Update on hepatic stem cells

The liver, like most organs in an adult healthy body, maintains a perfect balance between cell gain and cell loss. Though normally proliferatively quiescent, simple hepatocyte loss such as that caused by partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and 'wear and tear' renewal is largely achieved by hepatocyte self-replication. Furthermore, cell transplant models have shown that hepatocytes can undergo significant clonal expansion. Such observations indicate that hepatocytes are the functional stem cells of the liver. More severe liver injury activates a facultative stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of biliary epithelia within the lobules before these cells differentiate into hepatocytes. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells can contribute to the albeit low renewal rate of hepatocytes, make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate to achieve a homeostatic balance is not understood. This review focuses on three aspects of liver stem cell biology: 1) the hepatic stem cell candidates; 2) models of cell transplantation into the liver; and 3) the therapeutic potential of hepatic stem cells.     

Characterization of the differentiation capacity of rat-derived hepatic stem cells

The liver in an adult rat maintains a balance between cell gain and cell loss. Although normally proliferatively quiescent, hepatocyte loss such as that caused by partial hepatectomy (PH) invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and "wear and tear" renewal is largely achieved by hepatocyte self-replication. Furthermore, hepatocyte transplants in rats, in which a selective pressure for the transplanted cells can be applied, have shown that a certain proportion of hepatocytes can undergo significant clonal expansion, suggesting that hepatocytes themselves are the functional stem cells of the liver. Fetal liver may also harbor bipotential stem cells capable of sustained clonal expansion. More severe liver injury activates a potential stem cell compartment located within the canals of Hering, giving rise to cords of bipotential oval cells that can differentiate into hepatocytes and biliary epithelial cells. Other cell populations with hepatic potential reside in the bone marrow; whether these hematopoietic cells can function as stem cells for the rat liver remains to be confirmed. Pancreatic cells have also been found to have hepatocytic potential.     

Hepatic stem cells： existence and origin

Stem cells are not only units of biological organization,responsible for the development and the regeneration of tissue and organ systems, but also are units in evolution by natural selection. It is accepted that there is stem cell potential in the liver. Like most organs in a healthy adult,the liver maintains a perfect balance between cell gain and loss. It has three levels of cells that can respond to loss of hepatocytes: (1) Mature hepatocytes, which proliferate after normal liver tissue renewal, less severe liver damage, etc;they are numerous, unipotent, “committed“ and respond rapidly to liver injury. (2) Oval cells, which are activated to proliferate when the liver damage is extensive and chronic,or if proliferation of hepatocytes is inhibited; they lie within or immediately adjacent to the canal of Hering (Coil); they are less numerous, bipotent and respond by longer, but still limited proliferation. (3) Exogenous liver stem cells, which may derive from circulating hematopoietic stem cells (HSCs) or bone marrow stem cells; they respond to allyl alcohol injury or hepatocarcinogenesis; they are multipotent, rare,but have a very long proliferation potential. They make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate to achieve a homeostatic balance remains enigmatic. This review focuses on the location,activation, markers of the three candidates of liver stem cell, and the most importantly, therapeutic potential of hepatic stem cells.     

Liver regeneration in acute severe liver impairment: a clinicopathological correlation study.

