Cell lineage analysis during liver development using the spf(ash)-heterozygous mouse

Biliary epithelial cells differentiate from periportal hepatoblasts during fetal mouse liver development. It remains to be determined whether each hepatoblast is equivalent for differentiation into hepatocytes and biliary epithelial cells in normal liver development. To resolve this question, the mosaic pattern of ornithine transcarbamylase (OTC) expression was analyzed in the hepatoblast population of spf(ash) (sparse-fur with abnormal skin and hair)-heterozygous fetal mouse livers, in which random inactivation of either the X chromosome carrying the spf(ash) gene (causing OTC deficiency) or its wild-type gene occurs. Aggregates (patches) of OTC-positive hepatoblasts showed very complex patterns, and their shapes and size distributions were similar in sections from periportal regions and nonperiportal regions of the fetal liver in which bile duct differentiation by periportal hepatoblasts occurred. Average sizes of periportal patches were larger than those of nonperiportal patches because of the presence of more hemopoietic cells in the latter region. The OTC mosaicism in periportal bile duct progenitors and hepatoblast islands of other liver parenchyma was also similar. These results suggest that the growth patterns of hepatoblasts are similar in both periportal and nonperiportal regions. Isolated three-dimensional patches comprising hepatoblasts giving rise to only biliary epithelial cells or hepatoblasts giving rise to both hepatocytes and biliary epithelial cells were observed in periportal regions. In nonperiportal regions, patches consisting of hepatoblasts differentiating into hepatocytes were also seen. Thus, it is likely that there are three lineages for the developmental fates of hepatoblasts: hepatoblasts giving rise to only biliary epithelial cells, hepatoblasts giving rise to only hepatocytes, and hepatoblasts giving rise to both of them.     

FGF signaling segregates biliary cell-lineage from chick hepatoblasts cooperatively with BMP4 and ECM components in vitro.

Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.     

Developmental Changes of Cell Adhesion Molecule Expression in the Fetal Mouse Liver

Developmental changes of cell adhesion molecule expression, especially in nonparenchymal cells, have hardly ever been analyzed in the murine liver. The present study was undertaken to immunohistochemically examine the expression of NCAM, ICAM, VCAM, and N‐cadherin during mouse liver development and in fetal liver cell cultures. NCAM was transiently expressed in mesenchymal cells of the septum transversum and sinusoidal cells in liver development. In vitro studies demonstrated that desmin‐positive stellate cells expressed this cell adhesion molecule. NCAM expression in periportal biliary epithelial cells and connective tissue cells also coincided well with bile duct remodeling processes in the perinatal periods. Expression of ICAM and VCAM was transiently restricted to hepatoblasts, hepatocytes and hemopoietic cells in fetal stages. N‐cadherin was expressed not only in hepatoblasts and hepatocytes, but also in nonparenchymal cells such as endothelial cells, stellate cells and connective tissue cells, however the expression was weak. These results suggest that each cell adhesion molecule may play an important role during development in hepatic histogenesis, including hepatoblast/hepatocyte‐stellate cell interactions, hemopoiesis, and bile duct morphogenesis. Anat Rec 293:1698–1710, 2010. © 2010 Wiley‐Liss, Inc.     

Human hepatoblast phenotype maintained by hyaluronan hydrogels

Human hepatoblasts and hepatic stem cells, pluripotent hepatic progenitors that give rise to hepatocytes and biliary cells, were isolated from fetal livers and found to express hyaluronan receptors (CD44) in both the freshly isolated cells and after culture. This implicates an in vivo connection to hyaluronan (HA), an embryonic matrix component, as a candidate 3-dimensional (3-D) scaffold for hepatic progenitor cell expansion and/or differentiation. To assess HAs as scaffolds, hepatoblasts and hepatic stem cells were seeded into HA hydrogels with a serum-free, hormonally defined medium tailored for expansion of hepatic progenitors. Cell aggregates formed within the HA hydrogels and remained viable, proliferative, and demonstrated a stable phenotype intermediate between that of hepatic stem cells and hepatoblasts throughout more than 4 weeks of culturing, with little evidence of lineage restriction towards either hepatocytic or biliary pathways. The phenotype consisted of stable co-expression of both hepatocytic and biliary markers such as biliary-specific cytokeratin, CK19, low levels of expression of albumin, and urea synthesis. HA hydrogels are ideal as 3-D scaffolds for pluripotent hepatic progenitors and should be useful for generating cells to be used in bioartificial livers or tissue engineered liver grafts.     

