NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

Lineage-dependent effects of aryl hydrocarbon receptor agonists contribute to liver tumorigenesis.

UNLABELLED: Rodent cancer bioassays indicate that the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD), causes increases in both hepatocytic and cholangiocytic tumors. Effects of AHR activation have been evaluated on rodent hepatic stem cells (rHpSCs) versus their descendants, hepatoblasts (rHBs), two lineage stages of multipotent, hepatic precursors with overlapping but also distinct phenotypic traits. This was made possible by defining the first successful culture conditions for ex vivo maintenance of rHpScs consisting of a substratum of hyaluronans and Kubota's medium (KM), a serum-free medium designed for endodermal stem/progenitor cells. Supplementation of KM with leukemia inhibitory factor elicited lineage restriction to rHBs. Cultures were treated with various AHR agonists including TCDD, 6-formylindolo-[3,2-b]carbazole (FICZ), and 3-3'-diindolylmethane (DIM) and then analyzed with a combination of immunocytochemistry, gene expression, and high-content image analysis. The AHR agonists increased proliferation of rHpSCs at concentrations producing a persistent AHR activation as indicated by induction of Cyp1a1. By contrast, treatment with TCDD resulted in a rapid loss of viability of rHBs, even though the culture conditions, in the absence of the agonists, were permissive for survival and expansion of rHBs. The effects were not observed with FICZ and at lower concentrations of DIM. CONCLUSION: Our findings are consistent with a lineage-dependent mode of action for AHR agonists in rodent liver tumorigenesis through selective expansion of rHpSCs in combination with a toxicity-induced loss of viability of rHBs. These lineage-dependent effects correlate with increased frequency of liver tumors.

Identification of microRNAs specific for epithelial cell adhesion molecule-positive tumor cells in hepatocellular carcinoma.

UNLABELLED: Therapies that target cancer stem cells (CSCs) hold promise in eliminating cancer burden. However, normal stem cells are likely to be targeted owing to their similarities to CSCs. It is established that epithelial cell adhesion molecule (EpCAM) is a biomarker for normal hepatic stem cells (HpSCs), and EpCAM(+) AFP(+) hepatocellular carcinoma (HCC) cells have enriched hepatic CSCs. We sought to determine whether specific microRNAs (miRNAs) exist in hepatic CSCs that are not expressed in normal HpSCs. We performed a pair-wise comparison of the miRNA transcriptome of EpCAM(+) and corresponding EpCAM(-) cells isolated from two primary HCC specimens, as well as from two fetal livers and three healthy adult liver donors by small RNA deep sequencing. We found that miR-150, miR-155, and miR-223 were preferentially highly expressed in EpCAM(+) HCC cells, which was further validated. Their gene surrogates, identified using miRNA and messenger RNA profiling in a cohort of 292 HCC patients, were associated with patient prognosis. We further demonstrated that miR-155 was highly expressed in EpCAM(+) HCC cells, compared to corresponding EpCAM(-) HCC cells, fetal livers with enriched normal hepatic progenitors, and normal adult livers with enriched mature hepatocytes. Suppressing miR-155 resulted in a decreased EpCAM(+) fraction in HCC cells and reduced HCC cell colony formation, migration, and invasion in vitro. The reduced levels of identified miR-155 targets predicted the shortened overall survival and time to recurrence of HCC patients. CONCLUSION: miR-155 is highly elevated in EpCAM(+) HCC cells and might serve as a molecular target to eradicate the EpCAM(+) CSC population in human HCCs.

Successful transplantation of human hepatic stem cells with restricted localization to liver using hyaluronan grafts.

Cell therapies are potential alternatives to organ transplantation for liver failure or dysfunction but are compromised by inefficient engraftment, cell dispersal to ectopic sites, and emboli formation. Grafting strategies have been devised for transplantation of human hepatic stem cells (hHpSCs) embedded into a mix of soluble signals and extracellular matrix biomaterials (hyaluronans, type III collagen, laminin) found in stem cell niches. The hHpSCs maintain a stable stem cell phenotype under the graft conditions. The grafts were transplanted into the livers of immunocompromised murine hosts with and without carbon tetrachloride treatment to assess the effects of quiescent versus injured liver conditions. Grafted cells remained localized to the livers, resulting in a larger bolus of engrafted cells in the host livers under quiescent conditions and with potential for more rapid expansion under injured liver conditions. By contrast, transplantation by direct injection or via a vascular route resulted in inefficient engraftment and cell dispersal to ectopic sites. Transplantation by grafting is proposed as a preferred strategy for cell therapies for solid organs such as the liver.