BACKGROUND: Although normally quiescent, the adult mammalian liver possesses a great capacity to regenerate after different types of injury. Major players in the regeneration process are mature residual cells, including hepatocytes, cholangiocytes and stromal cells. However, if the regenerative capacity of mature cells is impaired, hepatic progenitor cells (HPCs) are activated and expand into the liver parenchyma. Upon transit amplification, the progenitor cells generate new hepatocytes and biliary cells to restore liver homeostasis. AIMS/METHODS: To study the relationship between different histopathological parameters as well as their correlations with clinical parameters and outcome, we examined liver specimens from 74 patients with acute or subacute severe liver impairment by immunohistochemistry for CK7/CK19 (evaluation of HPCs activation/differentiation), Mib1(Ki 67)/P21 (evaluation of proliferative activity/proliferation arrest of hepatocytes) and hematoxylin and eosin (evaluation of hepatocyte loss). RESULTS: Of the 74 patients, 32% survived without transplantation, 14% died without transplantation and 54% were transplanted. Our results show that a threshold of 50% loss of hepatocytes, associated with significant decrease in the proliferative activity of remaining mature hepatocytes, is needed for extensive hepatic progenitor cell activation. Such activation is a sign of disease severity and occurs early (within 1 week) in the disease course. However, development of intermediate hepatocytes, suggesting HPCs differentiation towards mature hepatocytes, takes at least 1 week's time. We found a positive correlation between histopathological parameters (percentage hepatocyte loss, number of proliferating hepatocytes and number of HPCs) and clinical parameters of liver impairment such as model for end stage liver diseases (MELD). Surviving patients compared with those who either died or were transplanted had significantly less hepatocyte loss, less HPCs activation and more mature hepatocyte proliferative activity. Hepatocyte proliferative activity and degree of hepatocyte loss were the most important independent histopathological parameters in predicting outcome. CONCLUSION: Liver biopsy can provide important additional information in a patient with severe acute liver impairment.     

细胞移植疗法在肝病中的研究与应用

人们对使用细胞移植疗法恢复肝脏功能的前景寄予厚望,期待其最终能够代替肝脏移植手术.众多学者对成人成熟肝细胞,不同来源干细胞的再生能力方面进行了大量研究,特别是间充质干细胞来源的肝细胞移植疗法在恢复肝脏再生方面的效果在动物模型实验中取得令人鼓舞的结果.本文将对近年来细胞移植治疗各种肝病的机制研究、动物实验以及临床试验等方...     

The diversity and plasticity of adult hepatic progenitor cells and their niche

The liver is a unique organ for homoeostasis with regenerative capacities. Hepatocytes possess a remarkable capacity to proliferate upon injury; however, in more severe scenarios liver regeneration is believed to arise from at least one, if not several facultative hepatic progenitor cell compartments. Newly identified pericentral stem/progenitor cells residing around the central vein is responsible for maintaining hepatocyte homoeostasis in the uninjured liver. In addition, hepatic progenitor cells have been reported to contribute to liver fibrosis and cancers. What drives liver homoeostasis, regeneration and diseases is determined by the physiological and pathological conditions, and especially the hepatic progenitor cell niches which influence the fate of hepatic progenitor cells. The hepatic progenitor cell niches are special microenvironments consisting of different cell types, releasing growth factors and cytokines and receiving signals, as well as the extracellular matrix (ECM) scaffold. The hepatic progenitor cell niches maintain and regulate stem cells to ensure organ homoeostasis and regeneration. In recent studies, more evidence has been shown that hepatic cells such as hepatocytes, cholangiocytes or myofibroblasts can be induced to be oval cell-like state through transitions under some circumstance, those transitional cell types as potential liver-resident progenitor cells play important roles in liver pathophysiology. In this review, we describe and update recent advances in the diversity and plasticity of hepatic progenitor cell and their niches and discuss evidence supporting their roles in liver homoeostasis, regeneration, fibrosis and cancers.     

