Selective tropism of liver stem cells to hepatocellular carcinoma in vivo

AIM： To investigate the selective tropism of liver stem cells to hepatocellular carcinoma （HCC） in an animal model and its feasibility as a vector to deliver therapeutic genes for targeted therapy of HCC.
METHODS： WB-F344, a kind of rat liver stem cell, was infected with recombinant virus to establish a cell line with stable, high-level expressing enhanced green fluorescent protein （EGFP）. An animal model of HCC in Wistar rats was established by implanting HCC cells （CBRH7919） combined with an immunosuppressive drug. EGFP labeled liver stern cells were injected into caudal veins of the animals and distribution was observed at different time points after injection. SDF-1 and c-kit expression in non-tumor liver and tumor tissue were analysed by immunohistochemistry for the relationshiop between the expression and migration of liver stem cells. Furthermore, hepatic stern cells were injected via the portal vein, hepatic artery, caudal vein, or directly into the pericancerous liver tissue, respectively, and effects on migration, localization, and proliferation of the hepatic stern cells within the tumor tissue were observed and analyzed.
RESULTS： Recombinant adenovirus could deliver the EGFP gene to hepatic stem cells. A new stem cell line, named WB-EGFP, was established that stably expressed EGFP. WB-EGFP cells still showed selective tropism towards HCC and EGFP expression was stable in vivo. According to immunohistochemistry results, SDF-1 may not be related to the mechanisms of tropism of hepatic stem cells. Different application sites affected the distribution of liver stem cells. Injection via the portal vein was superior with regard to selective migration, localization, and proliferation of the hepatic stem cells within the tumor tissue.
CONCLUSION： Liver stem cells have the biological behavior of selective migration to HCC in vivo and they could localize and proliferate within HCC tissue stably expressing the target gene. Liver stem cells are a potential tool for a targeted gene therapy o     

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.     

Role of stem cells in repair of liver injury:Experimental and clinical benefit of transferred stem cells on liver failure

Although the liver has a high regenerative capacity,as a result of massive hepatocyte death,liver failure occurs. In addition to liver failure,for acute,chronic and hereditary diseases of the liver,cell transplantation therapies can stimulate regeneration or at least ensure sufficient function until liver transplantation can be performed. The lack of donor organs and the risks of rejection have prompted extensive experimental and clinical research in the field of cellular transplantation. Transplantation of cell lineages involved in liver regeneration,including mature hepatocytes,fetal hepatocytes,fetal liver progenitor cells,fetal stem cells,hepatic progenitor cells,hepatic stem cells,mesenchymal stem cells,hematopoietic stem cells,and peripheral blood and umbilical cord blood stem cells,have been found to be beneficial in the treatment of liver failure.In this article,the results of experimental and clinical cell transplantation trials for liver failure are reviewed,with an emphasis on regeneration.     

Role of BMSCs in liver regeneration and metastasis after hepatectomy

Hepatocellular carcinoma(HCC),which develops from liver cirrhosis,is highly prevalent worldwide and is a malignancy that leads to liver failure and systemic metastasis.While surgery is the preferred treatment for HCC,intervention and liver transplantation are also treatment options for end-stage liver disease.However,the success of partial hepatectomy and intervention is hindered by the decompensation of liver function.Conversely,liver transplantation is difficult to carry out due to its high cost and the lack of donor organs.Fortunately,research into bone-marrow stromal cells(BMSCs)has opened a new door in this field.BMSCs are a type of stem cell with powerful proliferative and differential potential that represent an attractive tool for the establishment of successful stem cell-based therapy for liver diseases.A number of different stromal cells contribute to the therapeutic effects exerted by BMSCs because BMSCs can differentiate into functional hepatic cells and can produce a series of growth factors and cytokines capable of suppressing inflammatory responses,reducing hepatocyte apoptosis,reversing liver fibrosis and enhancing hepatocyte functionality.Additionally,it has been shown that BMSCs can increase the apoptosis rate of cancer cells and inhibit tumor metastasis in some microenvironments.This review focuses on BMSCs and their possible applications in liver regeneration and metastasis after hepatectomy.     

