Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

The role of hepatocytes and oval cells in liver regeneration and repopulation

The liver has the unique capacity to regulate its growth and mass. In rodents and humans, it grows rapidly after resection of more than 50% of its mass. This growth process, as well as that following acute chemical injury is known as liver regeneration, although growth takes place by compensatory hyperplasia rather than true regeneration. In addition to hepatocytes and non-parenchymal cells, the liver contains intra-hepatic "stem" cells which can generate a transit compartment of precursors named oval cells. Liver regeneration after partial hepatectomy does not involve intra or extra-hepatic (hemopoietic) stem cells but depends on the proliferation of hepatocytes. Transplantation and repopulation experiments have demonstrated that hepatocytes, which are highly differentiated and long-lived cells, have a remarkable capacity for multiple rounds of replication. In this article, we review some aspects of the regulation of hepatocyte proliferation as well as the interrelationships between hepatocytes and oval cells in different liver growth processes. We conclude that in the liver, normally quiescent differentiated cells replicate rapidly after tissue resection, while intra-hepatic precursor cells (oval cells) proliferate and generate lineage only in situations in which hepatocyte proliferation is blocked or delayed. Although bone marrow stem cells can generate oval cells and hepatocytes, transdifferentiation is very rare and inefficient.     

Adult stem cells: seing is not being

Recent unexpected observations in adult rodents that stem/progenitor cells located in the bone marrow, but also in other tissues, could, after their transplantation to an irradiated host contribute to the regeneration of damaged organs such as brain, liver, pancreas or muscle, have raised much hope for future therapeutic applications. These data have also initially been interpreted as a proof of a possible transdifferentiation or plasticity of adult stem cells located in these tissues. Additional experiments rigorously analyzed have tempered initial enthusiasm, by showing that if marrow cells do migrate in damaged muscles and liver, their contribution to organ repair is low, and in some cases, explained by cell fusion. Nevertheless, among bone marrow cells, two categories of stem cells now emerge that have a potentially tremendous interest in cell therapy, if we succeed in understanding how to purify, amplify and differentiate these more efficiently and reproducibly.     

Cell fusion and reprogramming: resolving our transdifferences

Bone marrow-derived cells (BMDCs) can contribute to the regeneration of diverse adult tissues, including brain, liver and heart, following bone marrow transplantation. These unexpected events were initially considered a result of transdifferentiation of BMDCs, supporting the emerging idea of extended plasticity of adult stem cells. Although studies have now clearly demonstrated that spontaneous cell fusion, rather than transdifferentiation, was the primary cause for unexpected cell fate-switches of BMDCs into hepatocytes, Purkinje cells and cardiac myocytes in vivo.     

Metamorphosis from Bone Marrow Derived Primitive Stem Cells to Functional Liver Cells

Both stem cell plasticity and cell fusion have been implicated as physiological responses to tissue injury. It remains the ultimate goal for the future to understand the regulatory control of each during regeneration. In our recent paper by Jang et al. we demonstrate the repair of damaged liver by bone marrow derived stem cells (SCs) in response to microenvironmental cues. Within 48 hrs after transplantation or co-culture, conversion of SCs into liver cells was observed. Fusion was ruled out as a major mechanism of this functional regeneration. Direct differentiation of SCs into liver epithelial cells may be clinically useful. However, if plasticity or fusion results in abnormal genetic changes they could be harmful. Before proceeding with therapeutic applications, the consequences of cellular therapy accompanying both plasticity and fusion must be examined in multiple animal models. Functional repair should also be demonstrated prior to treatment in patients.     

The role of some donor-host cell interactions in conditions of the regenerative process microenvironment

The review is devoted to the analysis of experimental data about possible mechanisms of transdifferentiation or plasticity of tissue specific stem cells. Considerable attention is focused on the mechanisms and genetic consequences of fusion of different types of donor cells with the cells of recipient tissues which investigated on the models of cellular therapy of liver and heart diseases. The role of various kinds of cell contacts and their role in stem cells integration, reparation and regeneration of injured tissue and horizontal genes transfer are considered.     

