Bile Duct-like Structures Research Articles

Effect of Valproic Acid on Promoting the Differentiation of Human Embryonic Stem Cells Into Cholangiocyte-Like Cells.

Cholangiocytes form a complex 3D network of bile ducts in the liver and contribute to liver function. The damage or destruction of cholangiocytes can lead to biliary diseases, and the shortage of cholangiocytes remains an obstacle for drug development targeting biliary diseases. Valproic acid (VPA) is a potent activator of Notch signaling pathway that is essential for cholangiocyte differentiation. Here, we report a VPA-based approach for cholangiocyte differentiation of human pluripotent stem cells. VPA activated Notch2 expression and upregulated HES-1, HEY-1, and Sox9 gene expression in hESC-derived hepatoblast. After 7 days treatment, VPA promoted successful differentiation of hepatoblast into cholangiocytes expressing cholangiocyte marker genes (AE2, AQP1, CFTR) and proteins (CK19 and CK7). In addition, the differentiated cholangiocytes formed bile duct-like structures after implantation into the spleen of NOD/SCID mice. Our results suggested that VPA can promote hESC differentiation to cholangiocyte-like cells. The induced cholangiocytes may serve as a potential cell source for both in vitro modeling and regenerative therapy of cholangiopathies. The findings can also support further development of small-molecule based differentiation protocols for cholangiocyte production.

Gelatin scaffold ameliorates proliferation & stem cell differentiation into the hepatic like cell and support liver regeneration in partial-hepatectomized mice model

Stem cell-based tissue engineering is an emerging tool for developing functional tissues of choice. To understand pluripotency and hepatic differentiation of mouse embryonic stem cells (mESCs) on a three-dimensional (3D) scaffold, we established an efficient approach for generating hepatocyte-like cells (HLCs) from hepatoblast cells. We developed porous and biodegradable scaffold, which was stimulated with exogenous growth factors and investigated stemness and differentiation capacity of mESCs into HLCs on the scaffold in-vitro. In animal studies, we had cultured mESCs-derived hepatoblast-like cells on the scaffold and then, transplanted them into the partially hepatectomized C57BL/6 male mice model to evaluate the effect of gelatin scaffold on hepatic regeneration. The 3D culture system allowed maintenance of stemness properties in mESCs. The step-wise induction of mESCs with differentiation factors leads to the formation of HLCs and expressed liver-specific genes, including albumin, hepatocyte nucleic factor 4 alpha, and cytokeratin 18. In addition, cells also expressed Ki67, indicating cells are proliferating. The secretome showed expression of albumin, urea, creatinine, alanine transaminase, and aspartate aminotransferase. However, the volume of the excised liver which aids regeneration has not been studied. Our results indicate that hepatoblast cells on the scaffold implanted in PH mouse indicates that these cells efficiently differentiate into HLCs and cholangiocytes, forming hepatic lobules with central and portal veins, and bile duct-like structures with neovascularization. The gelatin scaffold provides an efficient microenvironment for liver differentiation and regeneration both in-vitro and in-vivo. These hepatoblasts cells would be a valuable source for 3D liver tissue engineering/transplantation in liver diseases.

Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

Differentiation and isolation of iPSC-derived remodeling ductal plate-like cells by use of an AQP1-GFP reporter human iPSC line

Cholangiocytes are the epithelial cells that line bile ducts, and ductal plate malformation is a developmental anomaly of bile ducts that causes severe congenital biliary disorders. However, because of a lack of specific marker genes, methods for the stepwise differentiation and isolation of human induced pluripotent stem cell (hiPSC)-derived cholangiocyte progenitors at ductal plate stages have not been established. We herein generated an AQP1-GFP reporter hiPSC line and developed a combination treatment with transforming growth factor (TGF) β2 and epidermal growth factor (EGF) to induce hiPSC-derived hepatoblasts into AQP1+ cells in vitro. By confirming that the isolated AQP1+ cells showed similar gene expression patterns to cholangiocyte progenitors at the remodeling ductal plate stage around gestational week (GW) 20, we established a differentiation protocol from hiPSCs through SOX9+CK19+AQP1− ductal plate-like cells into SOX9+CK19+AQP1+ remodeling ductal plate-like cells. We further generated 3D bile duct-like structures from the induced ductal plate-like cells. These results suggest that AQP1 is a useful marker for the generation of remodeling ductal plate cells from hiPSCs. Our methods may contribute to elucidating the differentiation mechanisms of ductal plate cells and the pathogenesis of ductal plate malformation.