Heterogeneity and plasticity of hepatocyte lineage cells

It is hypothesized that the liver has 3 levels of cells in the hepatic lineage that respond to injury or carcinogenesis: 1) the mature hepatocyte, which responds to partial hepatectomy (PH), to centrolobular injury, such as that induced by carbon tetrachloride (CCl(4)), and to dimethylnitrosamine (DEN) hepatocarcinogenesis; 2) the ductular "bipolar" progenitor cell, which responds to centrolobular injury when the proliferation of hepatocytes is inhibited, and to N-2-acetylaminofluorene (AAF) hepatocarcinogenesis; and 3) the putative periductular stem cell, which responds to periportal injury, such as induced by allyl alcohol and to choline-deficiency models of hepatocarcinogenesis. Hepatocytes are numerous, respond rapidly by 1 or 2 cell cycles, but can only produce other hepatocytes. The ductular progenitor cells are less numerous, may proliferate for longer times than hepatocytes, and are generally considered "bipolar," i.e., can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential, and may be multipotent, but their full potential has yet to be defined. Extrahepatic (bone marrow) origin of the periductular stem cells is supported by recent data showing that hepatocytes may express genetic markers of donor hematopoietic cells after bone marrow transplantation. Thus, experimental models of liver injury and of hepatocarcinogenesis may call forth a cellular response at different levels in the hepatic lineage (heterogeneity), and these cells have different potential to form cells of other types (plasticity).     

ABC转运蛋白在大鼠肝脏卵圆细胞中的表达

目的研究ABC转运蛋白基因家族的三个主要成员MDR1、MRP1和Bcrp1基因在大鼠肝脏卵圆细胞中的表达及意义。方法建立大鼠2-乙酰氨基芴／三分之二肝切除模型，两步胶原酶灌注结合Percoll密度梯度离心分离大鼠肝脏卯圆细胞和肝细胞，采用免疫组织化学染色检测大鼠肝脏组织中MDR1、MRP1、Bcrp1转运蛋白的表达；采用荧光定量PCR方法检测MDR1、MRP1和Bcrp1基因mRNA在卵圆细胞和肝细胞中的表达水平。结果免疫组织化学染色显示大鼠肝脏组织中MDR1表达位于门静脉区附近，呈放射状分布，Bcrp1表达定位在细胞膜上。大鼠肝脏卵圆细胞MDR1、MRP1和Bcrp1基因mRNA的表达水平分别是肝细胞的9倍、1．5倍和13．8倍。结论卵圆细胞表达高水平的ABC转运蛋白，后者参与卵圆细胞免受外源性化学物质损伤的自我保护机制。     

Stem and progenitor cell systems in liver development and regeneration

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell‐specific surface markers including delta‐like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13⁺CD133⁺ fraction of human iPS‐derived cells contained numerous hepatic progenitor‐like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.     

人胚胎肝干细胞的形态特点

目的:观察胚胎发育早期肝干细胞的形态特征、时空分布及分化,以探讨肝干细胞的生物学特征．方法:运用发育第3-12 wk人胚标本47例(其中 3．5 wk各8例;6-8 wk各5例,9-12 wk各2例),石蜡切片,连续切片,免疫组化染色,光镜下观察人胚肝及肝干细胞的发育及其AFP、c-Met和 CK19的时空表达．结果:发育第3 wk,肝芽形成,第4 wk形成肝索,第5 wk出现原始肝血窦．第3-5 wk人胚肝芽和肝索细胞排列紧密,较小,形态不规则,核圆形或卵圆形,核质比例大,核深染,胞质颜色较淡,偏蓝色,显示出幼稚细胞的形态学特征,并呈甲胎蛋白(α-Fetoprotein,AFP)、c-Met 阳性反应．第6 wk,肝索内出现了体积大、核大、淡染的细胞,呈AFP、c-Met阴性反应．随胚龄增加,这类细胞数量增加．10-12 wk． AFP、c-Met阳性细胞主要分布于汇管区周围． CK19阳性反应在7 wk时开始出现于一些与 AFP、c-Met阳性反应的细胞形态类似的肝索细胞中．10-11 wk时,CK19阳性反应主要位于汇管区附近的肝索细胞、胆管板细胞及胆管上皮细胞,12 wk时,CK19阳性信号仅见于胆管板和胆管上皮细胞．此时所有的胆管板细胞及胆管上皮细胞均呈AFP、c-Met和CK19阳性．结论:人胚发育3-5 wk肝实质由肝干细胞组成,其表型为AFP /c-Met ．6 wk,肝干细胞开始向肝细胞系分化,7 wk向胆管系分化,10-12 wk,肝干细胞主要局限于汇管区周围的肝索, 与成年肝中卵圆细胞(成年肝干细胞)的分布一致．AFP ／c-Met／ CK19 细胞可能为胆管祖细胞．     

