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.     

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 comparison of multipotential for differentiation of progenitor mesenchymal-like stem cells obtained from livers of young and old rats

The presence of stem cells differentiating to hepatocytes and cholangiocytes has been previously reported in livers of young rats. Here, we have isolated, cultured, and characterized mesenchymal stem cells (MSCs) from livers of young and old rats and tested their multipotential for differentiation. The mesenchymal stem cells in liver sections were identified by the presence of markers, respectively for primary stem cells Thy-1 and CD34, for differentiation to early cholangiocytes GST and CK19, and for differentiation to hepatocytes GSTalpha and CK18. Ki67 was detected as the cell proliferation marker. Cells isolated from livers of either age group were tested in a culture for their viability following storage and were characterized for the presence of most of the markers detected in cells in situ. The results revealed age-dependent changes in the number of recovered primary MSCs. In both age groups we have observed cells changing under differentiating conditions to liver cell lineages, such as cholangiocytes and hepatocytes, as well as to non-liver cells such as adipocytes, astrocytes, neuroblasts, and osteoblasts. Our data revealed that from the livers of rats 20 months and older the primary MSCs could be isolated and expanded; however, they were significantly fewer, even though their differentiation multipotential was preserved. The mechanism involved in the differentiation of liver MSCs seemed to depend on a constellation of signals in Notch signalling pathways. Thus, our results support the idea of potential use of liver as a source of MSCs, not only for liver reconstruction but also for cell therapy in general.     

Identification and propagation of liver stem cells

Although the liver has been known for its enormous regenerative capacity, little is known about the mechanisms responsible for such regeneration.To provide evidence for the existence of liver stem cell, using FACS and single cell-based assays, cells with multi-lineage differentiation potential and self-renewal capability have been prospectively identified. These cells could be clonally propagated in culture where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When the cells clonally expanded in vitro were transplanted into mouse, they morphologically and functionally differentiated into hepatocytes and cholangiocytes. Furthermore, these cells differentiated into pancreatic acinar cells or intestinal epithelial cells upon transplantation into pancreas or duodenal wall. Manipulation of self-renewing liver stem cells may provide new insight into therapies for diseases of the digestive system.     

New differentiation markers of mouse liver epithelial cell lines

We describe two new markers of mouse liver epithelial cells detected by monoclonal antibodies. Immunomorphological localization of antigens was performed using light and electron microscopy. Antigen G7 is a marker of cholangiocytes and oval cells. Antigen A6 is present in cholangiocytes and oval cells; moreover, it is expressed in normal liver in single hepatocytes adjacent to the portal vein, in preneoplastic liver, in newly formed hepatocytes, and in certain hepatocarcinomas. Thus, antigen A6 is a marker of cholangiocytes, oval cells and of certain stages of hepatocyte differentiation. We also detected phenotypic heterogeneity of Gehring cells in terms of antigen A6 content. We have formulated problem of the relationship between A6-negative Gehring cells and liver stem cells. Both marker antigens are species-specific but are not specific for the liver. Antigen A6 is simultaneously a differentiation marker of cells belonging to the erythroid series. It is expressed in erythroblasts of fetal liver and is absent in erythroblasts of the yolk sac and erythrocytes. The relationship between antigen A6 and blood group antigens is discussed.     

Unbalanced distribution of materials: the art of giving rise to hepatocytes from liver stem/progenitor cells

Liver stem/progenitor cells (LSPCs) are able to duplicate themselves and differentiate into each type of cells in the liver, including mature hepatocytes and cholangiocytes. Understanding how to accurately control the hepatic differentiation of LSPCs is a challenge in many fields from preclinical to clinical treatments. This review summarizes the recent advances made to control the hepatic differentiation of LSPCs over the last few decades. The hepatic differentiation of LSPCs is a gradual process consisting of three main steps: initiation, progression and accomplishment. The unbalanced distribution of the affecting materials in each step results in the hepatic maturation of LSPCs. As the innovative and creative works for generating hepatocytes with full functions from LSPCs are gradually accumulated, LSPC therapies will soon be a new choice for treating liver diseases.     

E-cadherin as a reliable cell surface marker for the identification of liver specific stem cells

Oval cells are liver-specific bipotent stem cells which accumulate in injured liver when proliferation of mature hepatocytes and/or cholangiocytes is impaired. They represent an intermediary cell type with phenotypical characteristics of both, hepatocytes and cholangiocytes. Oval cells express specific cell surface proteins allowing their identification in situ. Most of these cell surface proteins, however, are recognized by antibodies in mouse liver tissue that are not commercially available or work only on frozen sections. We show herein the unequivocal identification of oval cells in paraffin-embedded mouse liver samples based on strong E-cadherin expression different from that of hepatocytes and bile duct cells. By comparing the pattern of E-cadherin expression with that of both, A6-antigen and CD44, we suggest a tight control of E-cadherin expression depending on the differentiation stage of the progenitor cells. In human cirrhotic liver samples E-cadherin expression was found as a common feature of both, typical and atypical reactions, and, thus, can also serve as an indication of the progenitor cell compartment activation.     

