Allelic Heterogeneity at the CRP Locus Identified by Whole-Genome Sequencing in Multi-ancestry Cohorts.

Whole-genome sequencing (WGS) can improve assessment of low-frequency and rare variants, particularly in non-European populations that have been underrepresented in existing genomic studies. The genetic determinants of C-reactive protein (CRP), a biomarker of chronic inflammation, have been extensively studied, with existing genome-wide association studies (GWASs) conducted in >200,000 individuals of European ancestry. In order to discover novel loci associated with CRP levels, we examined a multi-ancestry population (n = 23,279) with WGS (∼38× coverage) from the Trans-Omics for Precision Medicine (TOPMed) program. We found evidence for eight distinct associations at the CRP locus, including two variants that have not been identified previously (rs11265259 and rs181704186), both of which are non-coding and more common in individuals of African ancestry (∼10% and ∼1% minor allele frequency, respectively, and rare or monomorphic in 1000 Genomes populations of East Asian, South Asian, and European ancestry). We show that the minor (G) allele of rs181704186 is associated with lower CRP levels and decreased transcriptional activity and protein binding in vitro, providing a plausible molecular mechanism for this African ancestry-specific signal. The individuals homozygous for rs181704186-G have a mean CRP level of 0.23 mg/L, in contrast to individuals heterozygous for rs181704186 with mean CRP of 2.97 mg/L and major allele homozygotes with mean CRP of 4.11 mg/L. This study demonstrates the utility of WGS in multi-ethnic populations to drive discovery of complex trait associations of large effect and to identify functional alleles in noncoding regulatory regions.

Human MicroRNAs Attenuate the Expression of Immediate Early Proteins and HCMV Replication during Lytic and Latent Infection in Connection with Enhancement of Phosphorylated RelA/p65 (Serine 536) That Binds to MIEP.

Attenuating the expression of immediate early (IE) proteins is essential for controlling the lytic replication of human cytomegalovirus (HCMV). The human microRNAs (hsa-miRs), miR-200b-3p and miR-200c-3p, have been identified to bind the 3'-untranslated region (3'-UTR) of the mRNA encoding IE proteins. However, whether hsa-miRs can reduce IE72 expression and HCMV viral load or exhibit a crosstalk with the host cellular signaling machinery, most importantly the NF-κB cascade, has not been evaluated. In this study, argonaute-crosslinking and immunoprecipitation-seq revealed that miR-200b-3p and miR-200c-3p bind the 3'-UTR of UL123, which is a gene that encodes IE72. The binding of these miRNAs to the 3'-UTR of UL123 was verified in transfected cells stably expressing GFP. We used miR-200b-3p/miR-200c-3p mimics to counteract the downregulation of these miRNA after acute HCMV infection. This resulted in reduced IE72/IE86 expression and HCMV VL during lytic infection. We determined that IE72/IE86 alone can inhibit the phosphorylation of RelA/p65 at the Ser536 residue and that p-Ser536 RelA/p65 binds to the major IE promoter/enhancer (MIEP). The upregulation of miR-200b-3p and miR-200c-3p resulted in the phosphorylation of RelA/p65 at Ser536 through the downregulation of IE, and the binding of the resultant p-Ser536 RelA/p65 to MIEP resulted in a decreased production of pro-inflammatory cytokines. Overall, miR-200b-3p and miR-200c-3p-together with p-Ser536 RelA/p65-can prevent lytic HCMV replication during acute and latent infection.

Specific fate decisions in adult hepatic progenitor cells driven by MET and EGFR signaling.

The relative contribution of hepatocyte growth factor (HGF)/MET and epidermal growth factor (EGF)/EGF receptor (EGFR), two key signal transduction systems in the normal and diseased liver, to fate decisions of adult hepatic progenitor cells (HPCs) has not been resolved. Here, we developed a robust culture system that permitted expansion and genetic manipulation of cells capable of multilineage differentiation in vitro and in vivo to examine the individual roles of HGF/MET and EGF/EGFR in HPC self-renewal and binary cell fate decision. By employing loss-of-function and rescue experiments in vitro, we showed that both receptors collaborate to increase the self-renewal of HPCs through activation of the extracellular signal-regulated kinase (ERK) pathway. MET was a strong inducer of hepatocyte differentiation by activating AKT and signal transducer and activator of transcription (STAT3). Conversely, EGFR selectively induced NOTCH1 to promote cholangiocyte specification and branching morphogenesis while concomitantly suppressing hepatocyte commitment. Furthermore, unlike the deleterious effects of MET deletion, the liver-specific conditional loss of Egfr facilitated rather than suppressed progenitor-mediated liver regeneration by switching progenitor cell differentiation toward hepatocyte lineage. These data provide new insight into the mechanisms regulating the stemness properties of adult HPCs and reveal a previously unrecognized link between EGFR and NOTCH1 in directing cholangiocyte differentiation.

Cholesterol burden in the liver induces mitochondrial dynamic changes and resistance to apoptosis.

Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of histopathological changes ranging from non-inflammatory intracellular fat deposition to non-alcoholic steatohepatitis (NASH), which may progress into hepatic fibrosis, cirrhosis, or hepatocellular carcinoma. Recent data suggest that impaired hepatic cholesterol homeostasis and its accumulation are relevant to the pathogenesis of NAFLD/NASH. Despite a vital physiological function of cholesterol, mitochondrial dysfunction is an important consequence of dietary-induced hypercholesterolemia and was, subsequently, linked to many pathophysiological conditions. The aim in the current study was to evaluate the morphological and molecular changes of cholesterol overload in mouse liver and particularly, in mitochondria, induced by a high-cholesterol (HC) diet for one month. Histopathological studies revealed microvesicular hepatic steatosis and significantly elevated levels of liver cholesterol and triglycerides leading to impaired liver synthesis. Further, high levels of oxidative stress could be determined in liver tissue as well as primary hepatocyte culture. Transcriptomic changes induced by the HC diet involved disruption in key pathways related to cell death and oxidative stress as well as upregulation of genes related to glutathione homeostasis. Impaired liver function could be associated with a decrease in mitochondrial membrane potential and ATP content and significant alterations in mitochondrial dynamics. We demonstrate that cholesterol overload in the liver leads to mitochondrial changes which may render damaged hepatocytes proliferative and resistant to cell death whereby perpetuating liver damage.

Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells.

BACKGROUND & AIMS: Currently, much effort is directed towards the development of new cell sources for clinical therapy using cell fate conversion by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. METHODS: Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by 2 small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. RESULTS: We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of the fate conversion process. Based on functional evidence, activation of the HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through the activation of the ERK pathway. hCdHs expressed hepatic progenitor markers and could self-renew for at least 10 passages while retaining a normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Gene expression profiling using RNAseq confirmed the transcriptional reprogramming of hCdHs towards a progenitor state and the suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation in several models of therapeutic liver repopulation, hCdHs effectively repopulated the damaged parenchyma. CONCLUSION: Our study is the first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a novel tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and the repair of diseased livers. LAY SUMMARY: Human primary hepatocytes were reprogrammed towards hepatic progenitor cells by a combined treatment with 2 small molecules, A83-01 and CHIR99021, and HGF. Chemically derived hepatic progenitors exhibited a high proliferation potential and the ability to differentiate into hepatocytes and biliary epithelial cells both in vitro and in vivo. This approach enables the generation of patient-specific hepatic progenitors and provides a platform for personal and stem cell-based regenerative medicine.

The Brain-Enriched MicroRNA miR-9-3p Regulates Synaptic Plasticity and Memory.

UNLABELLED: MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression in many tissues. Although a number of brain-enriched miRNAs have been identified, only a few specific miRNAs have been revealed as critical regulators of synaptic plasticity, learning, and memory. miR-9-5p/3p are brain-enriched miRNAs known to regulate development and their changes have been implicated in several neurological disorders, yet their role in mature neurons in mice is largely unknown. Here, we report that inhibition of miR-9-3p, but not miR-9-5p, impaired hippocampal long-term potentiation (LTP) without affecting basal synaptic transmission. Moreover, inhibition of miR-9-3p in the hippocampus resulted in learning and memory deficits. Furthermore, miR-9-3p inhibition increased the expression of the LTP-related genes Dmd and SAP97, the expression levels of which are negatively correlated with LTP. These results suggest that miR-9-3p-mediated gene regulation plays important roles in synaptic plasticity and hippocampus-dependent memory. SIGNIFICANCE STATEMENT: Despite the abundant expression of the brain-specific microRNA miR-9-5p/3p in both proliferating and postmitotic neurons, most functional studies have focused on their role in neuronal development. Here, we examined the role of miR-9-5p/3p in adult brain and found that miR-9-3p, but not miR-9-5p, has a critical role in hippocampal synaptic plasticity and memory. Moreover, we identified in vivo binding targets of miR-9-3p that are involved in the regulation of long-term potentiation. Our study provides the very first evidence for the critical role of miR-9-3p in synaptic plasticity and memory in the adult mouse.

Targeting ODC1 inhibits tumor growth through reduction of lipid metabolism in human hepatocellular carcinoma.

Ornithine decarboxylase 1 (ODC1), a metabolic enzyme critically involved in the polyamine biosynthesis, is commonly upregulated in hepatocellular carcinoma (HCC). Despite its altered expression in human HCC tissues, the molecular mechanism by which ODC1 alters the course of HCC progression and functions in HCC cell survival is unknown. Here we identified that silencing of ODC1 expression with small interfering (si) RNA causes inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. Next, to obtain insights into the molecular changes in response to ODC1 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that 119 genes show same directional regulation (76 up- and 43 down-regulated) in both Huh1 and Huh7 cells and were considered as a common ODC1 knockdown signature. Particularly, we found through a network analysis that KLF2, which is known to inhibit PPARγ expression and adipogenesis, was commonly up-regulated. Subsequent Western blotting affirmed that the downregulation of ODC1 was accompanied by a decrease in the levels of PPARγ as well as of PARP-1, cyclin E1 and pro-caspase 9 delaying cell cycle progression and accelerating apoptotic signaling. Following the down-regulation of PPARγ expression, ODC1 silencing resulted in a strong inhibition in the expression of important regulators of glucose transport and lipid biogenesis, and caused a marked decrease in lipid droplet accumulation. In addition, ODC1 silencing significantly inhibited the growth of human HCC xenografts in nude mice. These findings indicate that the function of ODC1 is correlated with HCC lipogenesis and suggest that targeting ODC1 could be an attractive option for molecular therapy of HCC.