Culture System of Bile Duct-Like Cystic Structures Derived from Human-Inducible Pluripotent Stem Cells.

Inducible pluripotent stem (iPS) cells are multipotent stem cells that are produced by gene transfer of reprogramming factors to somatic cells. They are thought to be an important source of regenerative medicine because of their pluripotency and self-renewal ability. Although the liver has high regeneration ability, continuous death of hepatocytes due to chronic inflammation leads to liver cirrhosis and liver carcinoma. With regard to such serious liver diseases, liver transplantation is used as a complete cure, but there is a problem of donor shortage. Therefore, transplantation therapy using liver tissue generated from stem cells in vitro is expected.We are developing a system to induce the differentiation of cholangiocytes, one of important non-parenchymal cells in living liver tissue, from human iPS cells. Bile duct-like cystic structures can be induced by purifying human iPS cell-derived hepatoblasts expressing hepatic progenitor cell surface markers and inducing differentiation under appropriate culture conditions. These cells are considered to be useful in constructing a hepatic organoid that reproduces the liver structure of the living body.

Human hepatic organoids for the analysis of human genetic diseases.

We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct-like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor.

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures.

Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells

Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14–deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14–deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14–deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14–deficient livers. We demonstrate that MMP-14–mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes.

Generation of Self-Renewing Hepatoblasts From Human Embryonic Stem Cells by Chemical Approaches.

Somatic stem cells play crucial roles in organogenesis and tissue homeostasis and regeneration and may ultimately prove useful for cell therapy for a variety of degenerative diseases and injuries; however, isolation and expansion of most types of somatic stem cells from tissues are technically challenging. Human pluripotent stem cells are a renewable source for any adult cell types, including somatic stem cells. Generation of somatic stem cells from human pluripotent stem cells is a promising strategy to get these therapeutically valuable cells. Previously, we developed a chemically defined condition for mouse hepatoblast self-renewal through a reiterative screening strategy. In the present study, we efficiently generated hepatoblasts from human embryonic stem cells by a stepwise induction strategy. Importantly, these human embryonic stem cell-derived hepatoblasts can be captured and stably maintained using conditions previously established for mouse hepatoblast self-renewal, which includes basal media supplemented with insulin, transferrin, sodium selenite, epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor β receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate. The cells can stably retain hepatoblast phenotypes during prolonged culture and can differentiate into mature hepatocytes through in vitro provision of hepatocyte lineage developmental cues. After being embedded into three-dimensional Matrigel, these cells efficiently formed bile duct-like structures resembling native bile duct tissues. These human embryonic stem cell-derived hepatoblasts would be useful as a renewable source for cell therapy of liver diseases. Somatic stem cells have been proposed as promising candidates for cell-based therapy; however, isolation of somatic stem cells from adult tissues is usually invasive and technically challenging. In the present study, hepatoblasts from human embryonic stem cells were efficiently generated. These human hepatoblasts were then stably captured and maintained by a growth factor and small molecule cocktail, which included epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor β receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate. These human embryonic stem cell-derived hepatoblasts would be useful as a renewable source for cell therapy of liver diseases.

Self-renewal of hepatoblasts under chemically defined conditions by iterative growth factor and chemical screening

Tissue-specific stem/progenitor cells are essential to mediate organogenesis and tissue homeostasis. In addition, these cells have attracted significant interest for their therapeutic potential. However, it remains challenging to expand most types of these cells in vitro. In this study we devised a screening strategy aimed at identifying growth factors and small molecules that can sustain self-renewal of mouse hepatoblasts. This approach began with a defined basal condition, on top of which collections of growth factors and bioactive small molecules were screened for maintaining self-renewal of primary hepatoblasts. The initially identified proteins and small molecules were then combined in the basal media for subsequent screening to identify additional molecules that can synergistically promote hepatoblast self-renewal. This strategy was performed iteratively to eventually define a small molecule and growth factor cocktail, including epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor β receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate, which was sufficient to sustain long-term self-renewal of the murine hepatoblasts under chemically defined conditions. These expanded hepatoblasts retain the ability to respond to liver developmental cues and produce functional hepatocytes and form bile duct-like structures. Our work established a chemically defined condition that allows long-term expansion of hepatoblasts, improved our understanding of hepatoblast self-renewal, and highlights the power of phenotypic screening to enable self-renewal of somatic stem/progenitor cells.