Adipose‐derived stem cells: A candidate for liver regeneration

The scarcity of donor livers and the impracticality of hepatocyte transplantation represent the biggest obstacles for the treatment of liver failure. Adipose‐derived stem cells, with their ability to differentiate into the hepatic lineage, provide a reliable alternative cell source with clear ethical and practical advantages. Moreover, adipose‐derived stem cells can effectively repair liver damage by the dominant indirect pattern and increase the number of hepatocytes by the secondary direct pattern. In recent years, the development of the indirect pattern, which mainly includes immunomodulatory and trophic effects, has become a hot topic in the field of cell engineering. Therefore, adipose‐derived stem cells are considered to be ideal therapeutic stem cells for human liver regeneration. In this article, we reviewed the advantages of adipose‐derived stem cells in liver regeneration, and explore their underlying mechanisms.     

Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure

Background and Aim:  Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT-MSC), known as adipose-derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo .
Methods:  In vitro generated ASC-derived hepatocytes were checked for hepatocyte-specific markers and functions. Afterwards, they were transplanted into nude mice with liver injury. Twenty-four hours after transplantation, biochemical parameters were evaluated in blood serum.
Results:  We have shown here that ASC can be differentiated into hepatocytes within 13 days and can reach the functional properties of primary human hepatocytes. After transplantation into mice with acute liver failure, ASC-derived hepatocytes can restore such liver functions as ammonia and purine metabolism. Markers of liver injury, alanine aminotransferase, aspartate aminotransferase, as well as ammonia, were decreased after ASC-derived hepatocyte transplantation.
Conclusions:  Our data highlight the properties of ASC as having a special affinity for hepatocyte differentiation in vitro and liver regeneration in vivo . Thus, ASC may be a superior choice for the establishment of a therapy for injured liver.     

Expression of liver‐specific markers in naïve adipose‐derived mesenchymal stem cells

Background: Increasing evidence suggests that adipose tissue contains mesenchymal stem cells (MSC) that possess the ability to transdifferentiate into other cell types including hepatocytes, similar to bone marrow‐derived stem cells. The existence of precommitted cells in the MSC population may explain transdifferentiation. Aims: Our aim was to identify a population of putative hepatocyte‐like precursor cells in human adipose tissue. Methods: We analysed the ‘basal’ hepatic potential of undifferentiated, naïve human adipose‐derived mesenchymal stem cells (hADMSC). hADMSC were isolated from human adipose tissue and characterized for cell surface markers and for liver‐specific gene expression. Results: The isolated undifferentiated naïve hADMSCs expressed MSC surface markers. They also expressed α‐fetoprotein, CK18, CK19 and HNF4, which are known as early liver expressing genes. Interestingly, the undifferentiated naïve hADMSC were also positive for albumin, G‐6‐P and α‐1‐antitrypsin (AAT), which are all known to be predominantly expressed in adult liver cells. These cells acquired a hepatocyte‐specific phenotype and function upon treatment with a differentiation medium, resulting in the upregulation of albumin, G‐6‐P and AAT. Moreover, urea production, glycogen storage ability and cellular uptake of indocyanine green, which were absent in the basal state, were evident in the treated cells. Conclusions: Our findings suggest the presence of cells with hepatocyte‐like properties that are isolated from human adipose tissue and that can readily acquire hepatocyte‐like functions. Adipose tissue could thus be an exciting alternative means for repopulating the liver after various injuries, and might serve as a source for the transplantation of liver cells.     

Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.     