Immunohistochemical analysis of development of desmin-positive hepatic stellate cells in mouse liver

Development of desmin-positive hepatic stellate cells was studied in mice using double immunofluorescent techniques and in vitro cultures with special attention given to their cell lineages. Several studies recently reported on the presence of cells that are immunologically reactive with both antidesmin and anticytokeratin antibodies in young fetal rat livers, and suggested the possibility that these cells give rise to hepatocytes and hepatic stellate cells. At early stages of mouse liver development, stellate cells with desmin-positive filaments were scattered in the liver parenchyma. However, the stellate cells definitely differed from hepatoblasts and hepatocytes in terms of their morphology and expression of desmin and hepatoblast and hepatocyte-specific E-cadherin in the liver. Fetal hepatoblasts and hepatocytes did not react with antidesmin antibodies, nor did desmin-positive stellate cells express E-cadherin in vivo and in vitro. Thus it is likely that desmin-positive stellate cells and hepatoblasts belong to different cell lineages. In the fetal liver, the desmin-positive stellate cells surrounded blood vessels, and extended their processes to haematopoietic cells and megakaryocytes. Many, but not all, hepatoblasts and hepatocytes were observed to be associated with the stellate cells. At fetal stages, cellular processes positive for desmin in the stellate cells were also thick compared with those in the adult liver, in which desmin-positive stellate cells lay in Disse's space and were closely associated with all hepatocytes. These developmental changes in the geography of desmin-positive cells in the liver parenchyma and their morphology may be associated with their maturation and interactions with other cell types.     

Ultrastructural studies of hepatoblast junctions and liver hematopoiesis of the mouse embryo]

To clarify the morphological changes in hepatoblast connections during the development of fetal liver hematopoiesis, ICR mouse livers of 11 to 19 days of gestation were studied by means of three-dimensional reconstruction, immunohistochemistry, electron microscopy and freeze fracture replica method. Embryonic liver weight showed rapid increase until 19 days of gestation, and an initial steep increase, due to hematopoietic development, was observed at 13 to 15 days of gestation. Hepatoblast volume appeared to be constant until 13 days of gestation, and, thereafter, showed a gradual increase. An 11-day primitive hepatic cord contained a few immature hematopoietic cells among hepatoblasts, and the hepatoblasts made contact with one another by short cytoplasmic projections. The area of the contact surface had a diameter of 4-5 microns, where E-cadherin-mediated adherens junctions were found. At 12-13 days of gestation, hepatoblasts surrounded large ellipsoidal hematopoietic foci, with long cytoplasmic projections. In addition to the adherens junctions, small desmosomes appeared to bind hepatoblasts together, and biliary canaliculi could be recognized between hepatoblasts. At peak stage of liver hematopoiesis at 14 days of gestation, both tight junctions and gap junctions appeared around the biliary canaliculi, and four types of specialized junctions, i.e., adherens junctions, desmosomes, tight junctions and gap junctions, appeared to be fully developed. After 15 days of gestation, hepatocyte volume showed rapid increase, and the surface areas between adjacent hepatocytes were markedly enlarged. As a result, the involuted hematopoietic foci were forced to move from interhepatocytic spaces to perisinusoidal space at the end of the intrauterine life.     

Phthalazinone Pyrazole Enhances the Hepatic Functions of Human Embryonic Stem Cell-Derived Hepatocyte-Like Cells via Suppression of the Epithelial-Mesenchymal Transition

During liver development, nonpolarized hepatic progenitor cells differentiate into mature hepatocytes with distinct polarity. This polarity is essential for maintaining the intrinsic properties of hepatocytes. The balance between the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) plays a decisive role in differentiation of polarized hepatocytes. In this study, we found that phthalazinone pyrazole (PP), a selective inhibitor of Aurora-A kinase (Aurora-A), suppressed the EMT during the differentiation of hepatocyte-like cells (HLCs) from human embryonic stem cells. The differentiated HLCs treated with PP at the hepatoblast stage showed enhanced hepatic morphology and functions, particularly with regard to the expression of drug metabolizing enzymes. Moreover, we found that these effects were mediated though suppression of the AKT pathway, which is involved in induction of the EMT, and upregulation of hepatocyte nuclear factor 4α expression rather than Aurora-A inhibition. In conclusion, these findings provided insights into the regulatory role of the EMT on in vitro hepatic maturation, suggesting that inhibition of the EMT may drive transformation of hepatoblast cells into mature and polarized HLCs.     

