Bipotential Hepatoblasts Research Articles

Cell fate decisions of human iPSC-derived bipotential hepatoblasts depend on cell density

During embryonic development bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes- the two main cell types within the liver. Cell fate decision depends on elaborate interactions between distinct signalling pathways, namely Notch, WNT, TGFβ, and Hedgehog. Several in vitro protocols have been established to differentiate human pluripotent stem cells into either hepatocyte or cholangiocyte like cells (HLC/CLC) to enable disease modelling or drug screening. During HLC differentiation we observed the occurrence of epithelial cells with a phenotype divergent from the typical hepatic polygonal shape- we refer to these as endoderm derived epithelial cells (EDECs). These cells do not express the mature hepatocyte marker ALB or the progenitor marker AFP. However they express the cholangiocyte markers SOX9, OPN, CFTR as well as HNF4α, CK18 and CK19. Interestingly, they express both E Cadherin and Vimentin, two markers that are mutually exclusive, except for cancer cells. EDECs grow spontaneously under low density cell culture conditions and their occurrence was unaffected by interfering with the above mentioned signalling pathways.

Single-cell murine genetic fate mapping reveals bipotential hepatoblasts and novel multi-organ endoderm progenitors.

The definitive endoderm (DE) is the embryonic germ layer that forms the gut tube and associated organs, including thymus, lungs, liver and pancreas. To understand how individual DE cells furnish gut organs, genetic fate mapping was performed using the Rosa26lacZ Cre-reporter paired with a tamoxifen-inducible DE-specific Cre-expressing transgene. We established a low tamoxifen dose that infrequently induced heritable lacZ expression in a single cell of individual E8.5 mouse embryos and identified clonal cell descendants at E16.5. As expected, only a fraction of the E16.5 embryos contained lacZ-positive clonal descendants and a subset of these contained descendants in multiple organs, revealing novel ontogeny. Furthermore, immunohistochemical analysis was used to identify lacZ-positive hepatocytes and biliary epithelial cells, which are the cholangiocyte precursors, in each clonally populated liver. Together, these data not only uncover novel and suspected lineage relationships between DE-derived organs, but also illustrate the bipotential nature of individual hepatoblasts by demonstrating that single hepatoblasts contribute to both the hepatocyte and the cholangiocyte lineage in vivo.

A single-cell transcriptomic analysis reveals precise pathways and regulatory mechanisms underlying hepatoblast differentiation.

Our studies have defined distinct pathways for hepatocyte and cholangiocyte development in vivo, which are critically important for understanding basic liver biology and developing effective strategies to induce stem cells to differentiate toward specific hepatic cell fates in vitro. (Hepatology 2017;66:1387-1401).

Feeder-Free and Serum-Free Production of Hepatocytes, Cholangiocytes, and Their Proliferating Progenitors from Human Pluripotent Stem Cells: Application to Liver-Specific Functional and Cytotoxic Assays

We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.

Liver stem/progenitor cells: their characteristics and regulatory mechanisms

Liver stem cells give rise to both hepatocytes and bile duct epithelial cells also known as cholangiocytes. During liver development hepatoblasts emerge from the foregut endoderm and give rise to both cell types. Colony-forming cells are present in the liver primordium and clonally expanded cells differentiate into either hepatocytes or cholangiocytes depending on culture conditions, showing stem cell characteristics. The growth and differentiation of hepatoblasts are regulated by various extrinsic signals. For example, periportal mesenchymal cells provide a cue for bipotential hepatoblasts to become cholangiocytes, and mesothelial cells covering the parenchyma support the expansion of foetal hepatocytes by producing growth factors. The adult liver has an extraordinary capacity to regenerate, and after 70% hepatectomy the liver recovers its original mass by replication of the remaining hepatocytes without the activation of liver stem cells. However, in certain types of liver injury models, liver stem/progenitor-like cells, known as oval cells in rodents, proliferate around the portal vein, while the roles of such cells in liver regeneration remain a matter of debate. Clonogenic and bipotential cells are also present in the normal adult liver. In this minireview we describe recent studies on liver stem/progenitor cells by focusing on extracellular signals.

Epiplakin1 is expressed in the cholangiocyte lineage cells in normal liver and adult progenitor cells in injured liver

We have previously identified Epiplakin1 (Eppk1) as a gene expressed in pancreatic progenitor cells. Here we studied the expression of Eppk1 in developing and regenerating livers in mice. Eppk1 is initially expressed in the early bipotential hepatoblasts and is later confined to the cholangiocytes. After birth, Eppk1 is expressed in the bile duct. In the livers of mice fed with a choline-deficient ethionine-supplemented (CDE) diet, Eppk1-positive cells dramatically increase in number. The Eppk1-positive cells express A6, thereby indicating that they are hepatic progenitor cells. Other cholangiocyte markers, such as Cytokeratins, E-cadherin, osteopontin and Sox9, are also co-expressed in the hepatic progenitor cells. Some of the Eppk1-positive cells express PCNA, a proliferation marker, thereby suggesting their identities as transient amplifying cells. In conclusion, we have shown that Eppk1 serves as a useful marker for detecting the hepatic progenitor population in the developing and adult liver. The use of Eppk1 as a marker will facilitate studies of mouse hepatic progenitor cells.

Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: Drastic change of EpCAM expression during liver development

Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1 + cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM +DLK1 + hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1 + cells of E11.5 liver were fractionated into EpCAM + and EpCAM − cells; one forth of EpCAM +DLK1 + cells formed a colony in vitro whereas EpCAM −DLK1 + cells rarely did it. Moreover, EpCAM +DLK1 + cells contained cells capable of forming a large colony, indicating that EpCAM +DLK1 + cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM +DLK1 + and EpCAM −DLK1 + cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM +DLK1 + cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM −DLK1 + cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.