Cultured Hepatocytes Adopt Progenitor Characteristics and Display Bipotent Capacity to Repopulate the Liver

Clinical studies have proved the therapeutic potential of hepatocyte transplantation as a promising alternative to whole organ liver transplantation in the treatment of hereditary or end-stage liver disease. However, donor shortage seriously restricts cell availability, and the lack of appropriate cell culture protocols for the storage and maintenance of donor cells constitutes a significant obstacle. The aim of this study was to stimulate mature hepatocytes in culture to multiply in vitro and track their fate on transplantation. Rat hepatocytes isolated nonenzymatically were cultured serum free for up to 10 days. They were stimulated into proliferation in the presence of growth factors and conditioned media from nonparenchymal and hepatocyte culture supernatants, as well as 10 mM lithium chloride (LiCl). Cell proliferation was assessed by determining DNA content. Additionally, the extent of cell differentiation was estimated using immunofluorescence staining of hepatic, biliary, progenitor, and mesenchymal markers and gene expression analyses. Transplantation studies were performed on the Fischer CD26-mutant rat following pretreatment with retrorsine and partial hepatectomy. Proliferating hepatocytes increasingly adopted precursor characteristics, expressing progenitor (OV6, CD133), hepatic lineage (CK18), biliary (CD49f, CK7, CK19), and mesenchymal (vimentin) markers. The supplement of LiCl further enhanced the proliferative capacity by 30%. Transplantation studies revealed extensive repopulation by large donor hepatocyte clusters. Furthermore, bile duct-like structures deriving from donor cells proved to be immunoreactive to ductular markers and formed in close proximity to endogenous bile ducts. Mature hepatocytes reveal their potential to "switch" between phenotypes, adopting progenitor characteristics during proliferation in vitro. Following transplantation, these "retrodifferentiated" cells further expanded in vivo, thereby generating bipotentially differentiated progenies (hepatocytes and bile duct-like structures). This apparent plasticity of mature hepatocytes may open new approaches for cell-based strategies to treat liver disease.

Portocaval shunt for hepatocyte package

In developing therapeutic alternatives to liver transplantation, we have used the strategy of applying a small intestinal segment as a scaffold for hepatocyte transplantation and also as a portocaval shunt (PCS) system to address both liver dysfunction and portal hypertension. The aim of this study was to investigate the feasibility of such an intestinal segment in animal models. Hepatocytes isolated from luciferase-transgenic Lewis rats were transplanted into jejunal segments of wild-type Lewis rats with mucosa removal without PCS application. Luciferase-derived luminescence from transplanted hepatocytes was stably detected for 30 days. Then, we performed autologous hepatocyte transplantation into the submucosal layer of an isolated and vascularized small intestinal segment in pigs. Transplanted hepatocytes were isolated from the resected left-lateral lobe of the liver. On day 7, hepatocyte clusters and bile duct-like structures were observed histologically. To create an intestinal PCS system in pigs, an auto-graft of the segmental ileum and interposing vessel graft were anastomosed to the portal vein trunk and inferior vena cava. However, thrombi were observed in vessels of the intestinal PCSs. We measured the correlation between infusion pressure and flow volume in whole intestines ex vivo in both species and found that the high pressure corresponding to portal hypertension was still insufficient to maintain the patency of the intestinal grafts. In conclusion, we demonstrated the feasibility of the small intestine as a scaffold for hepatocyte transplantation in rat and pig models, but PCS using an intestinal graft failed to maintain patency in a pig model.

