Genes and Pathways Promoting Long-Term Liver Repopulation by Ex Vivo hYAP-ERT2 Transduced Hepatocytes and Treatment of Jaundice in Gunn Rats.

Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV- (dipeptidyl peptidase-4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)-deficient Gunn rats (model of Crigler-Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti-hYAP-ERT2 (mutated estrogen receptor ligand-binding domain 2)-transduced hepatocytes (YAP-Hc). Yap (yes-associated protein) nuclear translocation/function in YAP-Hc was regulated by tamoxifen. Repopulating YAP-Hc and host hepatocytes were fluorescence-activated cell sorting-purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP-Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte-specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up-regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP-Hc. In Gunn rats transplanted with YAP-Hc+tamoxifen, there was a 65%-81% decline in serum bilirubin over 6 months versus 8%-20% with control-Hc, representing a 3-4-fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1-positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen-regulated nuclear translocation/function of hYAP-ERT2 enabled long-term repopulation of DPPIV-/- and Gunn rat livers by hYAP-ERT2-transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.

Knockdown of miR-23, miR-27, and miR-24 Alters Fetal Liver Development and Blocks Fibrosis in Mice.

MicroRNAs (miRNAs) regulate cell fate selection and cellular differentiation. miRNAs of the miR23b polycistron (miR-23b, miR-27b, and miR-24) target components of the TGF-ß signaling pathway and affect murine bile ductular and hepatocyte cell fate selection in vitro. Here we show that miR-23b polycistron miRNAs directly target murine Smad4, which is required for TGF-ß signaling. Injection of antagomirs against these miRNAs directly into E16.5 murine fetuses caused increased cytokeratin expression in sinusoids and primitive ductular elements throughout the parenchyma of newborn mice. Similar antagomir injection in newborn mice increased bile ductular differentiation in the liver periphery and reduced hepatocyte proliferation. Antagomir injection in newborn Alb/TGF-ß1 transgenic mice that develop fibrosis inhibited the development of fibrosis, and injection of older mice caused the resolution of existing fibrosis. Furthermore, murine stellate cell activation, including ColA1 and ACTA2 expression, is regulated by miR-23b cluster miRNAs. In summary, knockdown of miR-23b cluster miRNAs in fetal and newborn liver promotes bile duct differentiation and can block or revert TGF-ß-induced liver fibrosis that is dependent on stellate cell activation. These data may find practical application in the highly needed development of therapies for the treatment of fibrosis.

Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver.

Recent investigations have reported many markers associated with human liver stem/progenitor cells, "oval cells," and identified "niches" in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as "micro-niches." Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease.

Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location.

Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs.

Small RNAs derived from lncRNA RNase MRP have gene-silencing activity relevant to human cartilage-hair hypoplasia.

Post-transcriptional processing of some long non-coding RNAs (lncRNAs) reveals that they are a source of miRNAs. We show that the 268-nt non-coding RNA component of mitochondrial RNA processing endoribonuclease, (RNase MRP), is the source of at least two short (∼20 nt) RNAs designated RMRP-S1 and RMRP-S2, which function as miRNAs. Point mutations in RNase MRP cause human cartilage-hair hypoplasia (CHH), and several disease-causing mutations map to RMRP-S1 and -S2. SHAPE chemical probing identified two alternative secondary structures altered by disease mutations. RMRP-S1 and -S2 are significantly reduced in two fibroblast cell lines and a B-cell line derived from CHH patients. Tests of gene regulatory activity of RMRP-S1 and -S2 identified over 900 genes that were significantly regulated, of which over 75% were down-regulated, and 90% contained target sites with seed complements of RMRP-S1 and -S2 predominantly in their 3' UTRs. Pathway analysis identified regulated genes that function in skeletal development, hair development and hematopoietic cell differentiation including PTCH2 and SOX4 among others, linked to major CHH phenotypes. Also, genes associated with alternative RNA splicing, cell proliferation and differentiation were highly targeted. Therefore, alterations RMRP-S1 and -S2, caused by point mutations in RMRP, are strongly implicated in the molecular mechanism of CHH.

Overexpression of miR-21 promotes an in vitro metastatic phenotype by targeting the tumor suppressor RHOB.

Metastasis is a multistep process that involves the deregulation of oncogenes and tumor suppressors beyond changes required for primary tumor formation. RHOB is known to have tumor suppressor activity, and its knockdown is associated with more aggressive tumors as well as changes in cell shape, migration, and adhesion. This study shows that oncogenic microRNA, miR-21, represses RHOB expression by directly targeting the 3' untranslated region. Loss of miR-21 is associated with an elevation of RHOB in hepatocellular carcinoma cell lines Huh-7 and HepG2 and in the metastatic breast cancer cell line MDA-MB-231. Using in vitro models of distinct stages of metastasis, we showed that loss of miR-21 also causes a reduction in migration, invasion, and cell elongation. The reduction in migration and cell elongation can be mimicked by overexpression of RHOB. Furthermore, changes in miR-21 expression lead to alterations in matrix metalloproteinase-9 activity. Therefore, we conclude that miR-21 promotes multiple components of the metastatic phenotype in vitro by regulating several important tumor suppressors, including RHOB.

