Potential of bioengineering processes for therapeutic repopulation of the liver with cells

Multiple unique aspects of liver biology make this organ an excellent paradigm for novel cell and gene therapy applications. In recent years, insights were obtained into how transplanted cells engraft and proliferate in the liver, including in the context of pre-existing disease. Also, a variety of animal models were studied to establish the basis of cell and gene therapy applications in specific disorders. Through ongoing research activity, additional mechanisms in liver repopulation have been uncovered, where manipulation of specific cell compartments and cellular processes,e.g., those aimed at extracellular matrix component receptors or soluble signals in transplanted and native cells can be exploited for enhancing cell engraftment and proliferation. Such studies demonstrate the possibility of applying biotechnology and/or bioengineering principles to organ replacement aimed at cell and gene therapy. Joining of these disciplines with research in stem cell biology, particularly in efforts concerning targeting of transplanted stem cells to given organs with achievement of lineage-specific cell differentiation and function, will be particularly important for future cell and gene therapy applications. This review offers an overview of relevant mechanisms in liver repopulation.     

Pluripotent plasticity of stem cells and liver repopulation

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra‐hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self‐repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio‐artificial liver‐assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright © 2010 John Wiley & Sons, Ltd.     

Liver stem cells

The concept of a liver stem cell or progenitor cell has not been widely accepted until the last decade. Studies investigating liver regeneration under conditions which totally or substantially preclude hepatocyte proliferation report the proliferation of a subpopulation of small, oval-shaped cells, which are first observed in the portal triad, adjacent to the terminal ducts. These cells, termed liver progenitor oval cells (LPCs) are shown to participate in liver regeneration in a variety of rodent models of chronic liver damage. They express markers common to hepatocytes and cholangiocytes suggesting they are a common precursor of both liver cell lineages. Supporting evidence for liver stem cells has also come from cell tracing studies which show transdifferentiation of bone marrow cells into hepatocytes in both human and animal models. Another important issue is the link between LPCs and hepatocellular carcinoma (HCC). The widening liver donor-recipient gap; a consequence of poor donation rates coupled with increasing incidence of liver disease highlights the importance of establishing the utility of cell transplant as an alternative to treat liver disease. In this regard, liver stem cells and progenitor cells may have a significant role to play. To successfully utilize liver stem cells or LPCs for cell therapy, we have to first develop methods for maintaining and differentiating them in culture. This technology must be based on a thorough understanding of conditions which regulate their behaviour in vitro. In particular, we need to know which growth factors and cytokines affect them and their mechanism of action. Since they are a potential source of HCC, it is also necessary to understand the mechanisms which underlie their transformation to cancer.     

Extracellular matrix is required for the survival and differentiation of transplanted hepatic progenitor cells

Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.     

Cell turnover in the repopulated rat liver: distinct lineages for hepatocytes and the biliary epithelium

The dynamics of cell renewal in the normal adult liver remains an unresolved issue. We investigate the possible contribution of a common biliary precursor cell pool to hepatocyte turnover in the chimeric long-term repopulated rat liver. The retrorsine (RS)-based model of massive liver repopulation was used. Animals not expressing the CD26 marker (CD26^-) were injected with RS, followed by transplantation of 2 million syngeneic hepatocytes isolated from a normal CD26-expressing donor. Extensive (80-90 %) replacement of resident parenchymal cells was observed at 1 year post-transplantation and persisted at 2 years, as expected. A panel of specific markers, including cytokeratin 7, OV6, EpCAM, claudin 7 and α-fetoprotein, was employed to locate the in situ putative progenitor and/or biliary epithelial cells in the stably repopulated liver. No overlap was observed between any of these markers and the CD26 tag identifying transplanted cells. Exposure to RS was not inhibitory to the putative progenitor and/or biliary epithelial cells, nor did we observe any evidence of cell fusion between these cells and the transplanted cell population. Given the long-term (>2 years) stability of the donor cell phenotype in this model of liver repopulation, the present findings suggest that hepatocyte turnover in the repopulated liver is fuelled by a cell lineage distinct from that of the biliary epithelium and relies largely on the differentiated parenchymal cell population. These results support the solid biological foundation of liver repopulation strategies based on the transplantation of isolated hepatocytes.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Stem cells in liver regeneration and therapy

