyert DL, Xu J. Deaths: Preliminary data for 2011. Natl Vital Stat Rep. 2012; 61(6):1–51. [PubMed: 24984457] 2. Alqahtani SA. Update in liver transplantation. Curr Opin Gastroenterol. 2012; 28(3):230–238. [PubMed: 22450898] 3. Mokdad AA, Lopez AD, Shahraz S, et al. Liver cirrhosis mortality in 187 countries between 1980 and 2010: A systematic analysis. BMC Med. 2014; 12:145. [PubMed: 25242656] 4. Kim WR, Lake JR, Smith JM, et al. OPTN/SRTR 2013 Annual Data Report: Liver. Am J Transplant. 2015; 15(Suppl 2):1–28. [PubMed: 25626341] 5. U.S. Department of Health & Human Services HRaSA. Organ Procurement and Transplantation Network (OPTN). [cited 2015 Nov 3]. Available from: http://optn.transplant.hrsa.gov/ 6. Habka D, Mann D, Landes R, Soto-Gutierrez A. Future economics of liver transplantation: A 20year cost modeling forecast and the prospect of bioengineering autologous liver grafts. PLoS ONE. 2015; 10(7):e0131764. [PubMed: 26177505] 7. Busuttil RW, Tanaka K. The utility of marginal donors in liver transplantation. Liver Transpl. 2003; 9(7):651–663. [PubMed: 12827549] 8. Wong RJ, Aguilar M, Cheung R, et al. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology. 2015; 148(3):547–555. [PubMed: 25461851] 9. Jimenez-Romero C, Caso Maestro O, Cambra Molero F, et al. Using old liver grafts for liver transplantation: Where are the limits? World J Gastroenterol. 2014; 20(31):10691–10702. [PubMed: 25152573] 10. Zarrinpar A, Busuttil RW. Liver transplantation: Past, present and future. Nat Rev Gastroenterol Hepatol. 2013; 10(7):434–440. [PubMed: 23752825] 11. Graham JA, Guarrera JV. “Resuscitation” of marginal liver allografts for transplantation with machine perfusion technology. J Hepatol. 2014; 61(2):418–431. [PubMed: 24768755] 12. Geissler EK, Hutchinson JA. Cell therapy as a strategy to minimize maintenance immunosuppression in solid organ transplant recipients. Curr Opin Organ Transplant. 2013; 18(4): 408–415. [PubMed: 23838645] 13. Puppi J, Strom SC, Hughes RD, et al. Improving the techniques for human hepatocyte transplantation: Report from a consensus meeting in London. Cell Transplant. 2012; 21(1):1–10. [PubMed: 21457616] Am J Transplant. Author manuscript; available in PMC 2016 June 01.
de l’Hortet et al.
Page 11
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
14. Fox IJ, Daley GQ, Goldman SA, Huard J, Kamp TJ, Trucco M. Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease. Science. 2014; 345(6199):1247391. [PubMed: 25146295] 15. Soltys KA, Soto-Gutierrez A, Nagaya M, et al. Barriers to the successful treatment of liver disease by hepatocyte transplantation. J Hepatol. 2010; 53(4):769–774. [PubMed: 20667616] 16. Fox IJ, Chowdhury JR, Kaufman SS, et al. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med. 1998; 338(20):1422–1426. [PubMed: 9580649] 17. Zhou H, Dong X, Kabarriti R, et al. Single liver lobe repopulation with wildtype hepatocytes using regional hepatic irradiation cures jaundice in Gunn rats. PLoS ONE. 2012; 7(10):e46775. [PubMed: 23091601] 18. Chen Y, Li Y, Wang X, et al. Amelioration of hyperbilirubinemia in gunn rats after transplantation of human induced pluripotent stem cell-derived hepatocytes. Stem Cell Reports. 2015; 5(1):22–30. [PubMed: 26074313] 19. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861–872. [PubMed: 18035408] 20. Zhu S, Rezvani M, Harbell J, et al. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature. 2014; 508(7494):93–97. [PubMed: 24572354] 21. Kobayashi N, Ito M, Nakamura J, et al. Hepatocyte transplantation in rats with decompensated cirrhosis. Hepatology. 2000; 31(4):851–857. [PubMed: 10733539] 22. Navarro-Alvarez N, Soto-Gutierrez A, Chen Y, et al. Intramuscular transplantation of engineered hepatic tissue constructs corrects acute and chronic liver failure in mice. J Hepatol. 2010; 52(2): 211–219. [PubMed: 20022655] 23. Hoppo T, Komori J, Manohar R, Stolz DB, Lagasse E. Rescue of lethal hepatic failure by hepatized lymph nodes in mice. Gastroenterology. 2011; 140(2):656–666.e2. [PubMed: 21070777] 24. Soto-Gutierrez A, Wertheim JA, Ott HC, Gilbert TW. Perspectives on whole-organ assembly: Moving toward transplantation on demand. J Clin Invest. 