A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver.

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or alpha-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

Involvement of CCAAT/enhancer binding protein-beta (C/EBPbeta) in epigenetic regulation of mouse methionine adenosyltransferase 1A gene expression.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the main methyl donor in cellular transmethylation reactions and the aminopropyl moiety in polyamine biosynthesis. In mammals, two different genes, MAT1A and MAT2A, encode catalytic polypeptides of liver-specific MAT I/III and ubiquitous MAT II, respectively. Reverse transcription-polymerase chain reaction showed that MAT1A gene expression was at a detectable level in embryonic day 14 mouse fetal liver and subsequently increased. Bisulfite genomic sequencing indicated that the methylation status of 10CpG sites in the MAT1A promoter proximal region was appreciably correlated with the gene expression in mouse developing liver and in adult hepatic cells; hepatic stellate cells and hepatocytes. When mouse hepatoma-derived Hepa-1 cells showing extremely low expression of MAT1A gene were treated with 5-aza-2'-deoxycytidine and trichostatin A, MAT1A gene expression was enhanced. In addition, in vitro methylation of the MAT1A promoter region suppressed the MAT1A promoter activity in reporter assay. Next, we performed electrophoretic mobility shift assay and found that the transcriptional factor CCAAT/enhancer binding protein-beta (C/EBPbeta) specifically binds to a putative binding site of C/EBPbeta in the MAT1A promoter. Suppression of C/EBPbeta expression by short hairpin RNA decreased the MAT1A promoter activity and MAT1A gene expression, and inhibition of C/EBPbeta binding to MAT1A by site-directed mutagenesis also showed similar results. Western blot analysis and chromatin immunoprecipitation assay indicated that C/EBPbeta binding is dependent on DNA methylation status. Based on these findings, we conclude that C/EBPbeta plays an important role in epigenetic regulation of the mature hepatic gene MAT1A.

Hepatic progenitor cells in the mouse extrahepatic bile duct after a bile duct ligation.

The intrahepatic bile duct has been suggested to be a source of hepatic progenitor cells in the severely damaged liver. In contrast, little attention has been paid to the question of whether hepatic progenitor cells exist in the extrahepatic bile duct (EHBD). In the present study, we examined the phenotypic changes of the mouse EHBD following bile duct ligation. After bile duct ligation, the number of c-Kit-positive epithelial cells increased in the EHBD. The ligated EHBD expressed mRNA for hepatic progenitor cell markers, including c-Kit and Thy-1. Hepatocyte markers such as albumin and cytochrome P450 7a1 were also transiently detected in the EHBD after a bile duct ligation. In a culture of EHBD cells, we detected hepatic progenitor cells that were positive for both staining with anti-albumin antibodies and Dolichos biflorus agglutinin, a biliary epithelial cell-specific lectin. Furthermore, hepatic progenitor cells positive for both c-Kit and albumin were found in the cultured EHBD population. Additionally EHBD-derived hepatocyte-like cells were also observed in the culture. A transplantation study revealed that EHBD cells integrate into the parenchyma and are albumin positive. These data suggest that hepatic progenitor cells emerge in the EHBD following bile duct ligation, that subsequently give rise to hepatocyte-like cells. We also observed that the gall bladder transiently expressed hepatocyte markers after bile duct ligation. Our results suggest a potential of the EHBD and gall bladder as useful transplantable sources for liver injury.

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source.

BACKGROUND: We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. METHODS: On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. RESULTS: Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. CONCLUSIONS: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.

Human neutrophils isolated from peripheral blood contain Ku protein but not DNA-dependent protein kinase.

Ku protein, a heterodimer of 70kDa (Ku70) and 86kDa (Ku86) polypeptides, is involved in non-homologous DNA end-joining (NHEJ) of DNA double-strand break repair and V(D)J recombination in combination with the catalytic component of DNA-dependent protein kinase (p470). Although Ku protein is known to be ubiquitously present in eukaryotic cells, it was previously reported to be absent in mature neutrophils. Using a mixture of protease inhibitors in the isolation procedure of neutrophils from human peripheral blood, we were able to detect Ku in the neutrophils by immunoblot and flow-cytometric analyses. Transcripts of Ku70 and Ku86 genes were also detected by the reverse transcriptase-polymerase chain reaction (RT-PCR), and Ku protein was shown to be localized in the nucleus of neutrophils as a heterodimer. Like poly(ADP-ribose) polymerase-1, neither mRNA nor protein of p470 was detected in the neutrophils. These results suggest that Ku is involved independently of p470 in DNA metabolism and signal transduction.

Hepatic stellate cells mediate differentiation of dendritic cells from monocytes.

BACKGROUND: We have previously reported that human umbilical cord blood (UCB)-nucleated cells differentiate into hepatocyte-like cells when cultured in a 5-cytokine cocktail medium. We further found that UCB cells rather differentiated into dendritic-shaped cells by coculture with a human stellate cell (HSC) line, LI90. METHODS: Monocytes from UCB and adult peripheral blood were cocultured with LI90 or rat primary HSCs in a cell-culture insert. Monocytes were also cultured with LI90-conditioned medium containing secreted factors, which were analyzed by a cytokine array. RESULTS: In the coculture with LI90, resulting dendritic-shaped cells from monocytes expressed dendritic cell (DC) markers and activated allogeneic T cells, indicating that the dendritic-shaped cells were DCs. LI90 in the cytokine cocktail medium secreted various inflammatory factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Fibroblast growth factor-2 in the cytokine cocktail was responsible for GM-CSF production from LI90 cells and for differentiation of monocytes into DCs in the LI90 coculture. Moreover, the coculture of monocytes with activated HSCs derived from damaged rat liver induced the differentiation of DCs, whereas quiescent HSCs derived from normal liver scarcely induced such a change. CONCLUSION: These results suggest that activated HSCs are involved in differentiation of monocytes into DCs in the liver.