Role of epimorphin in bile duct formation of rat liver epithelial stem‐like cells: Involvement of small G protein RhoA and C/EBPβ

Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem-like cells (WB-F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67(EPM)) with WB-F344 cells. Here, we showed that EPM could promote WB-F344 cells differentiation into bile duct-like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin-1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA-WB-F344 cell line (WB(RhoA-DN)) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up-regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM-induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase-reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPβ. Taken together, these data demonstrated that EPM regulates bile duct formation of WB-F344 cells through effects on RhoA and C/EBPβ, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

Bile duct–like differentiation in teratoma: a clinicopathologic and immunohistochemical study

The presence of biliary differentiation as part of teratoma is very unusual and has been reported only once as part of hepatocellular component; however, its incidence and significance were not studied. We reviewed available mixed germ cell tumors and pure teratomas for the last 18 years at our institution. Data on age, tumor size, location, gross and microscopic findings, proper prior clinical history, and outcome were obtained. Histologic components of teratoma were evaluated; percentage of bile duct-like structures was calculated, and cases were immunohistochemically studied. Forty-five cases were included (19 testicular neoplasms, 20 retroperitoneal lymph node resections, 5 mediastinal tumors, and 1 pulmonary resection), obtained from male patients 15 to 48 years old (mean, 28.8 years; median, 29 years). Bile duct-like tubules were identified in 11 (24%) cases. In 10 cases, the foci of bile duct-like structures represented less than 1% of the tumor tissue, whereas 1 case was almost exclusively composed of these structures. In all the cases, immunostains of bile duct-like tubules were positive for cytokeratin 7 and 19 and negative for other markers. Our findings show a significant prevalence of bile duct-like structures in teratoma. Immunohistochemical studies, although not specific, support a biliary-like immunophenotype. The presence of these structures did not adversely affect patient's prognosis; however, diagnostic awareness of their existence is relevant, as treatment and prognosis may vary significantly between teratomas and yolk sac or Sertoli cell tumors.

T Cell-mediated biliary epithelial-to-mesenchymal transition in liver allograft rejection

Loss of bile duct epithelium is characteristic of early chronic rejection following liver transplantation. Recent studies have suggested that intrahepatic biliary epithelial cells can transform into myofibroblasts. This study examines the induction and molecular regulation of this transition during allograft rejection. Immortalized human cholangiocytes were stimulated with either transforming growth factor beta1 (TGFbeta1) or a T cell line, and they were examined for morphological, proteomic, and functional features. Posttransplant liver biopsy sections were also examined. Treatment of cholangiocytes with TGFbeta1 or TGFbeta-presenting T cells induced a bipolar morphology, reduced expression of E-cadherin and zona occludens 1 (ZO-1), and increased vimentin, fibronectin, matrix metalloproteinase 2 (MMP-2), MMP-9, and S100 calcium binding protein A4 (S100A4); treated cells invaded a model basement membrane. Chemokines induced T cell penetration of 3-dimensional, cultured bile duct-like structures and bile ducts in liver biopsy sections. A spatial association was observed between duct-infiltrating T cells and cholangiocyte expression of mesenchymal markers, including S100A4. Inhibition of S100A4 expression in vitro blocked TGFbeta1-mediated loss of E-cadherin and ZO-1 but did not reduce induction of fibronectin, MMP-2, or MMP-9. This study demonstrates the potential for T cells to induce an intrahepatic biliary epithelial-to-mesenchymal cell transition during chronic rejection. Furthermore, S100A4 expression by cholangiocytes was identified as a crucial regulator of this transition.

Direct hepatic differentiation of mouse embryonic stemcells induced by valproic acid and cytokines

To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.

