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。     

Interplay between Wnt2 and Wnt2bb controls multiple steps of early foregut-derived organ development

The vertebrate liver, pancreas and lung arise in close proximity from the multipotent foregut endoderm. Tissue-explant experiments uncovered instructive signals emanating from the neighbouring lateral plate mesoderm, directing the endoderm towards specific organ fates. This suggested that an intricate network of signals is required to control the specification and differentiation of each organ. Here, we show that sequential functions of Wnt2bb and Wnt2 control liver specification and proliferation in zebrafish. Their combined specific activities are essential for liver specification, as their loss of function causes liver agenesis. Conversely, excess wnt2bb or wnt2 induces ectopic liver tissue at the expense of pancreatic and anterior intestinal tissues, revealing the competence of intestinal endoderm to respond to hepatogenic signals. Epistasis experiments revealed that the receptor frizzled homolog 5 (fzd5) mediates part of the broader hepatic competence of the alimentary canal. fzd5 is required for early liver formation and interacts genetically with wnt2 as well as wnt2bb. In addition, lack of both ligands causes agenesis of the swim bladder, the structural homolog of the mammalian lung. Thus, tightly regulated spatiotemporal expression of wnt2bb, wnt2 and fzd5 is central to coordinating early liver, pancreas and swim bladder development from a multipotent foregut endoderm.     

Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm

A strain of axolotl, Ambystoma mexicanum, that carries the cardiac lethal or c gene presents an excellent model system in which to study inductive interactions during heart development. Embryos homozygous for gene c contain hearts that fail to beat and do not form sarcomeric myofibrils even though muscle proteins are present. Although they can survive for approximately three weeks, mutant embryos inevitably die due to lack of circulation. Embryonic axolotl hearts can be maintained easily in organ culture using only Holtfreter's solution as a culture medium. Mutant hearts can be induced to differentiate in vitro into functional cardiac muscle containing sarcomeric myofibrils by coculturing the mutant heart tube with anterior endoderm from a normal embryo. The induction of muscle differentiation can also be mediated through organ culture of mutant heart tubes in medium 'conditioned' by normal anterior endoderm. Ribonuclease was shown to abolish the ability of endoderm-conditioned medium to induce cardiac muscle differentiation. The addition of RNA extracted from normal early embryonic anterior endoderm to organ cultures of mutant hearts stimulated the differentiation of these tissues into contractile cardiac muscle containing well-organized sarcomeric myofibrils, while RNA extracted from early embryonic liver or neural tube did not induce either muscular contraction or myofibrillogenesis. Thus, RNA from anterior endoderm of normal embryos induces myofibrillogenesis and the development of contractile activity in mutant hearts, thereby correcting the genetic defect.     

Both Humoral Mesenchymal Factors and the Close Association between the Hepatic Endoderm and Mesenchyme can be Involved in Liver Formation of Mouse Embryos

Previous studies with tissue recombination experiments demonstrated that the splanchnic mesenchymes, including hepatic, pulmonary and stomach mesenchymes can support hepatocyte differentiation from the hepatic endoderm in 9.5-day mouse embryos. This phenomenon corresponds to the second hepatic induction. The present study was undertaken to determine whether direct cell-cell contacts between the hepatic endoderm and mesenchyme are required for hepatocyte differentiation, using transfilter experiments in which membrane filters with various pore sizes were inserted between the endoderm and the hepatocyte-inducing mesenchyme (the chick lung mesenchyme). Hepatocyte differentiation occurred even when the direct cell-cell contacts between the hepatic endoderm and the mesenchyme were absent, suggesting that humoral factors may work in this interaction. However, growth of hepatocytes was most prominent in the transfilter experiments with filters having pore sizes of 0.2 and 0.8 mum, which permitted mesenchymal cells or their cell processes to penetrate to the side of the endoderm. These results suggest that two types of tissue interactions, including humoral mesenchymal factors and very local tissue interactions such as direct cell-cell contacts, may be involved in the second step of hepatic induction.     

Functional,persistent, and extended liver to pancreas transdifferentiation

Pancreatic and duodenal homeobox gene-1 (PDX-1) regulates pancreas development during embryogenesis, whereas in the adult it controls beta-cell function. Here we analyze whether PDX-1 functions as a pancreatic differentiation factor and a bona fide master regulator when ectopically expressed in mature fully differentiated liver in vivo. By ectopic and transient PDX-1 expression in liver in vivo, using the first generation recombinant adenoviruses, we demonstrate that PDX-1 induces in liver a wide repertoire of both exocrine and endocrine pancreatic gene expression. Moreover, PDX-1 induces its own expression (auto-induction), which in turn may explain the long lasting nature of the "liver to pancreas" transdifferentiation. Insulin as well glucagon-producing cells are mainly located in the proximity of hepatic central veins, possibly allowing direct hormone release into the bloodstream, without affecting normal hepatic function. Importantly, we demonstrate that hepatic insulin production triggered by Ad-CMV-PDX-1 recombinant adenovirus administration is functional and prevents streptozotocin-induced hyperglycemia in Balb/c mice even 8 months after the initial treatment. We conclude that PDX-1 plays an important instructive role in pancreas differentiation, not only from primitive gut endoderm but also from mature liver. Transconversion of liver to pancreas may serve as a novel approach for generating endocrine-pancreatic tissue that can replace malfunctioning beta-cells in diabetics.     

