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.     

Hepatic oval 'stem' cell in liver regeneration

Hepatic oval cell activation, proliferation, and differentiation has been observed under certain physiological conditions, mainly when the proliferation of existing hepatocytes has been inhibited followed by severe hepatic injury. 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. Bone marrow stem cells have recently been shown to be a potential source of the hepatic oval cells and that reconstitution of an injured liver from a purified stem cell population is possible. The focus of this review is on the studies involving the activation, proliferation, and differentiation of these hepatic oval cells and the role that they play in regeneration of the damaged liver. In order to present the potentiality of the hepatic oval cell, an experimental model that involves the inhibition of normal hepatic growth and division as well as severe hepatic injury via chemical or surgical means has been employed. In this model, an as yet undetermined signal or perhaps the lack of regenerative capability in the hepatocytes activates the hepatic oval cell compartment. However, other than understanding a potential origin of these cells and some of the markers that characterize them, it still remains unclear as to how these cells migrate ('home') into the damaged areas and how they begin their differentiation into mature and functioning hepatic cells.     

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.     

Generation,characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end‐stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end‐stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra‐hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra‐hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. J. Cell. Physiol. 228: 298–305, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

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.     

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.     

The therapeutic potential of bone marrow-derived mesenchymal stem cells on hepatic cirrhosis

Hepatic cirrhosis is the end-stage of chronic liver diseases. The majority of patients with hepatic cirrhosis die from life-threatening complications occurring at their earlier ages. Liver transplantation has been the most effective treatment for these patients. Since liver transplantation is critically limited by the shortage of available donor livers, searching for an effective alternative therapy has attracted great interest in preclinical studies. The transplantation of autologous bone marrow-derived mesenchymal stem cells holds great potential for treating hepatic cirrhosis. Mesenchymal stem cells can differentiate to hepatocytes, stimulate the regeneration of endogenous parenchymal cells, and enhance fibrous matrix degradation. Experimental and clinical studies have shown promising beneficial effects. This review is intended to translate the bench study results to the patients' bedside. The potential interventions of mesenchymal stem cells on cirrhosis are illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.     

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。     

Progenitor cells of the biliary epithelial cell lineage

Stem-like cells have been identified in liver that are able to differentiate in vivo and in culture to biliary epithelial cells (BEC), hepatocytes and oval cells. The growth factors/cytokines and signal pathways required for the differentiation processes are beginning to be evaluated. There is increasing evidence to suggest that these stem-like cells may originate from both the bone marrow population or from a precursor remnant from liver embryogenesis, as they share many of the same markers (CD34, c-kit, CD45). Most recently, it has been shown that a population of progenitor cells can copurify with mesenchymal bone marrow cells and differentiate under specific culture conditions to form both hepatic epithelial and also endothelial cells. The interaction of haemopoietic and mesenchymal stem cells needs further evaluation. The close association of ductular reactive cells and neovessels in end-stage cholestatic liver diseases and the relation to Jagged/Notch signalling pathway may be important in the regulation of stem cells to form both biliary epithelial and endothelial cells.     

胚胎肝干细胞的研究进展

近年来的研究表明在胚胎肝脏中存在大量的肝干细胞,它们在肝脏的发育中起着重要作用,并且受到各种时序性表达基因的调控。几个研究组采用不同的方法,分别从小鼠、大鼠和灵长类动物的胚胎肝脏分离并鉴定了具有双潜能的肝干细胞。就胚胎肝脏的发育、调控机制以及胚胎肝干细胞的分离、鉴定等方面的研究进展作一综述,并对胚胎肝干细胞的应用前景和今后的研究方向作了展望。     

Liver transplantation of hepatic stem cells: potential use for treating liver diseases

Hepatic stem cells are an alternative means for repopulating the liver after various injuries instead of liver transplantation. The first step before use is to select stem cells that will be good candidates. This review discusses the different candidates including fetal progenitor bipotential hepatic stem cells; adult hepatocytes, which can be considered as unipotential committed stem cells; and oval cells, a type of nonparenchymal pluripotential hepatic stem cell. The advantages and disadvantages of each type of cell are discussed and several other possible alternatives, such as the use of hematopoietic stem cells are analyzed. This revised version was published online in July 2006 with corrections to the Cover Date.     