MicroRNA-23b cluster microRNAs regulate transforming growth factor-beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads.

UNLABELLED: Transforming growth factor-beta / bone morphogenetic protein (TGFbeta/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFbeta ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR-23b cluster miRNAs comprising miR-23b, miR-27b, and miR-24-1 and miR-10a, miR-26a, and miR-30a as up-regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR-23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR-23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFbeta signaling molecules, as putative miR-23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3' UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down-regulation of constructs containing Smad 3, 4, or 5, 3' UTRs by a mixture of miR-23b cluster mimics. Knockdown of miR-23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC-3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR-23b mimics during bile duct differentiation of HBC-3 cells blocked the process. CONCLUSION: Our data provide a model in which miR-23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down-regulating Smads and consequently TGFbeta signaling. Concomitantly, low levels of the miR-23b miRNAs are needed in cholangiocytes to allow TGFbeta signaling and bile duct formation.

Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype.

Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs--miRNAs with known roles in cancer--the miR-17-92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17-92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.

Clonal, cultured, murine fetal liver hepatoblasts maintain liver specification in chimeric mice.

UNLABELLED: Recent studies have shown a pluripotential nature of stem cells that were previously thought to be committed to specific lineages. HBC-3 cells are a clonal fetal murine hepatoblast cell line derived from an e9.5 murine embryo, and these cells can be induced to form hepatocytes and bile ducts in vitro and when transplanted into adult mouse livers. To determine whether HBC-3 cells can exhibit a pluripotential phenotype, we created chimeric mice by injection of enhanced green fluorescent protein (EGFP)-marked HBC-3 cells into wild-type or dipeptidyl dipeptidase IV (DPPIV) knockout blastocysts. Genetically labeled HBC-3 cells were identified by EGFP polymerase chain reaction (PCR) in all the major organs of many chimeric mice and visualized in chimeras as bright red DPPIV-positive cells in the DPPIV knockout chimeric mice. Strikingly, the HBC-3 cells maintained phenotypic and biochemical features of liver specification in every case in which they were identified in nonliver organs, such as brain, mesenchyme, and bone. In adult liver they were present as small foci of hepatocytes and bile ducts in the chimeras. Additional major histocompatibility complex (MHC) marker analysis and X and Y chromosome content analysis further demonstrated that HBC-3 cells did not acquire the phenotype of the organs in which they resided and that they were not present because of fusion with host cells. CONCLUSION: In contrast to other stem cell types, these data demonstrate that cultured murine fetal liver stem cells appear to maintain their liver specification in the context of nonliver organs in chimeric mice.

New cell surface markers for murine fetal hepatic stem cells identified through high density complementary DNA microarrays.

UNLABELLED: Isolation of hepatic stem cells from the adult liver (AL) has not yet been achieved due to the lack of specific cell surface markers. To identify new surface markers for hepatic stem cells, we analyzed differences in the gene expression profile of embryonic day (ED) 13.5 fetal liver stem/progenitor cells (FLSPC) versus AL by complementary DNA (cDNA) microarray technology. Using FLSPC purified to >90% by immunomagnetic selection for E-cadherin and high density (27k) mouse cDNA microarrays, we identified 474 genes that are more strongly expressed in FLSPC (FLSPC-up genes) and 818 genes that are more strongly expressed in AL (AL-up genes). The most highly overrepresented gene ontology (GO) categories for FLSPC-up genes are nucleus, cellular proliferation, and cell cycle control. AL-up genes are overrepresented for genes in metabolic pathways for specific hepatic functions. We identified 24 FLSPC-up gene surface markers and 69 AL-up gene surface markers. Western blot studies confirmed the expression of the FLSPC-up gene neighbor of Punc E11 (Nope) in fetal liver, but expression was not detectable in AL. Immunohistochemistry, confocal microscopy, and fluorescence-activated cell sorting (FACS) analysis of fetal liver demonstrated that Nope is specifically expressed on the surface of FLSPC within the fetal liver. CONCLUSION: This is the first microarray study to analyze the specific gene expression profile of purified murine FLSPC. Our analysis identified 24 new/potential cell surface markers for murine fetal hepatic stem cells, of which Nope may be particularly useful in future studies to identify, characterize and isolate hepatic stem cells from the AL.

Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver.