Liver specification and early morphogenesis

The classically defined induction of the liver from the endoderm, elicited by the cardiac mesoderm, has recently been discovered to involve signaling by fibroblast growth factors (FGFs). Multiple FGFs induce hepatic gene expression independent of an effect on growth. A subset of these FGFs cooperates with other factors to promote morphogenesis of the newly specified hepatocytes. Subsequent to the formation of the liver bud, distinct mesenchymal signals and hepatic response pathways stimulate further growth and differentiation of the hepatic parenchymal cells and prevent apoptosis. The initial stages of hepatogenesis are therefore beginning to be understood, and serve as a paradigm for the development of other tissues from the endoderm.     

Crucial role of vHNF1 in vertebrate hepatic specification

Mouse liver induction occurs via the acquisition of ventral endoderm competence to respond to inductive signals from adjacent mesoderm, followed by hepatic specification. Little is known about the regulatory circuit involved in these processes. Through the analysis of vHnf1 (Hnf1b)-deficient embryos, generated by tetraploid embryo complementation, we demonstrate that lack of vHNF1 leads to defective hepatic bud formation and abnormal gut regionalization. Thickening of the ventral hepatic endoderm and expression of known hepatic genes do not occur. At earlier stages, hepatic specification of vHnf1(-/-) ventral endoderm is disrupted. More importantly, mutant ventral endoderm cultured in vitro loses its responsiveness to inductive FGF signals and fails to induce the hepatic-specification genes albumin and transthyretin. Analysis of liver induction in zebrafish indicates a conserved role of vHNF1 in vertebrates. Our results reveal the crucial role of vHNF1 at the earliest steps of liver induction: the acquisition of endoderm competence and the hepatic specification.     

Embryonic stem cells: hepatic differentiation and regenerative medicine for the treatment of liver disease

Hepatocyte transplantation is considered a potential treatment for liver diseases and a bridge for patients awaiting liver transplantation, but its application has been hampered by a limited supply of hepatocytes. Embryonic stem (ES) cells established from early mouse and human embryos are pluripotent, and proliferate indefinitely in an undifferentiated state in vitro. Since differentiation from ES cells seems to recapitulate early embryonic development, if hepatocytes could be efficiently generated in vitro, ES cells might become a source of transplantable hepatocytes for cell replacement therapy. Hepatocytes have been generated from ES cells in vitro, and the hepatocytes differentiated from ES cells have been found to express many hepatocyte-related genes and perform hepatic functions. However, it remains unclear whether the hepatocytes differentiated from ES cells are derived from definitive endoderm or primitive endoderm. Because visceral endoderm, which expresses many hepatocyte-related genes, is derived from primitive endoderm and is fated to form extraembryonic yolk sac tissues, not to form hepatocytes, ES cells must be directed to a definitive endoderm lineage in vitro. This article discusses the differentiation of ES cells into hepatocytes in vitro in comparison with early embryogenesis, and describes the efficacy of ES cell-derived hepatocyte transplantation.     

Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation

The liver and pancreas arise from a common multipotent population of endoderm cells and share many aspects of their early development. Yet each tissue originates from multiple spatial domains of the endoderm, under the influence of different genes and inductive cues, and obtains different regenerative capacities. Emerging genetic evidence is illuminating the ability of newly specified hepatic and pancreatic progenitors to reverse their course and develop into gut progenitors. Understanding how tissue programming can be reversed and how intrinsic regenerative capacities are determined should facilitate the discovery of the basis of cellular plasticity and aid in the targeted programming and growth of stem cells.     

