Eliane Wauthier

Human hepatic stem cells from fetal and postnatal donors

Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies.

The Phenotypes of Pluripotent Human Hepatic Progenitors

Human livers contain two pluripotent hepatic progenitors, hepatic stem cells and hepatoblasts, with size, morphology, and gene expression profiles distinct from that of mature hepatocytes. Hepatic stem cells, the precursors to hepatoblasts, persist in stable numbers throughout life, and those isolated from the livers of all age donors from fetal to adult are essentially identical in their gene and protein expression profiles. The gene expression profile of hepatic stem cells throughout life consists of high levels of expression of cytokeratin 19 (CK19), neuronal cell adhesion molecule (NCAM), epithelial cell adhesion molecule (EpCAM), and claudin‐3 (CLDN‐3); low levels of albumin; and a complete absence of expression of α‐fetoprotein (AFP) and adult liver‐specific proteins. By contrast, hepatoblasts, the dominant cell population in fetal and neonatal livers, decline in numbers with age and are found as <0.1% of normal adult livers. They express high levels of AFP, elevated levels of albumin, low levels of expression of adult liver‐specific proteins, low levels of CK19, and a loss of NCAM and CLDN‐3. Mature hepatocytes lack expression altogether of EpCAM, NCAM, AFP, CLDN‐3, cytokeratin 19, and have acquired the well‐known adult‐specific profile that includes expression of high levels of albumin, cytochrome P4503A4, connexins, phosphoenolpyruvate carboxykinase, and transferrin. Thus, hepatic stem cells have a unique stem cell phenotype, whereas hepatoblasts have low levels of expression of both stem cell genes and genes expressed in high levels in mature hepatocytes.

Human hepatic stem cell and maturational liver lineage biology.

Livers are comprised of maturational lineages of cells beginning extrahepatically in the hepato‐pancreatic common duct near the duodenum and intrahepatically in zone 1 by the portal triads. The extrahepatic stem cell niches are the peribiliary glands deep within the walls of the bile ducts; those intrahepatically are the canals of Hering in postnatal livers and that derive from ductal plates in fetal livers. Intrahepatically, there are at least eight maturational lineage stages from the stem cells in zone 1 (periportal), through the midacinar region (zone 2), to the most mature cells and apoptotic cells found pericentrally in zone 3. Those found in the biliary tree are still being defined. Parenchymal cells are closely associated with lineages of mesenchymal cells, and their maturation is coordinated. Each lineage stage consists of parenchymal and mesenchymal cell partners distinguishable by their morphology, ploidy, antigens, biochemical traits, gene expression, and ability to divide. They are governed by changes in chromatin (e.g., methylation), gradients of paracrine signals (soluble factors and insoluble extracellular matrix components), mechanical forces, and feedback loop signals derived from late lineage cells. Feedback loop signals, secreted by late lineage stage cells into bile, flow back to the periportal area and regulate the stem cells and other early lineage stage cells in mechanisms dictating the size of the liver mass. Recognition of maturational lineage biology and its regulation by these multiple mechanisms offers new understandings of liver biology, pathologies, and strategies for regenerative medicine and treatment of liver cancers. (HEPATOLOGY 2011;)

Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets

Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato‐pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum‐free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2‐3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger‐2‐AE2 and CFTR), or regulatable C‐peptide secreting neoislet‐like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin‐induced diabetic mice, results in functional islets secreting glucose‐regulatable human C‐peptide. Conclusion: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes. (HEPATOLOGY2011;)

Lineage Restriction of Human Hepatic Stem Cells to Mature Fates Is Made Efficient by Tissue-Specific Biomatrix Scaffolds

Current protocols for differentiation of stem cells make use of multiple treatments of soluble signals and/or matrix factors and result typically in partial differentiation to mature cells with under‐ or overexpression of adult tissue‐specific genes. We developed a strategy for rapid and efficient differentiation of stem cells using substrata of biomatrix scaffolds, tissue‐specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with a serum‐free, hormonally defined medium (HDM) tailored for the adult cell type of interest. Biomatrix scaffolds were prepared by a novel, four‐step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffolds maintained native histology, patent vasculatures, and ≈1% of the tissues proteins but >95% of its collagens, most of the tissues collagen‐associated matrix components, and physiological levels of matrix‐bound growth factors and cytokines. Collagens increased from almost undetectable levels to >15% of the scaffolds proteins with the remainder including laminins, fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix‐bound cytokines and growth factors in patterns that correlate with histology. Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffolds and in an HDM tailored for adult liver cells, lost stem cell markers and differentiated to mature, functional parenchymal cells in ≈1 week, remaining viable and with stable mature cell phenotypes for more than 8 weeks. Conclusion: Biomatrix scaffolds can be used for biological and pharmaceutical studies of lineage‐restricted stem cells, for maintenance of mature cells, and, in the future, for implantable, vascularized engineered tissues or organs. (HEPATOLOGY 2011.)