The liver has adapted to the inflow of ingested toxins by the evolutionary development of unique regenerative properties and responds to injury or tissue loss by the rapid division of mature cells. Proliferation of the parenchymal cells, i.e. hepatocytes and epithelial cells of the bile duct, is regulated by numerous cytokine/growth-factor-mediated pathways and is synchronised with extracellular matrix degradation and restoration of the vasculature. Resident hepatic stem/progenitor cells have also been identified in small numbers in normal liver and implicated in liver tissue repair. Their putative role in the physiology, pathophysiology and therapy of the liver, however, is not yet precisely known. Hepatic stem/progenitor cells also known as “oval cells” in rodents have been implicated in liver tissue repair, at a time when the capacity for hepatocyte and bile duct replication is exhausted or experimentally inhibited (facultative stem/progenitor cell pool). Although much more has to be learned about the role of stem/progenitor cells in the physiology and pathophysiology of the liver, experimental analysis of the therapeutic value of these cells has been initiated. Transplantation of hepatic stem/progenitor cells or in vivo pharmacological activation of the pool of hepatic stem cells may provide novel modalities for the therapy of liver diseases. In addition, extrahepatic stem cells (e.g. bone marrow cells) are being investigated for their contribution to liver regeneration. Hepatic progenitor cells derived from embryonic stem cells are included in this review, which also discusses future perspectives of stem cell-based therapies for liver diseases.     

Cell transplantation causes loss of gap junctions and activates GGT expression permanently in host liver

Cell transplantation into hepatic sinusoids, which is necessary for liver repopulation, could cause hepatic ischemia. To examine the effects of cell transplantation on host hepatocytes, we transplanted Fisher 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. Within 24 h of cell transplantation, areas of ischemic necrosis, along with transient disruption of gap junctions, appeared in the liver. Moreover, host hepatocytes expressed gamma-glutamyl transpeptidase (GGT) extensively, which was observed even 2 years after cell transplantation. GGT expression was not associated with alpha-fetoprotein activation, which is present in progenitor cells. Increased GGT expression was apparent after transplantation of nonparenchymal cells and latex beads but not after injection of saline, fragmented hepatocytes, hepatocyte growth factor, or turpentine. Some host hepatocytes exhibited apoptosis, as well as DNA synthesis, between 24 and 48 h after cell transplantation. Changes in gap junctions, GGT expression, DNA synthesis, and apoptosis after cell transplantation were prevented by vasodilators. The findings indicated the onset of ischemic liver injury after cell transplantation. These hepatic perturbations must be considered when transplanted cells are utilized as reporters for biological studies.     

Therapeutic potential of bone marrow stem cells for liver diseases

Stem cells of the bone marrow, including hematopoietic stem cells (HSC), mesenchymal stem cells (MSC) and hepatic progenitors were reported to give rise to hepatocytes by both transdifferentiation and cellular fusion. Transdifferentiation was observed without liver damage although significant numbers of stem cell derived hepatocytes were not described. Cellular fusion was demonstrated in the presence of a proliferation stimulus in conjunction with impaired intrinsic liver regeneration capacity. Here, we review potential therapeutic applications of stem cell derived hepatocytes depending on how they emerge. Stem cells turning into hepatocytes by transdifferentiation introduce new functioning liver cells into a diseased organ, which can support intrinsic liver regeneration or bridge the time gap until a definitive treatment is available. When cellular fusion is the mechanism behind stem cell plasticity, however, no new cells emerge in the first place, whereas new genetic material is introduced. The fusion cell thereby acquires a selective advantage over resident hepatocytes allowing for extensive proliferation and liver repopulation. Therefore genetic deficiencies might be the predominant target for cell fusion therapies. We conclude that transdifferentiation and cellular fusion might be powerful tools for the therapy of liver diseases in the future and we propose the introduction of artificial cell fusion as well as stem cell differentiation as therapeutic options.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Liver repopulation with hepatocyte transplantation: new avenues for gene and cell therapy

Liver-directed gene therapy is appropriate for many conditions. Recent work established that liver repopulation with transplanted cells can be effective in treating genetic disorders. Although hepatocytes express therapeutic genes with considerable efficiency, correction of genetic disorders is constrained by limitations in permanent gene transfer into hepatocytes and repopulation of the liver with transplanted cells. Adenoviral vectors are highly efficient for hepatic gene transfer but the onset of deleterious host immune responses against adenoviral vectors, along with clearance of transduced hepatocytes have caused problems. Nonetheless, recent work concerning engraftment and proliferation of transplanted hepatocytes in the liver has provided significant new information, which should refocus interest in hepatocyte-based therapies. Moreover, hepatocyte transplantation systems offer creative tools for defining critical mechanisms in gene regulation and survival of transduced cells.     