Hepatic stem cells: from inside and outside the liver?

The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass. Moreover, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes in foetal and adult liver can clonally expand, suggesting that hepatoblasts/hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.     

Stem cell biology: a never ending quest for understanding

Stem cells (SC) research is an important part of biotechnology that could lead to the development of new therapeutic strategies. A lot of effort has been put to understand biology of the stem cells and to find genes and subsequently proteins that are responsible for their proliferation, self-renewal and differentiation. Different cytokines and growth factors has been used to expand stem cells, but no combination of these factors was identified that could effectively expand the most primitive stem cells. Recently, however, genes and receptors responsible for SC proliferation and differentiation have been described. Ligands for these receptors or these genes themselves are being already used for ex vivo expansion of stem cells and the first data are very promising. New markers, such as CXCR4 and CD133, have been discovered and shown to be present on surface of hematopoietic stem cells. The same markers were recently also found to be expressed on neuronal-, hepatic- or skeletal muscle-stem cells. By employing these markers several laboratories are trying to isolate stem cells for potential clinical use. New characteristics of stem cells such as transdifferentiation and cell fusion have been described. Our team has identified a population of tissue committed stem cells (TCSC). These cells are present in a bone marrow and in other tissues and they can differentiate into several cell types including cardiac, neural and liver cells.     

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.     

Fetal and adult liver stem cells for liver regeneration and tissue engineering

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.     

The role of cytokines in liver failure and regeneration: potential new molecular therapies

The liver is a unique organ, and first in line, the hepatocytes encounter the potential to proliferate during cell mass loss. This phenomenon is tightly controlled and resembles in some way the embryonal co-inhabitant cell lineage of the liver, the embryonic hematopoietic system. Interestingly, both the liver and hematopoietic cell proliferation and growth are controlled by various growth factors and cytokines. IL-6 and its signaling cascade inside the cells through STAT3 are both significantly important for liver regeneration as well as for hematopoietic cell proliferation. The process of liver regeneration is very complex and is dependent on the etiology and extent of liver damage and the genetic background. In this review we will initially describe the clinical relevant condition, portraying a number of available animal models with an emphasis on the relevance of each one to the human condition of fulminant hepatic failure (FHF). The discussion will then be focused on the role of cytokines in liver failure and regeneration, and suggest potential new therapeutic modalities for FHF. The recent findings on the role of IL-6 in liver regeneration and the activity of the designer IL-6/sIL-6R fusion protein, hyper-IL-6, in particular, suggest that this molecule could significantly enhance liver regeneration in humans, and as such could be a useful treatment for FHF in patients.     

Nuclear reprogramming and adult stem cell potential

Cell-based therapy may represent a new strategy to treat a vast array of clinical disorders including neurodegenerative diseases. Recent observations indicate that adult somatic stem cells have the capacity to contribute to the regeneration of different tissues, suggesting that differentiative restrictions are not completely irreversible and can be reprogrammed. Cell fusion might account for some changed phenotype of adult cells but it seems to be biologically irrelevant for its extreme rarity. Other experimental evidences are compatible with the hypothesis of wide multipotency of well-defined stem cell populations, but also with transdifferentiation and/or dedifferentiation. Further studies on nuclear reprogramming mechanisms are necessary to fulfil the promise for developing autologous cellular therapies.     

The role of some donor-host cell interactions under a microenvironmental influence during regeneration processes

This review of publications is dedicated to the analysis of experimental data concerning the possible mechanisms constituting the basis of transdifferentiation or plasticity of tissue-specific stem cells. In the review, we focused on the mechanisms and genetic consequences of fusion between donor cells and recipient tissue cells that were investigated using models of cell therapy for liver and heart diseases. The role of intercellular contacts of different types and horizontal gene transfer during the heart tissue regeneration process was also considered.     

SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.     