hDlk-1: a cell surface marker common to normal hepatic stem/progenitor cells and carcinomas

Advances in stem cell biology have clarified that a tumour is a collection of heterogeneous cell populations, and that only a small fraction of tumour cells possesses the potential to self-renew. Delta-like 1 protein (Dlk-1) is a surface antigen present on foetal hepatic stem/progenitor cells but absent from mature hepatocytes in neonatal and adult rodent liver. Using a monoclonal antibody (mAb) against hDlk-1, Yanai et al. (Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J. Biochem. 2010;148:85-92) have shown that human (h) Dlk-1 is expressed in human foetal, but not adult, liver and that 20% of all hepatocellular carcinomas (HCCs) are hDlk-1(+). Importantly, an even higher percentage of HCCs in younger patients are hDLK-1(+). These authors also found that hDlk-1 is present at high frequency in colon adenocarcinomas, pancreatic islet carcinomas and small cell lung carcinomas. Here, I discuss the implications of the expression of foetal hepatic stem/progenitor cell antigens on carcinoma cells.     

骨髓干细胞移植治疗肝硬化的基础与临床研究现状

肝硬化是一种临床常见的肝病良性终末期表现。目前临床上尚缺乏有效的治疗措施。肝脏移植是最理想的治疗方法,但受供体肝脏来源限制,且费用昂贵。近年来开展的自体骨髓干细胞(BMSCs)移植治疗,为肝硬化的治疗带来了新的希望。BMSCs主要包括造型血干细胞和间充质干细胞,其具有可塑性,体外通过生长因子,体内利用特定微环境均可诱导BMSCs分化为肝前体细胞和成熟肝细胞,并明显改善肝功能。从动物实验到临床研究亦表明,BMSCs具有来源丰富、费用低廉、损伤小、自体移植不栓塞、无排斥反应等优点,为治疗肝病带来了新思路,有望成为生物人工肝的细胞来源。本文就BMSCs移植治疗肝硬化的研究现状,尤其是移植途径以及在肝脏内定居、迁移和分化机制的示踪观察方法和存在的问题作一综述,以期为从事肝病研究的同仁提供参考依据。通过对BMSCs移植从基础研究及临床应用的最新进展的描述,展示BMSCs在肝硬化治疗方面良好的治疗前景。     

Robust differentiation of fetal hepatocytes from human embryonic stem cells and iPS

Hepatocyte transplantation is considered as an alternative to organ transplantation in particular for the treatment of liver metabolic diseases. However, due to the difficulties to obtain a large number of hepatocytes, new sources of cells are needed. These cells could be either of hepatic origin (hepatic stem cells) or extrahepatic such as mesenchymal stem cells or pluripotent stem cells (human embryonic stem cells [hESC] or iPS). We developed a new method to differentiate hESCs into fetal hepatocytes. These conditions recapitulate the main liver developmental stages, using fully defined medium devoid of animal products or unknown factors. The differentiated cells express many fetal hepatocytes markers (cytochrome P450 3A7, albumin, alpha-1-antitrypsin, etc.). The cells display specific hepatic functions (ammonia metabolism, excretion of indocyanin green) and are capable to engraft and express hepatic proteins two months after transplantation into newborn uPAxrag2gc-/- mouse liver. We have also showed that this approach is transposable to human iPS, and further studies on animal models will allow us to compare the in vivo potential of these two sources of pluripotent cells. Finally, only studies on large animals such as nonhuman primates will validate an eventual clinical application.     

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.     

胎肝干细胞的分离、培养与鉴定

目的体外扩增培养大鼠胎肝干细胞,研究其形态、生物学特性及表面标志物,探讨胎肝干细胞的性质。方法分离培养胎龄12-16d的胎肝细胞,SABC法检测原代、传代后及细胞克隆中的肝干细胞特异表面标志物OV-6、CK-19及nestin的表达。结果原代、传代培养的胎肝细胞部分表达OV-6、CK-19及nestin;培养3d开始出现小细胞团,1个月即形成肉眼可见的细胞集落,5-7d传代一次;细胞克隆几乎全部为干细胞标志阳性细胞。结论胎肝干细胞可通过克隆筛选法进行体外扩增,胎肝内存在nestin阳性干细胞,可能是一种更为原始的干细胞,在胚胎发育中起重要作用。     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.     

Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.     

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.