Selective bipotential differentiation of mouse embryonic hepatoblasts in vitro.

A line of hepatic endoderm cells, hepatoblast cell line 3 (HBC-3), was derived from the liver diverticulum of the mouse on day 9.5 of gestation by culture on a mitomycin C treated STON+ feeder layer in a hepatoblast culture medium consisting of Dulbecco's modified Eagle's medium, nonessential amino acids, fetal calf serum, and beta-mercaptoethanol. This line, HBC-3, stains positively for alpha-fetoprotein, albumin, and cytokeratin 14 (CK-14), protein markers expressed by the embryonic liver diverticulum, indicating that HBC-3 cells retain an undifferentiated hepatoblast phenotype. HBC-3 cells acquire hepatocyte-like ultrastructural characteristics, including bile canaliculi, peroxisomes, and glycogen granules, when maintained in culture for 3 weeks without passage. Treatment with dimethylsulfoxide or sodium butyrate induces a rapid hepatocytic differentiation. The cells cease to express alpha-fetoprotein and CK-14, maintain albumin expression, and become positive for glucose-6-phosphatase activity (a profile consistent with differentiation along the hepatocyte lineage). On Matrigel, HBC-3 cells form elaborate ductular structures, which are positive for gamma-glutamyl transpeptidase and CK-14 and CK-19 and do not express detectable amounts of albumin, a phenotypic change consistent with differentiation along the bile ductular lineage. Thus, HBC-3 cells behave in culture as bipotential hepatoblasts and provide a model system to identify factors that regulate bipotential differentiation in the liver.     

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes of the fetal mouse in vitro

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes was analysed in vitro. When fetal mouse liver fragments containing immature hepatocytes but no bile ducts were cultured organotypically, the immature hepatocytes differentiated into large hepatocytes. Some of these expressed bile duct markers such as cytokeratin and Dolichos biflorus agglutinin-binding sites, though only to a small extent, and typical intrahepatic bile duct cells failed to differentiate. Dexamethasone stimulated immature hepatocytes to differentiate into both mature hepatocyte and biliary epithelial cell lineages. Especially in the liver fragments cultured on Matrigel, dexamethasone stimulated the expression of bile duct markers (such as cytokeratin and binding sites for two types of lectin) in the immature hepatocytes. These results support the idea that immature hepatocytes can differentiate into both mature hepatocytes and biliary epithelial cells during normal development of the mouse liver, and suggest that glucocorticoids stimulate both these differentiation pathways. It also seems that basal laminar components may play a role in bile duct differentiation.     

Progenitor cell characterization and location in the developing human liver

Tissue-derived stem cells may offer future liver disease therapies. The developing human liver provides an excellent model to examine normal hepatic progenitor cell maturation, but candidate populations are poorly characterized. We sought to identify putative progenitor phenotypes in first-trimester human liver, by characterizing the architectural relationship between developing epithelial, mesenchymal, and hematopoietic lineages. Bipotential hepatoblasts were identified by co-expression of hepatocytic (cytokeratin 18, albumin) and biliary(cytokeratin 19) specific markers and epithelial-specific E-cadherin. Restriction of dlk/pref-1 expression to hepatoblasts identifies this as a novel human marker allowing for hepatoblast sorting for in vitro analysis. Furthermore, the liver stem cell and haematopoietic marker Thy-1 was co-expressed with markers of hematopoietic (CD34) and mesenchymal (vimentin) lineage restriction on portal vein endothelium. Therefore, this structure may constitute a novel progenitor compartment with hemangioblast-like properties.     

Transforming growth factor-beta induces loss of epithelial character and smooth muscle cell differentiation in epicardial cells.