2012; 122(11):3817–3823. [PubMed: 23114604] 25. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartificial heart. Nat Med. 2008; 14(2):213–221. [PubMed: 18193059] 26. Soto-Gutierrez A, Zhang L, Medberry C, et al. A whole-organ regenerative medicine approach for liver replacement. Tissue Eng Part C Methods. 2011; 17(6):677–686. [PubMed: 21375407] 27. Baptista PM, Siddiqui MM, Lozier G, Rodriguez SR, Atala A, Soker S. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology. 2011; 53(2):604– 617. [PubMed: 21274881] 28. Zhou P, Lessa N, Estrada DC, et al. Decellularized liver matrix as a carrier for the transplantation of human fetal and primary hepatocytes in mice. Liver Transpl. 2011; 17(4):418–427. [PubMed: 21445925] 29. Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, Ott HC. Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat Med. 2013; 19(5):646–651. [PubMed: 23584091] 30. Yagi H, Fukumitsu K, Fukuda K, et al. Human-scale whole-organ bioengineering for liver transplantation: A regenerative medicine approach. Cell Transplant. 2013; 22(2):231–242. [PubMed: 22943797] 31. Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med. 2010; 16(7): 814–820. [PubMed: 20543851] 32. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science. 2010; 329(5991):538–541. [PubMed: 20576850] 33. Ott HC, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med. 2010; 16(8):927–933. [PubMed: 20628374] 34. Ko IK, Peng L, Peloso A, et al. Bioengineered transplantable porcine livers with re-endothelialized vasculature. Biomaterials. 2015; 40:72–79. [PubMed: 25433603] 35. Yoder MC. Differentiation of pluripotent stem cells into endothelial cells. Curr Opin Hematol. 2015; 22(3):252–257. [PubMed: 25767955] Am J Transplant. Author manuscript; available in PMC 2016 June 01.
de l’Hortet et al.
Page 12
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
36. Prasain N, Lee MR, Vemula S, et al. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells. Nat Biotechnol. 2014; 32(11):1151–1157. [PubMed: 25306246] 37. Sampaziotis F, Cardoso de Brito M, Madrigal P, et al. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nat Biotechnol. 2015; 33(8):845– 852. [PubMed: 26167629] 38. Ogawa M, Ogawa S, Bear CE, et al. Directed differentiation of cholangiocytes from human pluripotent stem cells. Nat Biotechnol. 2015; 33(8):853–861. [PubMed: 26167630] 39. Takebe T, Sekine K, Enomura M, et al. Vascularized and functional human liver from an iPSCderived organ bud transplant. Nature. 2013; 499(7459):481–484. [PubMed: 23823721] 40. Takebe T, Enomura M, Yoshizawa E, et al. Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell. 2015; 16(5):556–565. [PubMed: 25891906] 41. Atala, A.; Yoo, JJ. Essentials of 3D biofabrication and translation. 1st. Atlanta, GA: Elsevier; 2015. 42. LeCluyse EL, Witek RP, Andersen ME, Powers MJ. Organotypic liver culture models: Meeting current challenges in toxicity testing. Crit Rev Toxicol. 2012; 42(6):501–548. [PubMed: 22582993] 43. Godoy P, Hewitt NJ, Albrecht U, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013; 87(8):1315–1530. [PubMed: 23974980] 44. Chang R, Emami K, Wu H, Sun W. Biofabrication of a three-dimensional liver micro-organ as an in vitro drug metabolism model. Biofabrication. 2010; 2(4):045004. [PubMed: 21079286] 45. Skardal A, Devarasetty M, Kang HW, et al. A hydrogel bioink toolkit for mimicking native tissue biochemical and mechanical properties in bioprinted tissue constructs. Acta Biomater. 2015; 25:24–34. [PubMed: 26210285] 46. Jakab K, Norotte C, Marga F, Murphy K, Vunjak-Novakovic G, Forgacs G. Tissue engineering by self-assembly and bio-printing of living cells. Biofabrication. 2010; 2(2):022001. [PubMed: 20811127] 47. Norotte C, Marga FS, Niklason LE, Forgacs G. Scaffold-free vascular tissue engineering using bioprinting. Biomaterials. 2009; 30(30):5910–5917. [PubMed: 19664819] 48. Robbins JB, Gorgen V, Min P, Sheperd B, Presnell S. A novel in vitro three-dimensional bioprinted liver tissue system for drug development. FASEB J. 2013; 27(872):812. 49. Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol. 2006; 7(3):211–224. [PubMed: 16496023] 50. Lancaster MA, Knoblich JA. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science. 2014; 345(6194):1247125. [PubMed: 25035496] 51. Kobayashi T, Yamaguchi T, Hamanaka S, et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell. 2010; 142(5):787–799. [PubMed: 20813264] 52. Usui J, Kobayashi T, Yamaguchi T, Knisely AS, Nishinakamura R, Nakauchi H. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am J Pathol. 2012; 180(6): 2417–2426. [PubMed: 22507837] 53. Matsunari H, Nagashima H, Watanabe M, et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc Natl Acad Sci USA. 2013; 110(12):4557–4562. [PubMed: 23431169] 54. Ekser B, Ezzelarab M, Hara H, et al. Clinical xenotransplantation: The next medical revolution? Lancet. 2012; 379(9816):672–683. [PubMed: 22019026] 55. Gines P, Cardenas A, Arroyo V, Rodes J. Management of cirrhosis and ascites. N Engl J Med. 2004; 350(16):1646–1654. [PubMed: 15084697] 56. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet. 2008; 371(9615):838–851. [PubMed: 18328931] 57. Lin TY, Lee CS, Chen CC, Liau KY, Lin WS. Regeneration of human liver after hepatic lobectomy studied by repeated liver scanning and repeated needle biopsy. Ann Surg. 1979; 190(1):48–53. [PubMed: 464678] Am J Transplant. Author manuscript; available in PMC 2016 June 01.
de l’Hortet et al.
Page 13
Author Manuscript Author Manuscript
58. Pack GT, Islami AH, Hubbard JC, Brasfield RD. Regeneration of human liver after major hepatectomy. Surgery. 1962; 52:617–623. [PubMed: 14483060] 59. Huang P, Zhang L, Gao Y, et al. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell. 2014; 14(3):370–384. [PubMed: 24582927] 60. Song K, Nam YJ, Luo X, et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 2012; 485(7400):599–604. [PubMed: 22660318] 61. Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature. 2008; 455(7213):627–632. [PubMed: 18754011] 62. Muraoka N, Ieda M. Direct reprogramming of fibroblasts into myocytes to reverse fibrosis. Annu Rev Physiol. 2014; 76:21–37. [PubMed: 24079415] 63. Liu L, Yannam GR, Nishikawa T, et al. The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis. Hepatology. 2012; 55(5):1529–1539. [PubMed: 22109844] 64. Nishikawa T, Bellance N, Damm A, et al. A switch in the source of ATP production and a loss in capacity to perform glycolysis are hallmarks of hepatocyte failure in advance liver disease. J Hepatol. 2014; 60(6):1203–1211. [PubMed: 24583248] 65. Nishikawa T, Bell A, Brooks JM, et al. Resetting the transcription factor network reverses terminal chronic hepatic failure. J Clin Investig. 2015; 125(4):1533–1544. [PubMed: 25774505] 66. Lechler RI, Sykes M, Thomson AW, Turka LA. Organ transplantation–how much of the promise has been realized? Nat Med. 2005; 11(6):605–613. [PubMed: 15937473]
Author Manuscript Author Manuscript Am J Transplant. Author manuscript; available in PMC 2016 June 01.
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Figure 1. Schematic representation of different approaches under investigation in regenerative medicine for liver replacement and regeneration
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Cell source for liver repopulation (A) and liver engineering (B) should be facilitated by an unlimited supply of induced pluripotent stem–derived hepatocytes, but the underlying tumor potential would remain to be elucidated. Successful liver repopulation will require optimizing functional hepatic cells to achieve high engraftment and repopulation efficiency. “Resetting” liver functions (C) to normal will require improvement of in vivo genetic delivery systems and better understanding of complex molecular networks associated with liver diseases. Important health and ethical issues associated with blastocyst complementation (D) will need to be addressed. The liver may contain host cells and pathogens that could pose a threat of immunological rejection and transmission of interspecies diseases. Ethical concerns could be solved once the genetic toolbox makes it possible to restrict differentiation toward particular organs (brain and gonads). 3D, threedimensional.
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