Vascular endothelial growth factor promotes proliferation and function of hepatocyte-like cells in embryoid bodies formed from mouse embryonic stem cells.

BACKGROUND/AIMS: Embryoid bodies (EBs) formed from embryonic stem cells (ESCs) differentiate into hepatocyte-like cells (HLCs), and are thus thought to be a useful cell source for drug testing and bioartificial liver. The aim of this study was to induce proliferation and function of ESC-derived HLCs in EBs using HLC-endothelial cell interaction. METHODS: EBs were cultured in the presence of vascular endothelial growth factor (VEGF) and/or VEGF receptor (VEGFR) inhibitors. To reproduce HLC-endothelial cell interaction, we overexpressed VEGF in ESC-derived HLCs under the control of Cyp7a1 gene in EBs. RESULTS: VEGF added to the cultured EBs increased the proliferation of ESC-derived endothelial cells, resulting in the promotion of proliferation and function of ESC-derived HLCs. In EBs, the VEGFR2 inhibitor suppressed expression of albumin and endothelial cell marker genes, whereas the inhibitor for both VEGFR1 and VEGFR2 suppressed expression of Cyp7a1 and hepatocyte growth factor (Hgf) genes. Upon exposure to VEGF, the endothelial cells in EBs increased Hgf mRNA expression. Forced VEGF expression in ESC-derived HLCs in EBs induced angiogenesis around the HLCs and resulted in an increase in the amount of HLCs. CONCLUSIONS: VEGF indirectly induces the proliferation and function of ESC-derived HLCs through VEGFR1 and VEGFR2 signaling in endothelial cells.

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver.

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and alpha1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.

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 coexpressed 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.

Human umbilical cord blood as a source of transplantable hepatic progenitor cells.

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21-day cultures expressed ALB, and the ALB(+) cells coexpressed hepatocyte lineage markers. The ALB-expressing cells were able to proliferate in the culture system. Moreover, in the cell-transplantation model into liver-injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB-derived cell transplantation as a novel therapeutic option for liver failure.

Expression of the liver-specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells.

Hepatic differentiation from mouse embryonic stem (ES) cells via the formation of embryoid bodies (EBs) has been revealed by the expression of hepatocyte-related genes such as alpha-fetoprotein and albumin. It is known, however, that the visceral endoderm differentiates in early EBs and expresses these hepatocyte-related genes. Thus, it remains unclear whether ES cells are capable of differentiating into hepatocytes derived from definitive endoderm in vitro. In the present study, yolk sac tissues isolated from the foetal mouse were found to express many hepatocyte-related genes. Among the hepatocyte-related genes examined, cytochrome P450 7A1 (Cyp7a1) was identified as a liver-specific gene that was not expressed in the yolk sac. Cyp7a1 was induced in developing EBs, and hepatic differentiation was preferentially observed in the developing EBs in attached culture as compared to those in suspension culture. Leukaemia inhibitory factor permitted the differentiation of visceral endoderm, but inhibited the expression of gastrulation-related genes and the hepatic differentiation in cultured EBs. ES cells expressing green fluorescent protein (GFP) under the control of the Cyp7a1 enhancer/promoter showed that cultured EBs contained GFP-positive epithelial-like cells. These results demonstrate that ES cells can differentiate in vitro into hepatocytes derived from definitive endoderm.

Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes.

Embryonic stem (ES) cells have a potential to differentiate into various progenitor cells. Here we investigated the differentiation capacity of mouse ES cells into hepatocytes both in vitro and in vivo. During the culture of embryoid bodies (EBs) derived from ES cells, albumin (ALB) messenger RNA (mRNA) was expressed within 12 days after removal of leukemia inhibitory factor, and alpha-fetoprotein (AFP) mRNA was observed within 9 days without additional exogenous growth factors. In ES cells and early EBs, by contrast, neither ALB mRNA nor AFP mRNA was observed. ALB protein was first detected at day 15 and the level increased with the culture period. The differentiation of EBs facilitated the synthesis of urea with the culture period, whereas early EBs and ES cells produced no urea. These results suggest that cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from EBs were transplanted through portal vein to the liver after 30% partial hepatectomy of female mice pretreated with 2-acetylaminofluorene. Four weeks after transplantation with isolated cells from day-9 EBs, ES-derived cells containing Y-chromosome in the liver were positive for ALB (0.2% of total liver cells), whereas teratoma was found in mice transplanted with ES cells or EBs up to day 6. The incidence of teratoma was decreased with the culture duration and no teratoma was observed in the liver transplanted with isolated cells from day-9 EBs. In conclusion, our in vitro and in vivo experiments revealed that cultured EBs contain functional hepatocytes or hepatocyte-like cells.