Application of liver stem cells for cell therapy

The worldwide shortage of donor livers to transplant end stage liver disease patients has prompted the search for alternative cell therapies for intractable liver disease. Embryonic stem cells can be readily differentiated into hepatocytes, and their transplantation into animals has improved liver function in the absence of teratoma formation: their use in bioartificial liver support is an obvious application. In animal models of liver disease, adopting strategies to provide a selective advantage for transplanted foetal or adult hepatocytes have proved highly effective in repopulating recipient livers, but the poor success of today's hepatocyte transplants can be attributed to the lack of a clinically applicable procedure to force a similar repopulation of the human liver. The activation of bipotential hepatic progenitor cells is clearly vital for survival in many cases of acute liver failure, but surprisingly little progress has been made with these cells in terms of transplantation. Finally there is the controversial subject of autologous bone marrow, and while the contribution of these indigenous cells to liver turnover seems at best, trivial, results from a small number of phase 1 studies of transplantation of bone marrow to cirrhotic patients have been moderately encouraging.     

Therapeutic liver repopulation for the treatment of metabolic liver diseases

Orthotopic liver transplantation is the treatment of choice for many inherited and acquired liver diseases. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. In contrast, hepatocyte suspensions can be isolated from a single donor liver can be transplanted into several hosts, and this procedure may help overcome the shortage in donor livers. In classic hepatocyte transplantation, however, only 1% of the liver mass or less can be replaced by donor cells. Recently though, we have used a mouse model of hereditary tyrosinemia to show that > 90% of host hepatocytes can be replaced by a small number of transplanted donor cells in a process we term "therapeutic liver repopulation". This phenomenon is analogous to repopulation of the hematopoietic system after bone marrow transplantation. Liver repopulation occurs when transplanted cells have a growth advantage in the setting of damage to recipient liver cells. Here we will review the current knowledge of this process and discuss the hopeful implications for treatment of liver diseases.     

Liver stem/progenitor cells: their characteristics and regulatory mechanisms

Liver stem cells give rise to both hepatocytes and bile duct epithelial cells also known as cholangiocytes. During liver development hepatoblasts emerge from the foregut endoderm and give rise to both cell types. Colony-forming cells are present in the liver primordium and clonally expanded cells differentiate into either hepatocytes or cholangiocytes depending on culture conditions, showing stem cell characteristics. The growth and differentiation of hepatoblasts are regulated by various extrinsic signals. For example, periportal mesenchymal cells provide a cue for bipotential hepatoblasts to become cholangiocytes, and mesothelial cells covering the parenchyma support the expansion of foetal hepatocytes by producing growth factors. The adult liver has an extraordinary capacity to regenerate, and after 70% hepatectomy the liver recovers its original mass by replication of the remaining hepatocytes without the activation of liver stem cells. However, in certain types of liver injury models, liver stem/progenitor-like cells, known as oval cells in rodents, proliferate around the portal vein, while the roles of such cells in liver regeneration remain a matter of debate. Clonogenic and bipotential cells are also present in the normal adult liver. In this minireview we describe recent studies on liver stem/progenitor cells by focusing on extracellular signals.     

Cholangiocytes: Cell transplantation

Background

Due to significant limitations to the access to orthotropic liver transplantation, cell therapies for liver diseases have gained large interest worldwide.

Evidence for a common progenitor of epithelial and mesenchymal components of the liver

Tissues of the adult organism maintain the homeostasis and respond to injury by means of progenitor/stem cell compartments capable to give rise to appropriate progeny. In organs composed by histotypes of different embryological origins (e.g. the liver), the tissue turnover may in theory involve different stem/precursor cells able to respond coordinately to physiological or pathological stimuli. In the liver, a progenitor cell compartment, giving rise to hepatocytes and cholangiocytes, can be activated by chronic injury inhibiting hepatocyte proliferation. The precursor compartment guaranteeing turnover of hepatic stellate cells (HSCs) (perisinusoidal cells implicated with the origin of the liver fibrosis) in adult organ is yet unveiled. We show here that epithelial and mesenchymal liver cells (hepatocytes and HSCs) may arise from a common progenitor. Sca+ murine progenitor cells were found to coexpress markers of epithelial and mesenchymal lineages and to give rise, within few generations, to cells that segregate the lineage-specific markers into two distinct subpopulations. Notably, these progenitor cells, clonally derived, when transplanted in healthy livers, were found to generate epithelial and mesenchymal liver-specific derivatives (i.e. hepatocytes and HSCs) properly integrated in the liver architecture. These evidences suggest the existence of a ‘bona fide'' organ-specific meso-endodermal precursor cell, thus profoundly modifying current models of adult progenitor commitment believed, so far, to be lineage-restricted. Heterotopic transplantations, which confirm the dual differentiation potentiality of those cells, indicates as tissue local cues are necessary to drive a full hepatic differentiation. These data provide first evidences for an adult stem/precursor cell capable to differentiate in both parenchymal and non-parenchymal organ-specific components and candidate the liver as the instructive site for the reservoir compartment of HSC precursors as yet non-localized in the adult.     