During embryogenesis, epicardial cells undergo epithelial-mesenchymal transformation (EMT), invade the myocardium, and differentiate into components of the coronary vasculature, including smooth muscle cells. We tested the hypothesis that transforming growth factor-beta (TGFbeta) stimulates EMT and smooth muscle differentiation of epicardial cells. In epicardial explants, TGFbeta1 and TGFbeta2 induce loss of epithelial morphology, cytokeratin, and membrane-associated Zonula Occludens-1 and increase the smooth muscle markers calponin and caldesmon. Inhibition of activin receptor-like kinase (ALK) 5 blocks these effects, whereas constitutively active (ca) ALK5 increases cell invasion by 42%. Overexpression of Smad 3 did not mimic the effects of caALK5. Inhibition of p160 rho kinase or p38 MAP kinase prevented the loss of epithelial morphology in response to TGFbeta, whereas only inhibition of p160 rho kinase blocked TGFbeta-stimulated caldesmon expression. These data demonstrate that TGFbeta stimulates loss of epithelial character and smooth muscle differentiation in epicardial cells by means of a mechanism that requires ALK5 and p160 rho kinase.     

Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFbeta.

Cells derived from the epicardium are required for coronary vessel development. Transforming growth factor beta (TGFbeta) induces loss of epithelial character and smooth muscle differentiation in chick epicardial cells. Here, we show that epicardial explants from embryonic day (E) 11.5 mouse embryos incubated with TGFbeta1 or TGFbeta2 lose epithelial character and undergo smooth muscle differentiation. To further study TGFbeta Signaling, we generated immortalized mouse epicardial cells. Cells from E10.5, 11.5, and 13.5 formed tightly packed epithelium and expressed the epicardial marker Wilm's tumor 1 (WT1). TGFbeta induced the loss of zonula occludens-1 (ZO-1) and the appearance of SM22alpha and calponin consistent with smooth muscle differentiation. Inhibition of activin receptor-like kinase (ALK) 5 or p160 rho kinase activity prevented the effects of TGFbeta while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data demonstrate that TGFbeta induces epicardial cell differentiation and that immortalized epicardial cells provide a suitable model for differentiation.     

Contribution of mesothelium-derived cells to liver sinusoids in avian embryos.

The developing liver is vascularized through a complex process of vasculogenesis that leads to the differentiation of the sinusoids. The main structural elements of the sinusoidal wall are endothelial and stellate (Ito) cells. We have studied the differentiation of the hepatic sinusoids in avian embryos through confocal colocalization of differentiation markers, in ovo direct labeling of the liver mesothelium, induced invasion of the developing chick liver by quail proepicardial cells, and in vitro culture of chimeric aggregates. Our results show that liver mesothelial cells give rise to mesenchymal cells which intermingle between the growing hepatoblast cords and become incorporated to the sinusoidal wall, contributing to both endothelial and stellate cell populations. We have also shown that the proepicardium, a mesothelial tissue anatomically continuous with liver mesothelium, is able to form sinusoid-like vessels into the hepatic primordium as well as in cultured aggregates of hepatoblasts. Thus, both intrinsic or extrinsic mesothelium-derived cells have the developmental potential to contribute to the establishment of liver sinusoids.     

Immunomagnetic exclusion of E-cadherin-positive hepatoblasts in fetal mouse liver cell cultures impairs morphogenesis and gene expression of sinusoidal endothelial cells

Previous studies have shown that various cell-cell interactions between hepatoblasts and nonparenchymal cells, including sinusoidal endothelial cells and stellate cells, are indispensable for the development of fetal murine hepatic architecture. The present study was undertaken to determine the effects of hepatoblasts on the sinusoidal structural formation using a culture system of fetal mouse livers. Primitive sinusoidal structures extensively developed in fetal livers, and were composed of LYVE-1- and PECAM-1-positive endothelial cells, desmin-positive stellate cells and F4/80-positive macrophages. When fetal liver cells at 12.5 days of gestation were cultured in vitro, hepatoblasts spread on glass slides and gave rise to hepatocytes on day 5. Desmin-positive stellate cells also spread on the glass slides. PECAM-1-positive endothelial cells became slender and developed into anastomosing capillary networks. When fetal liver cells were cultured without hepatoblasts, which were excluded by an immunomagnetic method using anti-E-cadherin antibodies, endothelial cells had impaired growth and capillary formation. These results demonstrated that capillary formation of endothelial cells was induced by the presence of hepatoblasts. VEGF and the conditioned medium containing humoral factors produced by hepatoblasts/hepatocytes did not induce capillary formation of endothelial cells in cultures of nonparenchymal cells, although they significantly increased the number of endothelial cells on the glass slides. The presence of hepatoblasts also significantly stimulated expression of CD32b mRNA, which is a sinusoidal endothelial marker. Hepatoblasts may work as a positive stimulator of sinusoid morphogenesis and maturation in liver development, in which a signal other than VEGF may play a decisive role, together with VEGF.     