Hematopoietic Progenitors from Early Murine Fetal Liver Possess Hepatic Differentiation Potential

Bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes during liver development. It is believed that hepatoblasts originate from endodermal tissue. Here, we provide evidence for the presence of hepatic progenitor cells in the hematopoietic compartment at an early stage of liver development. Flow cytometric analysis showed that at early stages of liver development, approximately 13% of CD45(+) cells express Delta-like protein-1, a marker of hepatoblasts. Furthermore, reverse transcriptase-PCR data suggest that many hepatic genes are expressed in these cells. Cell culture experiments confirmed the hepatic differentiation potential of these cells with the loss of the CD45 marker. We observed that both hematopoietic activity in Delta-like protein-1(+) cells and hepatic activity in CD45(+) cells were high at embryonic day 10.5 and declined thereafter. Clonal analysis revealed that the hematopoietic fraction of fetal liver cells at embryonic day 10.5 gave rise to both hepatic and hematopoietic colonies. The above results suggest a common source of these two functionally distinct cell lineages. In utero transplantation experiments confirmed these results, as green fluorescent protein-expressing CD45(+) cells at the same stage of development yielded functional hepatocytes and hematopoietic reconstitution. Since these cells were unable to differentiate into cytokeratin-19-expressing cholangiocytes, we distinguished them from hepatoblasts. This preliminary study provides hope to correct many liver diseases during prenatal development via transplantation of fetal liver hematopoietic cells.

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.

Plasticity of Hepatic Cell Differentiation: Bipotential Adult Mouse Liver Clonal Cell Lines Competent to Differentiate In Vitro and In Vivo

In fetal liver, bipotential hepatoblasts differentiate into hepatocytes and bile duct cells (cholangiocytes). The persistence of such progenitor cells in adult mouse liver is still debated. In damaged liver of adult murine animals, when hepatocyte proliferation is compromised, bipotential oval cells emerge, probably from bile ducts, proliferate, and differentiate to regenerate the liver. However, treatment to elicit oval cell proliferation is not necessary to obtain bipotential stem cells from adult mouse liver. Here, we have isolated bipotential clonal cell lines from healthy liver of 8-10-week-old C57BL/6 mice. Primary cultures established from hepatocyte-enriched suspensions were characterized by time-lapse image acquisition, immunocytology, and RNA transcript analysis. Although hepatocytes dedifferentiated with loss of apical polarity and other hepatocyte markers, they rapidly activated expression of bile duct/oval cell markers. Reversibility of these processes was achieved in part by culture under dilute Matrigel or by aging of confluent cultures. Cell lines were obtained at high frequency from mass cultures, from isolated colonies, and by primary cloning of the hepatocyte-enriched suspension. Cells of the clonal cell lines do not grow in soft agar and are nontumorigenic, and they express cytokeratin 19, A6 antigen, and alpha6 integrin, as well as a large panel of hepatocyte functions. Furthermore, they can participate in liver regeneration in albumin-urokinase-type plasminogen activator/severe combined immune-deficient mice, where they differentiate in clusters of hepatocytes and occasionally bile ducts. These results demonstrate the existence, in normal adult mouse liver, of a significant pool of clonogenic cells that are (or can become) bipotential.

Id3 is important for proliferation and differentiation of the hepatoblasts during the chick liver development

The specified hepatic endoderm (hepatoblasts), the bipotential progenitor for hepatocytes and bile duct epithelial cells, proliferates during the primordial stages of liver development. Despite extensive studies, the mechanism that regulates proliferation of bipotential hepatoblasts is not fully understood. Here we show that Id3, a negative regulator of helix–loop–helix transcription factors, is an important regulator of hepatoblast proliferation in the developing chick liver. Id3 was expressed in hepatoblasts at early developmental stages (stages 12–29) but not in hepatocytes at later developmental stages (stage 34 onwards). Depletion of Id3 in hepatoblasts by siRNA results in failure of cell proliferation, but is not associated with either cell death or failure of expression of Hhex and Fibrinogen, the earliest hepatoblast markers. These observations suggest that at early developmental stages, Id3 functions as a positive regulator of hepatoblast proliferation, independent of cell death or maintenance of the non-terminally differentiated state. Interestingly at later developmental stages, the expression pattern of Id3 is complementary to that of Albumin, a marker of mature hepatocytes. Overexpression of Id3 in liver explants delayed the initiation of Albumin expression. Taken together, our observations show that Id3 is not only a positive regulator of hepatoblast proliferation, but also an inhibitor of their differentiation into hepatocytes in the developing chick liver.

Hepatocytes Deficient in CCAAT/Enhancer Binding Protein α (C/EBPα) Exhibit both Hepatocyte and Biliary Epithelial Cell Character

To further elucidate the role of CCAAT/Enhancer Binding Protein α (C/EBPα) in hepatocyte differentiation, we investigated fetal and newborn C/EBPα-deficient (C/EBPα −/−) mice using confocal microscopy and markers specific for hepatocyte (AFP) and biliary epithelial cell (A6) differentiation. Histologically, in fetal liver of C/EBPα −/− mice, pseudoglandular structures appeared starting at 16.5 days of gestation. In newborn livers, the diameters of these structures greatly increased. They were randomly distributed between portal and central veins and interfered with the establishment of normal hepatic plates. However, the portal bile ducts developed normally. The pseudoglandular structures were lined with small hepatocytes with round nuclei and were positive for both AFP and A6 antigens. These data show that C/EBPα −/− hepatocytes exhibit biliary epithelial cell characters and suggest an involvement of C/EBPα in the control of the switch in the differentiation of bi-potential hepatoblasts along the hepatocyte lineage.