Progress in stem cell biology in regenerative medicine for liver disease

Regenerative medicine using stem cells has attracted much attention, since stem cells are responsible for highly proliferative activity and multipotential ability of differentiation. Induced pluripotent stem cells and embryonic stem cells or the adult stem cells such as bone marrow-derived stem cells and adipose tissue-derived stem cells have been expected as a cell source of regenerative medicine. Since differentiating methods of human stem cells into the defined lineage of cells remains to be developed, we focus on the differentiating strategies of pluripotent stem cells and mesenchymal stem cells into liver lineage, especially on cytokine function and gene expression during hepatic differentiation. The survey of previously published papers discloses that the protocols that mimic the liver developmental process seem to be effective in obtaining functional hepatocytes. However, in order to develop hepatic regenerative medicine that is useful in a clinical setting, more effective and potent strategies that obtain mature hepatocytes are required.     

Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro

Most studies on stem cell transplantation therapy on myocardially infarcted animal models and phase-I human clinical trials have focused on the use of undifferentiated stem cells. There is a strong possibility that some degree of cardiomyogenic differentiation of stem cells in vitro prior to transplantation would result in higher engraftment efficiency, as well as enhanced myocardial regeneration and recovery of heart function. Additionally, this may also alleviate the probability of spontaneous differentiation of stem cells into undesired lineages and reduces the risk of teratoma formation, in the case of embryonic stem cells. The development of efficient protocols for directing the cardiomyogenic differentiation of stem cells in vitro will also provide a useful model for molecular studies and genetic manipulation. This review therefore critically examines the various techniques that could possibly be used to direct and control the cardiomyogenic differentiation of stem cells in vitro.     

Origin of hepatocellular carcinoma: role of stem cells

The question of whether hepatocellular carcinoma (HCC) arises from the differentiation block of stem cells or dedifferentiation of mature cells remains controversial. Recently, researchers suggested that HCC may originate from the transdifferentiation of bone marrow cells. Interestingly, there are four levels of cells in the hepatic stem cell lineage: bone marrow cells, hepato-pancreas stem cells, oval cells and hepatocytes. Hematopoietic stem cells and the liver are known to have a close relationship in early development. Bone marrow stem cells could differentiate into oval cells, which could differentiate into hepatocytes and duct cells. The development of pancreatic and liver buds in embryogenesis suggests the existence of a common progenitor cell to both the pancreas and liver. Cellular events during hepatocarcinogenesis illustrate that HCC may arise from cells at various stages of differentiation in the hepatic stem cell lineage.     

间充质干细胞在肝脏的归巢及分化机制

间充质干细胞可通过旁分泌营养机制和分化为肝实质细胞等修复肝组织,移植间充质干细胞将成为临床治疗肝病的一种重要方法。本文介绍了间充质干细胞在肝损伤条件下,归巢到损伤肝脏并分化为肝实质细胞的可能机制。     

Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes

Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs). We used AT-MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105(+) fraction of AT-MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105(+) AT-MSC-derived hepatocyte-like cells revealed several liver-specific markers and functions, such as albumin production, low-density lipoprotein uptake, and ammonia detoxification. More importantly, CD105(+) AT-MSC-derived hepatocyte-like cells, after transplantation into mice incorporated into the parenchyma of the liver. CONCLUSION: Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction.     

Alternative sources of hepatocytes for cell therapy

There is an urgent need to search for alternatives to whole organ transplantation. Several methods have been proposed. Among these strategies, cell transplantation is currently one of the most promising. To achieve this aim, in addition to highly differentiated adult hepatocytes, the use of stem cells is considered a highly attractive therapeutic method for the treatment of liver disease and for temporary support of hepatic function until a liver becomes available for organ transplantation. This strategy is based on the ability of stem cells to differentiate into different cellular types according to their environment. Therefore, stem cells could be an unlimited source of hepatic cells for transplantation and gene therapy. Bone marrow is considered the most promising source of adult stem cells, partly due to the versatility of the cells obtained in repairing damaged tissues of several lineages. Several different types of stem cells have been described in bone marrow: hematopoietic, mesenchymal, side population and multipotent adult stem cells. Bone marrow cells have been hypothesized as a third recruitment source in liver regeneration in addition to hepatocytes and endogenous liver stem cells. Consequently, attempts have been made to differentiate them into hepatic lineage for their subsequent use in hepatic cell therapy. The present article reviews the progress made in this field or research.     

Hepatic differentiation of amniotic epithelial cells

Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, na?ve hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. CONCLUSION: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation.     

间充质干细胞分化为肝样细胞的研究进展

肝炎后肝硬化等慢性进展性肝病、急性肝功能衰竭、肝脏代谢性疾病及肝脏恶性肿瘤等终末期肝病的发病率日益上升,美国贝塞斯达回忆宣布肝移植是目前治疗终末期肝病最有效的方法,但由于肝源有限、移植后的免疫排斥反应以及高额费用等限制了这种治疗方法的开展。近年来,随着对干细胞研究的深入,发现间充质干细胞(MSCs)具有向肝样细胞分化的潜能,且安全性、可行性及疗效均显示了较好的应用前景,为终末期肝病的治疗提供了新的途径。本文就MSCs不同组织来源分化特点、体内外分化为肝样细胞研究、分化后的肝样细胞的生物学特性及MSCs分化为肝样细胞的机制、MSCs应用的一些问题作一概述。     

Directed differentiation of human embryonic stem cells into functional hepatic cells

The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A1, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low-density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition, we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION: We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests.     