Sal‐like protein 4 (SALL4), a stem cell biomarker in liver cancers

Liver cancers, including hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and fibrolamellar HCCs (FL‐HCCs) are among the most common cancers worldwide and are associated with a poor prognosis. Investigations of genes important in liver cancers have focused on Sal‐like protein 4 (SALL4), a member of a family of zinc finger transcription factors. It is a regulator of embryogenesis, organogenesis, pluripotency, can elicit reprogramming of somatic cells, and is a marker of stem cells. We found it expressed in normal murine hepatoblasts, normal human hepatic stem cells, hepatoblasts and biliary tree stem cells, but not in mature parenchymal cells of liver or biliary tree. It was strongly expressed in surgical specimens of human HCCs, CCs, a combined hepatocellular and cholangiocarcinoma, a FL‐HCC, and in derivative, transplantable tumor lines in immune‐compromised hosts. Bioinformatics analyses indicated that elevated expression of SALL4 in tumors is associated with poor survival of HCC patients. Experimental manipulation of SALL4′s expression results in changes in proliferation versus differentiation in human HCC cell lines in vitro and in vivo in immune‐compromised hosts. Virus‐mediated gene transfer of SALL4 was used for gain‐ and loss‐of‐function analyses in the cell lines. Significant growth inhibition in vitro and in vivo, accompanied by an increase in differentiation occurred with down‐regulation of SALL4. Overexpression of SALL4 resulted in increased cell proliferation in vitro, correlating with an increase in expression of cytokeratin19 (CK19), epithelial cell adhesion molecules (EpCAM), and adenosine triphosphate (ATP)‐binding cassette‐G2 (ABCG2). Conclusion: SALL4′s expression is an indicator of stem cells, a prognostic marker in liver cancers, correlates with cell and tumor growth, with resistance to 5‐FU, and its suppression results in differentiation and slowed tumor growth. SALL4 is a novel therapeutic target for liver cancers. (HEPATOLOGY 2013)

Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates.

The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult‐specific genes but not others, aberrant regulation of one or more genes, and variations in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage‐dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The subpopulations of liver‐derived mesenchymal cells, purified by immunoselection technologies, included (1) angioblasts, (2) mature endothelia, (3) hepatic stellate cell precursors, (4) mature stellate cells (pericytes), and (5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum‐free) conditions that we developed for short‐term cultures and were used as feeders with hHpSCs. Feeders of angioblasts yielded self‐replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse‐transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and three‐dimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. Conclusion: Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations. (HEPATOLOGY 2010;)

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

Mature human hepatocytes from ex vivo differentiation of alginate-encapsulated hepatoblasts.

Alginate gel was used to provide encapsulation to support the growth and eventually the differentiation of hepatic progenitors cells derived from human fetal livers. The encapsulated cells aggregated into spheroids within a few days in culture and continued to grow for at least 4 weeks in a serum-free medium. The hepatic progenitor cells in the spheroids undergo differentiation, as indicated by the appearance of functions of mature hepatocytes such as the detoxification of ammonia, albumin secretion, expression of the adult cytochrome P450 isozyme CYP3A4 and enzymatic activity typical of CYP2C9. Along with the expression of mature hepatic markers, these progenitor cells lost features typical of immature liver cells such as epithelial cell adhesion molecule. In addition to the acquisition of mature biochemical functions, the spheroids also developed a bile ducts, suggesting that they had differentiated into tissues resembling those in an intact liver.

Gradients in the liver's extracellular matrix chemistry from periportal to pericentral zones: influence on human hepatic progenitors.

Embryonic mesenchymal feeders produce paracrine signals requisite for ex vivo survival and expansion of hepatic progenitors. The signals consist of a subset of soluble factors found in conditioned medium, and a subset of insoluble factors found in extracellular matrix that include collagens and basal adhesion molecules. We have identified key matrix components required for ex vivo maintenance of human hepatic progenitors produced by biologically active feeders. These components are similar to those found in zone 1 of the liver acinus (e.g., space of Disse) between layers of parenchyma and endothelia. Within these layers are transition chemistry matrix gradients, from zone 1 to zone 3. Use of purified zone 1 matrix components enables attachment and expansion of human hepatic progenitors independent of feeders. Cells aggregated into spheroid-like structures on laminin or spread into monolayers on type III or IV collagens. Contrastingly, a zone 3 matrix component, type I collagen, elicited growth arrest and differentiation. Another zone 3 matrix component, fibronectin, inhibited attachment. Use of specific matrix components, along with soluble paracrine signals from feeders, should enable one to maintain hepatic progenitors ex vivo without feeders and under wholly defined conditions.