Wnt signaling in biliary development,proliferation, and fibrosis

Biliary fibrosis is an important pathological indicator of hepatobiliary damage. Cholangiocyte is the key cell type involved in this process. To reveal the pathogenesis of biliary fibrosis, it is essential to understand the normal development as well as the aberrant generation and proliferation of cholangiocytes. Numerous reports suggest that the Wnt signaling pathway is implicated in the physiological and pathological processes of cholangiocyte development and ductular reaction. In this review, we summarize the effects of Wnt pathway in cholangiocyte development from embryonic stem cells, as well as the underlying mechanisms of cholangiocyte responses to adult ductal damage. Wnt signaling pathway is regulated in a step-wise manner during each of the liver differentiation stages from embryonic stem cells to functional mature cholangiocytes. With the modulation of Wnt pathway, cholangiocytes can also be generated from adult liver progenitor cells and mature hepatocytes to repair liver damage. Non-canonical Wnt signaling is triggered in the active ductal cells during biliary fibrosis. Targeted control of the Wnt signaling may hold the great potential to reduce and/or reverse the biliary fibrogenic process.     

PECAM-1 (CD31) regulates a hydrogen peroxide-activated nonselective cation channel in endothelial cells

Using flow cytometry and single cell-based assays, we prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewing capability. These cells could be clonally propagated in culture where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When cells that expanded in vitro were transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. These data indicate that self-renewing pluripotent stem cells persist in the developing mouse liver and that such cells can be induced to become cells of other organs of endodermal origin under appropriate microenvironment. Manipulation of hepatic stem cells may provide new insight into therapies for diseases of the digestive system.     

Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a).Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli.We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.     

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.     

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.     

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.     

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.     

Wnt/β‐catenin signalling controls bile duct regeneration by regulating differentiation of ductular reaction cells

Recently, the incidence of bile duct‐related diseases continues to increase, and there is no effective drug treatment except liver transplantation. However, due to the limited liver source and expensive donations, clinical application is often limited. Although current studies have shown that ductular reaction cells (DRCs) reside in the vicinity of peribiliary glands can differentiate into cholangiocytes and would be an effective alternative to liver transplantation, the role and mechanism of DRCs in cholangiole physiology and bile duct injury remain unclear. A 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC)‐enriched diet was used to stimulate DRCs proliferation. Our research suggests DRCs are a type of intermediate stem cells with proliferative potential that exist in the bile duct injury. Meanwhile, DRCs have bidirectional differentiation potential, which can differentiate into hepatocytes and cholangiocytes. Furthermore, we found DRCs highly express Lgr5, and Lgr5 is a molecular marker for neonatal DRCs (P < .05). Finally, we confirmed Wnt/β‐catenin signalling achieves bile duct regeneration by regulating the expression of Lgr5 genes in DRCs (P < .05). We described the regenerative potential of DRCs and reveal opportunities and source for the treatment of cholestatic liver diseases.     

Autocrine regulation of biliary pathology by activated cholangiocytes

The bile duct system of the liver is lined by epithelial cells (i.e., cholangiocytes) that respond to a large number of neuroendocrine factors through alterations in their proliferative activities and the subsequent modification of the microenvironment. As such, activation of biliary proliferation compensates for the loss of cholangiocytes due to apoptosis and slows the progression of toxic injury and cholestasis. Over the course of the last three decades, much progress has been made in identifying the factors that trigger the biliary epithelium to remodel and grow. Because a large number of autocrine factors have recently been identified as relevant clinical targets, a compiled review of their contributions and function in cholestatic liver diseases would be beneficial. In this context, it is important to define the specific processes triggered by autocrine factors that promote cholangiocytes to proliferate, activate neighboring cells, and ultimately lead to extracellular matrix deposition. In this review, we discuss the role of each of the known autocrine factors with particular emphasis on proliferation and fibrogenesis. Because many of these molecules interact with one another throughout the progression of liver fibrosis, a model speculating their involvement in the progression of cholestatic liver disease is also presented.     

胚胎干细胞分化为肝细胞的研究进展

目前 ,细胞移植作为终末期肝病的辅助治疗方法 ,移植的细胞必须满足在受体肝脏中存活、增殖并可分化为成熟肝细胞两个重要条件 ,但目前应用的肝细胞来源有限 ,其功能随着培养时间的延长而逐渐下降等问题限制了这一治疗策略的广泛开展。作为具有发育全能性和无限增殖能力的细胞 ,胚胎干细胞向肝细胞的分化研究近年来引起了广泛的关注 ,并取得了较大的进展 ,寻找合适、高效的分化诱导方法是目前研究的热点之一。胚胎干细胞向肝细胞的分化研究既可以为临床细胞替代治疗提供合适的细胞来源 ,也可以在药物评估和肝脏发育分化基础研究方面起到重要的作用。通过概括肝脏和拟胚体分化发育的分子机制 ,对体外胚胎干细胞向肝细胞分化的几种诱导体系作了介绍 ,并对分化肝细胞的应用前景和存在的问题进行了讨论。