Multipotent mesenchymal stromal cells: A promising strategy to manage alcoholic liver disease

Chronic alcohol consumption is a major cause of liver disease. The term alcoholic liver disease (ALD) refers to a spectrum of mild to severe disorders including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. With limited therapeutic options, stem cell therapy offers significant potential for these patients. In this article, we review the pathophysiologic features of ALD and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), based on their potential to differentiate into hepatocytes, their immunomodulatory properties, their potential to promote residual hepatocyte regeneration, and their capacity to inhibit hepatic stellate cells. The perfect match between ALD pathogenesis and MSC therapeutic mechanisms, together with encouraging, available preclinical data, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage ALD onset and progression.     

Hepatic progenitor cells in liver regeneration: current advances and clinical perspectives

When there is a massive loss of hepatocytes and/or an inhibition in the proliferative capacity of the mature hepatocytes, activation of a dormant cell population of resident hepatic progenitor cells (HPCs) occurs. Depending on the type of liver damage HPCs generate new hepatocytes and biliary cells to repopulate the liver placing them as potential candidates for cell therapy in human liver failure. Liver injury specific mechanisms through which HPCs differentiate towards mature epithelial cell types are recently become understood. Such new insights will enable us not only to direct HPCs behaviour for therapeutic purposes, but also to develop clinically feasible methods for in vivo differentiation of other stem cell types towards functional hepatocytes. This review aimed to provide the current improved knowledge of the role of HPCs niche and its signals in directing the behaviour and fate of HPCs and to translate this basic knowledge of HPCs activation/differentiation into its clinical applications.     

Autologous Bone Marrow–Derived Cells in the Treatment of Liver Disease Patients

Liver transplantation is universally accepted as a “cure” procedure, and yet is not universally applicable for the treatment of end-stage liver diseases (ESLD) because of the shortage of donors, operative complications, risk of rejection, and high cost. Bioartificial liver device is an option to temporarily improve the liver function and to bridge the patients to liver transplantation. However, bioartificial liver device has many problems in clinical application, such as hepatocyte allograft rejection and maintenance of hepatocyte viability and function. Another therapeutic option is stem cell transplantation. There are two broad types of stem cells: Embryonic stem cells and adult stem cells. The latter are sourced from bone marrow (BM), adipose tissue, and blood. This review will concentrate on BM-derived cells. BM-derived cell transplantation, although not ideal, is theoretically an optimal modality for the treatment of ESLD. Autologous BM-derived cells have no graft rejection, have the capability of regeneration and self-renewal, and are multipotent stem cells that can differentiate into a variety of cell types which include hepatocytes. The pathway from BM-derived cell to hepatocyte is well documented. The present review summarizes the delivery routes of BM-derived cells to the liver, the evidences of engraftment of BM-derived cells in the liver, and the possible mechanisms of BM-derived cells in liver repair and regeneration, and finally, updates the clinical applications.     

Stem cells with decellularized liver scaffolds in liver regeneration and their potential clinical applications

End‐stage hepatic failure is a potentially life‐threatening condition for which orthotopic liver transplantation (OLT) is the only effective treatment. However, a shortage of available donor organs for transplantation each year results in the death of many patients waiting for liver transplantation. Cell‐based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation. However, primary hepatocyte transplantation has rarely produced therapeutic effects because mature hepatocytes cannot be effectively expanded in vitro, and the availability of hepatocytes is often limited by shortages of donor organs. Decellularization is an attractive technique for scaffold preparation in stem cell‐based liver engineering, as the resulting material can potentially retain the liver architecture, native vessel network and specific extracellular matrix (ECM). Thus, the reconstruction of functional and practical liver tissue using decellularized scaffolds becomes possible. This review focuses on the current understanding of liver tissue engineering, whole‐organ liver decellularization techniques, cell sources for recellularization and potential clinical applications and challenges.     

肝衰竭的细胞治疗:距离临床有多远?

肝衰竭患者因发病急、病死率高常须肝移植.除此之外,可能替代肝移植的细胞治疗是目前研究的热点.本文概述了近年来肝细胞移植、造血干细胞移植、间充质干细胞移植的基础研究进展和临床应用现况,及肝前体细胞和诱导性多能干细胞的特点和临床应用前景.