Hepatic stem cells: A viable approach for the treatment of liver cirrhosis

Liver cirrhosis is characterized by distortion of liver architecture, necrosis of hepatocytes and regenerative nodules formation leading to cirrhosis. Various types of cell sources have been used for the management and treatment of decompensated liver cirrhosis. Knowledge of stem cells has offered a new dimension for regenerative therapy and has been considered as one of the potential adjuvant treatment modality in patients with end stage liver diseases (ESLD). Human fetal hepatic progenitor cells are less immunogenic than adult ones. They are highly propagative and challenging to cryopreservation. In our earlier studies we have demonstrated that fetuses at 10-18 wk of gestation age contain a large number of actively dividing hepatic stem and progenitor cells which possess bi-potent nature having potential to differentiate into bile duct cells and mature hepatocytes. Hepatic stem cell therapy for the treatment of ESLD is in their early stage of the translation. The emerging technology of decellularization and recellularization might offer a significant platform for developing bioengineered personalized livers to come over the scarcity of desired number of donor organs for the treatment of ESLD. Despite these significant advancements long-term tracking of stem cells in human is the most important subject nowadays in order to answer several unsettles issues regarding the route of delivery, the choice of stem cell type(s), the cell number and the time-point of cell delivery for the treatment in a chronic setting. Answering to these questions will further contribute to the development of safer, noninvasive, and repeatable imaging modalities that could discover better cell therapeutic approaches from bench to bed-side. Combinatorial approach of decellularization and nanotechnology could pave a way towards the better understanding in determination of cell fate post-transplantation.     

Therapeutic plasticity of stem cells and allograft tolerance

Transplantation is the treatment of choice for many diseases that result in organ failure, but its success is limited by organ rejection. Stem cell therapy has emerged in the last years as a promising strategy for the induction of tolerance after organ transplantation. Here we discuss the ability of different stem cell types, in particular mesenchymal stromal cells, neuronal stem/progenitor cells, hematopoietic stem cells and embryonic stem cells, to modulate the immune response and induce peripheral or central tolerance. These stem cells have been studied to explore tolerance induction to several transplanted organs, such as heart, liver and kidney. Different strategies, including approaches to generating tolerance in islet transplantation, are discussed here.     

Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell

The liver progenitor cell (LPC) has enormous potential for use in cell therapy to treat liver disease. Since liver regenerates readily from pre-existing hepatocytes, a role for LPCs and, indeed, their existence have been questioned. Research during the last decade has established that LPCs are an important alternative source of cells for liver regeneration. Their utility for cell therapy lies in their ability to generate both hepatocytes and cholangiocytes. However, they are observed in liver diseases that often lead to cancer and there is experimental evidence that implicates LPCs as the source of tumours. This article provides a brief history of the studies that established the functional importance of LPCs in liver disease. It focuses on mouse models that have led to the identification of factors that regulate LPC growth and differentiation and discusses LPCs derived from different sources. Recent promising results from both in vitro and vivo studies suggest that LPCs could be useful for cell therapy. In the context of liver disease, LPCs may indeed be the cell of the future and understandably "our favourite cell".     

Hepatic progenitor cells

Liver diseases are associated with a marked reduction in the viable mass of hepatocytes. The most severe cases of liver disease (liver failure) are treated by orthotopic liver transplantation. One alternative to whole organ transplantation for patients with hepatic failure (and hereditary liver disease) is hepatocyte transplantation. However, there is a serious limitation to the treatment of liver diseases either by whole organ or hepatocyte transplantation, and that is the shortage of organ donors. Therefore, to overcome the problem of organ shortage, additional sources of hepatocytes must be found. Alternative sources of cells for transplantation have been proposed including embryonic stem cells, immortalised liver cells and differentiated cells. One other source of cells for transplantation found in the adult liver is the progeny of stem cells. These cells are termed hepatic progenitor cells (HPCs). The therapeutic potential of HPCs lies in their ability to proliferate and differentiate into hepatocytes and cholangiocytes. However, using HPCs as a cell therapy cannot be exploited fully until the mechanisms governing hepatocyte differentiation are elucidated. Here, we discuss the fundamental cellular and molecular elements required for HPC differentiation to hepatocytes.