Truncating mutations in the ACVR2 gene attenuates activin signaling in prostate cancer cells

Activins are classified as members of the TGFbeta superfamily of signaling molecules and both activin and TGFbeta ligands signal through structurally and functionally related serine/threonine kinase receptors. Defects in these signaling pathways have been associated with the initiation and progression of the cancer phenotype. Inactivating mutations in the TGFbeta type II receptor gene, TGFbetaR2, have been identified in a variety of tumors and cell lines, particularly those with microsatellite instability (MSI). More recently, mutations in the activin type II receptor gene, ACVR2, were identified in colon and pancreatic cell lines and tumors with MSI. Because prostate tumors appear to have a high incidence of MSI, we analyzed prostate cancer cell lines, with and without MSI, for ACVR2 and TGFbetaR2 mutations. Our analysis of 6 prostate cell lines revealed mutations in the ACVR2 gene in 22Rv-1, LAPC-4, DU145, and LNCaP cells and mutations in the TGFbetaR2 gene in 22Rv-1 and LAPC-4. PC3 and H660 cells were wild-type for ACVR2 and TFGbetaR2. All of the ACVR2 mutations were truncating mutations, and using an activin response assay, we demonstrate that truncating mutations of the ACVR2 gene result in a significant reduction in activin mediated cell signaling. Inactivation of ACVR2 is a common event in prostate cancer cells suggesting it may play an important role in the development of prostate cancer.     

Differentiation of mouse and human embryonic stem cells into hepatic lineages

We recently reported a novel method to induce embryonic stem (ES) cells differentiate into an endodermal fate, especially pancreatic, using a supporting cell line. Here we describe the modified culture condition with the addition and withdrawal of secreted growth factors could induce ES cells to selectively differentiate into a hepatic fate efficiently. The signaling of BMP and FGF that have been implicated in hepatic differentiation during normal embryonic development are shown to play pivotal roles in generating hepatic cells from the definitive endoderm derived from ES cells. Moreover, the expression of AFP, Albumin or a biliary molecular marker appeared sequentially thus suggested the differentiation of ES cells recapitulated normal developmental processes of liver. The ES cell-derived differentiated cells showed evidence of glycogen storage, secreted Albumin, exhibited drug metabolism activities and expressed a set of cytochrome or drug conjugate enzymes, drug transporters specifically expressed in mature hepatocytes. With the same procedure, human ES cells also gave rise to cells with mature hepatocytes' characteristics. In conclusion, this novel procedure for hepatic differentiation will be useful for elucidation of molecular mechanisms of hepatic fate decision at gut regionalization, and could represent an attractive approach for a surrogate cell source for pharmaceutical studies such as toxicology.     

Activation of a caspase-dependent oxidative damage response mediates TGFbeta1 apoptosis in rat hepatocytes

Activation of transforming growth factor-beta type 1- (TGFbeta1) mediated signaling occurs in response to cell injury affecting stem-type cells and hepatocytes in liver. In this work we used WB stemlike liver epithelial cells and p53-defective CWSV-1 nontumorigenic rat hepatocytes to investigate the possible roles of caspases and oxidative stress in TGFbeta1 signaling. TGFbeta1 significantly increased the level of 4-hydroxy-2-nonenal (4-HNE), a stable product of lipid peroxidation. In addition, TGFbeta1-treated cells exhibited activation of caspases that accompanied by enhanced cleavage of the caspase substrate poly(ADP)-ribose polymerase (PARP) and induction of apoptosis. WB cells were twice as sensitive as sensitive as CWSV-1 cells to induction of TGFbeta1 apoptosis. TGFbeta1-apoptosis was significantly reduced when cells were treated with TGFbeta1 in the presence of inhibitors of caspase-1, -3, -8, and -9. Importantly, in addition to suppression of apoptosis, treatment of cells with the caspase-3 inhibitor Z-DEVD-FMK in the presence of TGFbeta1 suppressed the formation 4-HNE and restored mitotic activity. Together, these data suggest TGFbeta1 induces activation of a caspase signaling cascade that includes an oxidative damage response, PARP cleavage, and apoptosis that do not require intact p53 in rat hepatocytes.