Tube Formation Potential of BMSCs and USSCs in Response to HIF-1α Overexpression under Hypoxia

Despite numerous studies on therapeutic effects of mesenchymal stem cells on ischemic tissue regeneration, including angiogenesis, their mechanism of action remains ambiguous. Due to the scarce of investigations based on different stem cell sources with known inherent molecular differences, present study compare tube formation of Bone marrow Mesenchymal Stem Cells and Unrestricted Somatic Stem Cells with known reported different Hox gene expression profile in response to HIF-1α overexpression under hypoxia. This might shed light on some parameters for selection of more responsive source with improved therapeutic effects. Superior in vitro tube formation on Matrigel substratum has been observed by Unrestricted Somatic Stem Cells compared to Bone marrow Mesenchymal Stem Cells which might possibly be due to the discriminating molecular properties of stem cell sources. It may help choosing the appropriate stem cell type for a given therapeutic expectations and also suggests some potential targets for future genetic modification of stem cells.     

Perspective on liver regeneration by bone marrow–derived stem cells—A scientific realization or a paradox

Bone marrow (BM)-derived stem cells are reported to have cellular plasticity, which provoked many investigators to use of these cells in the regeneration of nonhematopoietic tissues. However, adult stem cell plasticity contradicts our classic understanding on progressive restriction of the developmental potential of a cell type. Many alternate mechanisms have been proposed to explain this phenomenon; the working hypotheses for elucidating the cellular plasticity of BM-derived stem cells are on the basis of direct differentiation and/or fusion between donor and recipient cells. This review dissects the different outcomes of the investigations on liver regeneration, which were performed with the use of BM-derived stem cells in experimental animals, and reveals some critical factors to explain cellular plasticity. It has been hypothesized that the competent BM-derived stem/progenitor cells, under the influence of liver-regenerating cues, can directly differentiate into hepatic cells. This differentiation takes place as a result of genetic reprogramming, which may be possible in the chemically induced acute liver injury model or at the stage of fetal liver development. Cellular plasticity emerges as an important phenomenon in cell-based therapies for the treatment of many liver diseases in which tissue regeneration is necessary.     

Stem cells, cell transplantation and liver repopulation

Liver transplantation is currently the only therapeutic option for patients with end-stage chronic liver disease and for severe acute liver failure. Because of limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult mammalian liver contains hepatic stem cells is highly controversial. Part of the problem is that proliferation of mature adult hepatocytes is sufficient to regenerate the liver after two-thirds partial hepatectomy or acute toxic liver injury and participation of stem cells is not required. However, under conditions in which hepatocyte proliferation is blocked, undifferentiated epithelial cells in the periportal areas, called "oval cells", proliferate, differentiate into hepatocytes and restore liver mass. These cells are referred to as facultative liver stem cells, but they do not repopulate the normal liver after their transplantation. In contrast, epithelial cells isolated from the early fetal liver can effectively repopulate the normal liver, but they are already traversing the hepatic lineage and may not be true stem cells. Mesenchymal stem cells and embryonic stem cells can be induced to differentiate along the hepatic lineage in culture, but at present these cells are inefficient in repopulating the liver. This review will characterize these various cell types and compare the properties of these cells and the conditions under which they do or do not repopulate the liver following their transplantation.     

Stem/progenitor cells in liver injury repair and regeneration

Morbidity and mortality from cirrhosis is increasing rapidly in the world. Currently, orthotopic liver transplantation is the only definitive therapeutic option. However, its clinical use is limited, because of poor long‐term graft survival, donor organ shortage and high costs associated with the procedure. Stem cell replacement strategies are therefore being investigated as an attractive alternative approach to liver repair and regeneration. In this review we discuss recent preclinical and clinical investigations that explore the therapeutic potential of stem cells in repair of liver injuries. Several types of stem cells. including embryonic stem cells, haematopoietic stem cells and mesenchymal stem cells, can be induced to differentiate into hepatocyte‐like cells by defined culture conditions in vitro. Stem cell transplantation has been shown to significantly improve liver function and increase animal survival in experimentally‐induced liver‐injury models. Moreover, several pilot clinical studies have reported encouraging therapeutic effects in patients treated with stem cells. Although there remain many unresolved issues, the available data support the notion that stem cell technology may lead to the development of effective clinical modalities for human liver diseases.