Stepwise differentiation of human embryonic stem cells into early endoderm derivatives and their molecular characterization

Human embryonic stem cells have the potential to differentiate into all human cell types and therefore hold a great therapeutic promise. Differentiation into the embryonic endoderm and its derivatives is of special interest since it can provide a cure for severe widespread clinical conditions such as diabetes and hepatic failure. In this work we established a unique experimental outline that enables the study of early human endoderm development and can help improve and create new differentiation protocols. To this end we started with mesendoderm cells and separated them into early endoderm and mesoderm progenitor cells using CXCR4 and PDGFRA cell surface markers. We molecularly characterized the different lineages, and demonstrated the importance of the TGFβ pathway in definitive endoderm initiation. The endoderm progenitor cells were then purified creating an endodermal differentiation niche that is not affected by other cell populations. We followed the differentiation of these cells at different time points, and demonstrated an up regulation of genes indicative to differentiation into both foregut and hindgut. Surprisingly, upon continued culture, there was significant down regulation of the hepatic gene signature. This down regulation could be rescued with FGF2 treatment demonstrating its importance in hepatic cell maintenance. In conclusion, we suggest that isolating endoderm progenitor cells is crucial for the analysis of their fate, and enables the identification of factors involved in their differentiation and maintenance.     

Inducing the liver: Understanding the signals that promote murine liver budding

The endoderm emerges as an epithelial sheet that covers the surface of the developing murine embryo. This tissue will produce the entire gut tube as well as associated digestive and respiratory organs including the thyroid, thymus, lung, liver, and pancreas. The emergence of each endodermal organ occurs in a temporally distinct manner that is dependant upon reciprocal inductive interactions between the endoderm and the underlying mesoderm. The emergence of the hepatic endoderm, which occurs using a morphological process termed liver budding, initiates during early somitogenesis in the mouse at approximately 8.25 days post‐coitum (dpc). Explant and transplant studies performed in chicken and mouse have demonstrated that secreted signals from adjacent mesodermal tissues initiate the hepatic gene program from ventral‐fated endoderm. Here, we review the data in support of the roles of members of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and Wnt signaling pathways in liver budding and discover that little is known about the precise endogenous signals involved in the molecular and morphological induction of liver budding in the mouse. J. Cell. Physiol. 226: 1727–1731, 2011. © 2010 Wiley‐Liss, Inc.     

The POU domain protein spg (pou2/Oct4) is essential for endoderm formation in cooperation with the HMG domain protein casanova

The gastrulating vertebrate embryo develops three germlayers: ectoderm, mesoderm, and endoderm. Zebrafish endoderm differentiation starts with the activation of sox17 by casanova (cas). We report that spg (pou2/Oct4) is essential for endoderm formation. Embryos devoid of maternal and zygotic spg function (MZspg) lack endodermal precursors. Cell transplantations show that spg acts in early endodermal precursors, and cas mRNA-injection into MZspg embryos does not restore endoderm development. spg and cas together are both necessary and sufficient to activate endoderm development, and stimulate expression of a sox17 promoter-luciferase reporter. Endoderm and mesoderm derive from a common origin, mesendoderm. We propose that Spg and Cas commit mesendodermal precursors to an endodermal fate. The joint control of endoderm formation by spg and cas suggests that the endodermal germlayer may be a tissue unit with distinct genetic control, thus adding genetic support to the germlayer concept in metazoan development.     

Extracellular matrix bioengineering and systems biology approaches in liver disease

The extracellular matrix (ECM) in the liver as well as in many organs comprises a peripheral network linking numerous macromolecules typically classified into collagens, microfibrillar proteins, proteoglycans, chemokines, growth factors and glycoproteins. In addition to its role as an essential structural and physiological component, it plays a vital role in driving key cellular events such as cell adhesion, migration, proliferation, differentiation and survival. Any structural inherited or acquired defect and/or metabolic or pathologic alteration in the hepatic ECM may cause cellular and organ responses leading to the development or progression of liver disease. Therefore, the ECM molecules are key players in tissue engraftment and in the pathophysiology of liver disease. In this review we provide a snapshot on current efforts for understanding its role in physiological and non-physiological states, by describing how tissue engineering platforms can enhance in vitro and in vivo models of liver disease, by providing examples where bioengineered ECM can serve as systems biology approaches to study the ECM, and then by evaluating pathological protein regulatory networks in the liver using systems biology tools. These approaches hold great promise for future research.     

An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells

While particular combinations of mesodermal signals are known to induce distinct tissue-specific programs in the endoderm, there is little information about the response pathways within endoderm cells that control their specification. We have used signaling inhibitors on embryo tissue explants and whole-embryo cultures as well as genetic approaches to reveal part of an intracellular network by which FGF signaling helps induce hepatic genes and stabilize nascent hepatic cells within the endodermal epithelium. Specifically, we found that hepatic gene induction is elicited by an FGF/MAPK pathway. Although the PI3K pathway is activated in foregut endoderm cells, its inhibition does not block hepatic gene induction in explants; however, it does block tissue growth. We also found that at the onset of hepatogenesis, the FGF/MAPK and PI3K pathways do not crossregulate in the endoderm. The finding of separate pathways for endoderm tissue specification and growth provides insights for guiding cellular regeneration and stem cell differentiation.