Hepatocyte function and regeneration are severely compromised in severe liver disease, and a common sequela is cirrhosis. Structural changes caused by cirrhosis create a cellular environment conducive to the formation of ductular reactions (DRs). Ductular reactions are primarily composed of oval cells also known as "intermediate hepatobiliary cells". We have conducted single, double, and triple staining to study lineages of oval cells present in DRs. Staining with NCAM, CK19, and HepPar1 has revealed a distinctly bipolar structure to DRs that are embedded in cirrhotic tissue. Spatial analysis of cells that are singly HepPar1-positive, or CK19-positive, has revealed hepatocytic and biliary poles, respectively, in the DRs. Also, the location of singly NCAM-positive cells in DRs suggests that they may be bipotent liver stem/progenitor cells. The locations of other intermediate hepatobiliary cells, which have combinations of markers, suggest that CK19+/NCAM+ cells are transitional cells in the biliary lineage and that rare cells that are negative for all three markers are transitional cells in the hepatocytic lineage. A working cell lineage model for DRs is presented.

Transcriptional profiling implicates TGFbeta/BMP and Notch signaling pathways in ductular differentiation of fetal murine hepatoblasts.

Bile duct morphogenesis involves sequential induction of biliary specific gene expression, bilayer generation, cell proliferation, remodeling and apoptosis. HBC-3 cells are a model system to study differentiation of hepatoblasts along the hepatocytic or bile ductular lineage in vitro and in vivo. We used microarray to define molecular pathways during ductular differentiation in response to Matrigel. The temporal pattern of expression of marker genes induced was similar to that observed during bile duct formation in vivo. Notch, HNF1beta, Polycystic kidney disease 2, Bicaudal C 1 and beta-catenin were up regulated during the time course. Functional clustering analysis revealed significant up regulation of clusters of genes involved in extracellular matrix remodeling, ion transport, vacuoles, lytic vacuoles, pro-apoptotic and anti-apoptotic genes, transcription factors and negative regulators of the cell proliferation, while genes involved in the cell cycle were significantly down regulated. Notch signaling pathway was activated by treatment with Matrigel. In addition, TGFbeta/BMP signaling pathway members including the type I TGFbeta receptor and Smads 3, 4 and 5 were significantly up regulated, as were several TGFbeta/BMP responsive genes including Hey 1, a regulator of Notch pathway signaling. SMADS 3, 4 and 5 were present in the nuclear fraction of HBC-3 cells during ductular differentiation in vitro, but not during hepatocyte differentiation. SMAD 5 was preferentially expressed in hepatoblasts undergoing bile duct morphogenesis in the fetal liver, while the TGFbeta/BMP signaling antagonist chordin, was expressed throughout the liver suggesting a mechanism by which TGFbeta/BMP signaling is limited to hepatoblasts that contact portal mesenchyme in vivo.

cDNA microarray analysis of HBV transgenic mouse liver identifies genes in lipid biosynthetic and growth control pathways affected by HBV.

Hepatitis B virus (HBV) transgenic mice that replicate HBV in the liver generally do not exhibit gross liver pathology, while maintaining a high level (10(7) or greater) of viral titer in the blood. We have used this model to determine the minimum effects of HBV replication in the liver on cellular gene transcription, using cDNA microarrays. cDNA microarray data from sets of HBV versus control cDNA microarrays revealed a very small impact of HBV on the cellular transcriptome. After deletion of genes that were variable in control cDNA microarrays and applying significance analysis of microarrays (SAM), an application to detect statistically significantly regulated genes, we identified 18 upregulated genes and 14 downregulated genes. Most of the regulated genes show a change in expression with respect to control of less than 40% in either direction, demonstrating small effects of HBV. The largest functional category for upregulated genes was lipid biosynthesis, in which ATP citrate lyase, fatty acid synthase, sterol regulatory element binding factor 2, and retinol binding protein 1 were all upregulated. The most strongly downregulated genes were in the cytochrome p450 group, particularly p450, 4a14. Several growth regulatory genes including cyclin D1, IGF binding protein 3, and PCNA were moderately upregulated. These data are the first to specifically identify enzymes involved in fatty acid and NADPH-electron transport pathways that are altered by the presence of HBV. The data also demonstrates that HBV is well adapted to non-cytopathic replication in hepatocytes. Cellular genes expected to be affected by viral secretion from membranes are clearly upregulated, and upregulation of growth regulatory genes may facilitate replacement of dying hepatocytes during persistent infection.

RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples.

We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts at a sensitivity of four copies of LysA mRNA per liver cell equivalent. Furthermore, LysA cDNA detection varied linearly across a standard curve that matched the sensitivity of quantitative real-time PCR. In REMs with real samples, we detected organ-specific expression of albumin, Hnf-4 and Igfbp-1, in a set of mouse organ cDNA populations and c-Myc expression in tumor samples in paired tumor/normal tissue cDNA samples. REMs extend the use of classic microarrays in that a single REM can contain cDNAs from hundreds to thousands of cell or tissue samples each representing a specific physiological or pathophysiological state. REMs will extend the analysis of valuable samples by providing a common broad based platform for their analysis and will promote research aimed at defining gene functions, by broadening our understanding of their expression patterns in health and disease.