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

UNLABELLED: 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.

Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis.

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis.

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 approximately 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 approximately 36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are approximately 9 microm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for alpha-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.

Fibrolamellar carcinomas-growth arrested by paracrine signals complexed with synthesized 3-O sulfated heparan sulfate oligosaccharides.

Fibrolamellar carcinomas (FLCs), lethal tumors occurring in children to young adults, have genetic signatures implicating derivation from biliary tree stem cell (BTSC) subpopulations, co-hepato/pancreatic stem cells, involved in hepatic and pancreatic regeneration. FLCs and BTSCs express pluripotency genes, endodermal transcription factors, and stem cell surface, cytoplasmic and proliferation biomarkers. The FLC-PDX model, FLC-TD-2010, is driven ex vivo to express pancreatic acinar traits, hypothesized responsible for this model's propensity for enzymatic degradation of cultures. A stable ex vivo model of FLC-TD-2010 was achieved using organoids in serum-free Kubota's Medium (KM) supplemented with 0.1% hyaluronans (KM/HA). Heparins (10 ng/ml) caused slow expansion of organoids with doubling times of ∼7-9 days. Spheroids, organoids depleted of mesenchymal cells, survived indefinitely in KM/HA in a state of growth arrest for more than 2 months. Expansion was restored with FLCs co-cultured with mesenchymal cell precursors in a ratio of 3:7, implicating paracrine signaling. Signals identified included FGFs, VEGFs, EGFs, Wnts, and others, produced by associated stellate and endothelial cell precursors. Fifty-three, unique heparan sulfate (HS) oligosaccharides were synthesized, assessed for formation of high affinity complexes with paracrine signals, and each complex screened for biological activity(ies) on organoids. Ten distinct HS-oligosaccharides, all 10-12 mers or larger, and in specific paracrine signal complexes elicited particular biological responses. Of note, complexes of paracrine signals and 3-O sulfated HS-oligosaccharides elicited slowed growth, and with Wnt3a, elicited growth arrest of organoids for months. If future efforts are used to prepare HS-oligosaccharides resistant to breakdown in vivo, then [paracrine signal-HS-oligosaccharide] complexes are potential therapeutic agents for clinical treatments of FLCs, an exciting prospect for a deadly disease.

A postnatal network of co-hepato/pancreatic stem/progenitors in the biliary trees of pigs and humans.

A network of co-hepato/pancreatic stem/progenitors exists in pigs and humans in Brunner's Glands in the submucosa of the duodenum, in peribiliary glands (PBGs) of intrahepatic and extrahepatic biliary trees, and in pancreatic duct glands (PDGs) of intrapancreatic biliary trees, collectively supporting hepatic and pancreatic regeneration postnatally. The network is found in humans postnatally throughout life and, so far, has been demonstrated in pigs postnatally at least through to young adulthood. These stem/progenitors in vivo in pigs are in highest numbers in Brunner's Glands and in PDGs nearest the duodenum, and in humans are in Brunner's Glands and in PBGs in the hepato/pancreatic common duct, a duct missing postnatally in pigs. Elsewhere in PDGs in pigs and in all PDGs in humans are only committed unipotent or bipotent progenitors. Stem/progenitors have genetic signatures in liver/pancreas-related RNA-seq data based on correlation, hierarchical clustering, differential gene expression and principal component analyses (PCA). Gene expression includes representative traits of pluripotency genes (SOX2, OCT4), endodermal transcription factors (e.g. SOX9, SOX17, PDX1), other stem cell traits (e.g. NCAM, CD44, sodium iodide symporter or NIS), and proliferation biomarkers (Ki67). Hepato/pancreatic multipotentiality was demonstrated by the stem/progenitors' responses under distinct ex vivo conditions or in vivo when patch grafted as organoids onto the liver versus the pancreas. Therefore, pigs are logical hosts for translational/preclinical studies for cell therapies with these stem/progenitors for hepatic and pancreatic dysfunctions.

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.