人胎肝干细胞的体外培养及诱导分化

为体外分离培养和诱导分化人胎肝干细胞 ,从原代分离培养人胎肝干细胞集落 ,免疫细胞化学鉴定细胞集落分子标志物的表达 ;在体外特定细胞因子作用下诱导干细胞定向分化为成熟肝脏细胞 ,对其生物学特性进行初步鉴定。结果 ,从人胎肝组织中成功分离表达AFP、Albumin、Cytokeratin等标志物的胎肝干细胞集落 ,在体外特定细胞因子作用下肝干细胞可定向分化为成熟肝脏细胞。因此人胎肝中同样存在具有干细胞特性的原始细胞 ,体外可定向分化为成熟肝细胞。人胎肝干细胞的分离培养对于生物型人工肝的制备及其深入研究奠定了良好的基础     

Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells

BACKGROUND AND OBJECTIVES: The aim of this study was to determine whether Hoechst effluxing side population cells isolated from murine liver represent hepatic stem cells, and to examine whether hepatic side population cells arise from bone marrow side population cells. DESIGN AND METHODS: Side population cells were isolated from murine liver by flow cytometry after Hoechst staining and injected directly into murine livers of animals pre-treated with the hepatotoxin 3,5 diethoxy carbonyl-1, 4-dihydrocollidine (DDC). Y-chromosome in situ hybridization was used to track donor cells in the livers. In addition, bone marrow side population cells were stably engrafted into the hematopoietic system of sublethally irradiated recipients and CD45 alleleic staining and Y-chromosome in situ hybridization were used to track side population cell progeny in the liver. RESULTS: In vitro, CD45pos and CD45neg hepatic SP cells gave rise to hematopoietic colonies and mixed colonies of hematopoietic and hepatic differentiation. After orthotopic liver cell transplantation, donor hepatic side population cells contributed to the regeneration of mature liver parenchyma and bile duct epithelium. After transplantation of bone marrow side population cells, both CD45pos and CD45neg hepatic side population cells were partially derived from donor stem cells and could be recruited to repair liver damage after treatment with DDC. INTERPRETATION AND CONCLUSIONS: These findings introduce hepatic side population cells as a facultative liver-regenerating population, reveal interchangeability of tissue stem cells at the level of the side population, and suggest that bone marrow-derived side population cells might be exploited for the repair of diseased or damaged liver.     

The future of stem cells in liver diseases

Preliminary experience with clinical hepatocyte transplantation during the past decade has provided proof of concept that cell therapy can be effective for the treatment of some liver diseases. Recent progress in cell biology resulting in the isolation and characterization of hepatic stem cells and progenitor cells further increased the expectation for a new approach to the treatment of genetic and chronic liver disease. Several potential sources have been identified of hepatic stem/ progenitor cells exhibiting both differentiation towards the hepatic lineage in vitro and hepatic parenchymal repopulation with liver-specific metabolic activity in liver-injured animal models. However, a few of these results proved to be poorly reproducible in different laboratories, and it was recognized that some initial optimistic conclusions were drawn from incorrect interpretation of experimental data or from insufficient knowledge of the mechanisms involved in tissue regeneration. Moreover, only modest results have emerged so far from ongoing clinical experience involving the use of putative stem cells in liver disease. There is much need for a joined effort to concentrate the resources on a specific cell population, in order to better characterize its function, to assess its safety and to develop better focused clinical trials. In conclusion, while the biological features of stem cells still justify the hope for future clinical applications, hepatic stem cell therapy has still a long way to go from bench to bedside.     

干细胞移植治疗失代偿期肝硬化的临床研究

干细胞是具有自我更新和分化能力的细胞,可分化为肝系细胞。研究表明,干细胞移植治疗肝硬化患者取得良好的疗效。本文对各种干细胞移植治疗肝硬化的研究现状予以阐述,为肝硬化患者提供新的治疗手段。     

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

INTRODUCTIONMost liver diseases lead to hepatocyte dysfunction with the possibility of eventual organ failure. The replacement of diseased hepatocytes and the stimulation of endogenous or exogenous regeneration by stem cells are the main aims of liver-dir…     

Adipose tissue-derived stem cells: hepatic plasticity

Currently, the only effective treatment for end-stage liver disease is liver transplantation. The number of patients on the waiting list increases considerably each year, giving rise to a wide imbalance between supply and demand for healthy livers. Knowledge of stem cells and their possible use have awakened great interest in the field of hepatology, these cells being one of the most promising short-term alternatives. Hepatic stem cell therapy consists of the implantation of healthy cells capable of performing the functions that damaged cells are unable to carry out. Recent observations indicate that several stem cells can differentiate into distinct cell lineages. Hepatic differentiation of adult stem cells from several origins has yielded highly promising results. Adipose tissue in adults contains a reservoir of stem cells that can be induced and differentiated into different types of cells, showing a high degree of plasticity. Because of its abundance and easy access, adipose tissue is a promising source of adult stem cells for hepatic stem cell therapy. The present article reviews the progress made in the differentiation of adult stem cells from adipose tissue into cells with hepatic phenotype. We also discuss the potential application of this technique as a therapy for temporary metabolic support in patients with end-stage liver failure awaiting whole organ transplantation, as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and as a treatment in patients with genetic metabolic defects in vital liver functions.