Sall4 Regulates Cell Fate Decision in Fetal Hepatic Stem/Progenitor Cells

Fetal hepatic stem/progenitor cells, called hepatoblasts, differentiate into both hepatocytes and cholangiocytes. The molecular mechanisms regulating this lineage segmentation process remain unknown. Sall4 has been shown to be among the regulators of organogenesis, embryogenesis, maintenance of pluripotency, and early embryonic cell fate decisions in embryonic stem cells. The expression and functional roles of Sall4 during liver development have not been elucidated. We here provide their first description in hepatoblasts. To investigate functions of Sall4 in fetal liver development, Dlk(+)CD45(-)Ter119(-) hepatoblasts derived from embryonic day 14 mouse livers were purified, and in vitro gain and loss of function analyses and in vivo transplantation analyses were performed using retrovirus- or lentivirus-mediated gene transfer. We demonstrated that Sall4 was expressed in fetal hepatoblasts but not adult hepatocytes. The expression level of Sall4 gradually fell during liver development. Overexpression of Sall4 in hepatoblasts significantly inhibited maturation induced by oncostatin M and extracellular matrix in vitro, as evidenced by morphologic changes and suppression of hepatic maturation marker gene expression. When bile duct-like structures were induced by collagen gel-embedded culture, overexpression of Sall4 markedly augmented size and number of cytokeratin19(+)-branching structures. Knockdown of Sall4 inhibited formation of these branching structures. With in vivo transplantation, Sall4 enhanced differentiation of cytokeratin19(+)-bile ducts derived from transplanted hepatoblasts. These results suggest that Sall4 plays a crucial role in controlling the lineage commitment of hepatoblasts not only inhibiting their differentiation into hepatocytes but also driving their differentiation toward cholangiocytes.

Hepatic reconstruction from fetal porcine liver cells using a radial flow bioreactor

To examine the efficacy of the radial flow bioreactor (RFB) as an extracorporeal bioartificial liver (BAL) and the reconstruction of liver organoids using embryonic pig liver cells. We reconstructed the liver organoids using embryonic porcine liver cells in the RFB. We also determined the gestational time window for the optimum growth of embryonic porcine liver cells. Five weeks of gestation was designated as embryonic day (E) 35 and 8 wk of gestation was designated as E56. These cells were cultured for one week before morphological and functional examinations. Moreover, the efficacy of pulsed administration of a high concentration hepatocyte growth factor (HGF) was examined. Both cell growth and function were excellent after harvesting on E35. The pulsed administration of a high concentration of HGF promoted the differentiation and maturation of these fetal hepatic cells. Microscopic examination of organoids in the RFB revealed palisading and showed that bile duct-like structures were well developed, indicating that the organoids were mini livers. Transmission electron microscopy revealed microvilli on the luminal surfaces of bile duct-like structures and junctional complexes, which form the basis of the cytoskeleton of epithelial tissues. Furthermore, strong expression of connexin (Cx) 32, which is the main protein of hepatocyte gap junctions, was observed. With respect to liver function, ammonia detoxification and urea synthesis were shown to be performed effectively. Our system can potentially be applied in the fields of BAL and transplantation medicine.

Hepatocytes transdifferentiate into biliary epithelial cells following toxicant‐induced biliary epithelium injury

Previously we have reported that rats treated with a biliary epithelium (BE) toxicant, methylene dianiline (DAPM), followed by bile duct ligation exhibit extensive transdifferentiation of hepatocytes to biliary epithelial cells. To further characterize the hepatocyte to BE transdifferentiation, we treated male F344 rats with two doses of DAPM (50 and 75 mg/kg, ip) and studied the dynamics of injury and regenerative changes in the liver over a time course of 0 to 15 days. DAPM treatment resulted in dose dependent BE injury as indicated by increase in serum bilirubin and GGT levels. H&E staining revealed massive BE necrosis with extensive inflammation and portal triad edema. PCNA immunohistochemical analysis indicated significant proliferation of BE cells, which continued till day 7 in the high (75 mg/kg) dose. In addition to BE cells, periportal hepatocytes exhibited PCNA positivity indicating increase in hepatocyte proliferation. The periportal hepatocytes also stained positive for AE1/AE3, markers of BE. To further study which growth factor can stimulate transdifferentiation, primary rat hepatocytes were cultured in roller bottles and were stimulated with HGF, EGF, FGF, PDGF, VEGF, SCF, MSP alone, as well as HGF and EGF in combination. None of the growth factors tested except HGF and EGF alone, and HGF and EGF in combination, stimulated formation of bile duct-like structures. Western blot analysis revealed no change in EGFR. Taken together, these data provide evidence for hepatocyte to BE transdifferentiation after DAPM-induced injury and indicate involvement of HGF and EGF-stimulated signaling in hepatocyte transdifferentiation.