Use of mesenchymal stem cells to treat liver fibrosis:Current situation and future prospects

Progressive liver fibrosis is a major health issue for which no effective treatment is available,leading to cirrhosis and orthotopic liver transplantation.However,organ shortage is a reality.Hence,there is an urgent need to find alternative therapeutic strategies.Cellbased therapy using mesenchymal stem cells(MSCs) may represent an attractive therapeutic option,based ontheir immunomodulatory properties,their potential to differentiate into hepatocytes,allowing the replacement of damaged hepatocytes,their potential to promote residual hepatocytes regeneration and their capacity to inhibit hepatic stellate cell activation or induce their apoptosis,particularly via paracrine mechanisms.The current review will highlight recent findings regarding the input of MSC-based therapy for the treatment of liver fibrosis,from in vitro studies to pre-clinical and clinical trials.Several studies have shown the ability of MSCs to reduce liver fibrosis and improve liver function.However,despite these promising results,some limitations need to be considered.Future prospects will also be discussed in this review.     

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.     

Rationale for the potential use of mesenchymal stromal cells in liver transplantation

Mesenchymal stromal cells(MSCs) are multipotent and self-renewing cells that reside essentially in the bone marrow as a non-hematopoietic cell population, but may also be isolated from the connective tissues of most organs. MSCs represent a heterogeneous population of adult, fibroblast-like cells characterized by their ability to differentiate into tissues of mesodermal lineages including adipocytes, chondrocytes and osteocytes. For several years now, MSCs have been evaluated for their in vivo and in vitro immunomodulatory and ‘tissue reconstruction’ properties, which could make them interesting in various clinical settings, and particularly in organ transplantation. This paper aims to review current knowledge on the properties of MSCs and their use in pre-clinical and clinical studies in solid organ transplantation, and particularly in the field of liver transplantation. The first available clinical data seem to show that MSCs are safe to use, at least in the medium-term, but more time is needed to evaluate the potential adverse effects of long-term use. Many issues must be resolved on the correct use of MSCs. Intensive in vitro and pre-clinical research are the keys to a better understanding of the way that MSCs act, and to eventually lead to clinical success.     

Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP-cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.     

Current issues and innovations in stem cell based therapy

It is well established that stem cells can differentiate into cell types of the organ in which these are transplanted.However,the process is very slow due to lack of understanding of signals important for their survival and differentiation,most optimal stem cells and their plasticity.Limitations and advantages of various cell subtypes will be described. The rate of stem cells mobilization and their survival in the ischemic environment are major obstacles in engraftment and differentiation of stem cells for meaningful repair of the infarcted myocardium. Manipulation of stem cells with ischemic preconditioning,combined gene and cell therapy together with simultaneous activation of diverse signaling pathways for massive stem cell mobilization & regeneration has significant impact on the repair process by stem cells.These and other difficulties encountered in efficient use of various stem cells have resulted in invention of induced pluripotent stem cells which could revolutionize the stem cell based therapy and their applications for understanding of human disease and drug screening in the near future. Reprogramming of adult cells into iPS cells without the use of viral vectors is a major challenge towards getting iPS cells without viral integration into cells.To meet this challenge we have repro-grammed skeletal myoblasts into iPS cells with high efficiency using epigenetic modifiers.Transplantation of iPS cells derived pure cardiac progenitors into infarcted myocardium led to extensive repopulation of scar area with fully developed myocytes without tumor formation and resulting in marked improvement in cardiac function.Reprogramming with pure chemical means will make therapeutic use of these cells more safer.Targeting the induced pluripotent stem cells towards cardiac progenitors and their application towards transplantation is a major step forward in enhancing the myocardial repair capacity by these cells.     

Role of sex steroid receptors in pathobiology of hepatocellular carcinoma

The striking gender disparity observed in the incidence of hepatocellutar carcinoma （HCC） suggests an important role of sex hormones in HCC pathogenesis. Though the studies began as early as in 1980s, the precise role of sex hormones and the significance of their receptors in HCC still remain poorly understood and perhaps contribute to current controversies about the potential use of hormonal therapy in HCC. A comprehensive review of the existing literature revealed several shortcomings associated with the studies on estrogen receptor （ER） and androgen receptor （AR） in normal liver and HCC. These shortcomings include the use of less sensitive receptor ligand binding assays and immunohistochemistry studies for ERα alone until 1996 when ERβ isoform was identified. The animal models of HCC utilized for studies were primarily based on chemical-induced hepatocarcinogenesis with less similarity to virus-induced HCC pathogenesis. However, recent in vitro studies in hepatoma cells provide newer insights for hormonal regulation of key cellular processes including interaction of ER and AR with viral proteins. In light of the above facts, there is an urgent need for a detailed investigation of sex hormones and their receptors in normal liver and HCC. In this review, we systematically present the information currently available on androgens, estrogens and their receptors in normal liver and HCC obtained from in vitro, in vivo experimental models and clinical studies. This information will direct future basic and clinical research to bridge the gap in knowledge to explore the therapeutic potential of hormonal therapy in HCC.     

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.     

Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol

Liver transplantation is the only definitive treatment for end-stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient-specific disease modeling. Here, we describe an efficient and rapid three-step protocol that is able to rapidly generate hepatocyte-like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC-derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte-like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low-density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte-like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. Conclusion: We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte-like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases.     

New horizons for stem cell therapy in liver disease

There is an increasing range of potential applications of stem cells in liver diseases, with many clinical studies already undertaken. We identify four of the main areas which we propose stem cell therapy could be a realistic aim for in the future: (1) to improve regeneration and reduce scarring in liver cirrhosis by modulating the liver's own regenerative processes, (2) to down-regulate immune mediated liver damage, (3) supplying hepatocyte-like cells (HLCs) derived from stem cells for use in extracorporeal bio-artificial liver machines, and (4) to use stem cell derived HLCs for cell transplantation to supplement or replace hepatocyte function.     

Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells

With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpresed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.     

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。     

Peripheral blood monocytes from patients with HBV related decompensated liver cirrhosis can differentiate into functional hepatocytes

Peripheral blood monocytes (PBMCs) have the potential to differentiate into various progenitor cells. Here we have investigated the differentiation potential of PBMCs derived from patients with HBV related decompensated liver cirrhosis into hepatocyte-like cells. In our clinical trial, the PBMCs from 2 patients were mobilized by the recombinant human granulocyte colony stimulating factor, followed by leukapheresis and transplantation of PBMCs. PBMCs, induced by recombinant human hepatocyte growth factors, were identified by the expression of hepatocyte markers and specific biological functions with biochemical assays in vitro. Patients showed a lasting clinical amelioration for more than one year after transplantation, and hepatocyte-like cells were identified by expressing liver specific genes, synthesizing albumin, urea, aspirate transaminase, and glycogen, which were all similar to the human normal hepatic cell line QZG. Our results clearly demonstrated that mobilized PBMCs from patients with HBV related decompensated liver cirrhosis could differentiate into functional hepatocyte-like cells, indicating the possibility of autologous cell transplantation for treating patients with HBV related decompensated liver cirrhosis.     

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.     

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.     

骨髓来源干细胞门静脉移植治疗肝纤维化的实验研究

目的研究骨髓间充质千细胞（MSCs）诱导分化的肝样细胞对大鼠肝纤维化的治疗作用。方法在体外分离培养MSCs中加入细胞因子,诱导其分化成为肝样细胞,通过门静脉移植入肝纤维化模型大鼠体内后,观察病理组织学变化、评价纤维化分期并监测肝功能的变化。结果肝纤维化模型大鼠经门静脉移植诱导肝样细胞4周后,其肝脏纤维化明显改善。结论肝样细胞移植对肝纤维化模型大鼠的纤维化有明显的治疗作用。     

Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers

AIMS: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo. METHODS: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration. CONCLUSION: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.