Electro-hyperthermia up-regulates tumour suppressor Septin 4 to induce apoptotic cell death in hepatocellular carcinoma.

PURPOSE: Modulated electro-hyperthermia (mEHT) has been shown to be effective against various types of human tumours, including hepatocellular carcinoma (HCC). Here we aimed to investigate the molecular mechanism underlying the cytotoxic effects of mEHT to HCC cells. MATERIALS AND METHODS: Human liver cancer cell lines, Huh7 and HepG2, were treated with mEHT (42 °C/60 min) three times at 2-day intervals. Growth inhibition and apoptotic induction were evaluated using MTS, microscopic analysis, a clonogenic assay, annexin V/PI staining and a ccK18 ELISA. Global changes in gene expression were examined using RNA sequencing to obtain insights into molecular changes in response to mEHT. For in vivo evaluation of mEHT we used HepG2 HCC xenografts grown in nude mice. RESULTS: mEHT suppressed HCC cell proliferation and long-term colony formation through induction of apoptosis. The growth inhibitory effects are induced through a subset of molecular changes. Notably the expression level of septin 4 (SEPT4) (involved in pro-apoptotic activity and growth suppression) was up-regulated, whereas a key regulator of invasiveness G-Protein coupled receptor 64 (GPR64) was repressed. Subsequent Western blotting confirmed that the common increase in tumour suppressor SEPT4 in both Huh7 and HepG2 cells is accompanied by the restoration of cyclin-dependent kinase (CDK) inhibitor p21 and decrease in pro-caspase 7 and pro-caspase 3, thereby accelerating apoptotic signalling in HCC cells. Additionally, mEHT significantly inhibited the growth of human HCC xenografts in nude mice. CONCLUSIONS: These findings suggest that apoptotic cell death induced by mEHT is mediated by the up-regulation of tumour suppressor SEPT4 in human HCC cells.

Epigenetic reprogramming modulates malignant properties of human liver cancer.

UNLABELLED: Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3-day treatment of established and primary human HCC-derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self-renewal potential. Differences in self-renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1-G5 were examined. Transient ZEB exposure produced differential cell density-dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes characterized by a stable overexpression of cancer stem cell-related and key epithelial-mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis- and differentiation-related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self-renewal, irrespective of cell density or ZEB exposure. Spheres formed by low-density cells treated with ZEB or shDNMT1 displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. CONCLUSION: These results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors.

Electro-hyperthermia inhibits glioma tumorigenicity through the induction of E2F1-mediated apoptosis.

PURPOSE: Modulated electro-hyperthermia (mEHT), also known as oncothermia, shows remarkable treatment efficacies for various types of tumours, including glioma. The aim of the present study was to investigate the molecular mechanism underlying phenotypic changes in oncothermic cancer cells. MATERIALS AND METHODS: U87-MG and A172 human glioma cells were exposed to mEHT (42 °C/60 min) three times with a 2-day interval and subsequently tested for growth inhibition using MTS, FACS and microscopic analysis. To obtain insights into the molecular changes in response to mEHT, global changes in gene expression were examined using RNA sequencing. For in vivo evaluation of mEHT, we used U87-MG glioma xenografts grown in nude mice. RESULTS: mEHT inhibited glioma cell growth through the strong induction of apoptosis. The transcriptomic analysis of differential gene expression under mEHT showed that the anti-proliferative effects were induced through a subset of molecular alterations, including the up-regulation of E2F1 and CPSF2 and the down-regulation of ADAR and PSAT1. Subsequent Western blotting revealed that mEHT increased the levels of E2F1 and p53 and decreased the level of PARP-1, accelerating apoptotic signalling in glioma cells. mEHT significantly suppressed the growth of human glioma xenografts in nude mice. We also observed that mEHT dramatically reduced the portion of CD133(+) glioma stem cell population and suppressed cancer cell migration and sphere formation. CONCLUSIONS: These findings suggest that mEHT suppresses glioma cell proliferation and mobility through the induction of E2F1-mediated apoptosis and might be an effective treatment for eradicating brain tumours.

A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer.

The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy.

Sequential transcriptome analysis of human liver cancer indicates late stage acquisition of malignant traits.

BACKGROUND & AIMS: Human hepatocarcinogenesis is as a multi-step process starting from dysplastic lesions to early carcinomas (eHCC) that ultimately progress to HCC (pHCC). However, the sequential molecular alterations driving malignant transformation of the pre-neoplastic lesions are not clearly defined. This lack of information represents a major challenge in the clinical management of patients at risk. METHODS: We applied next-generation transcriptome sequencing to tumor-free surrounding liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, eHCC (n = 5) and pHCC (n = 3) from 8 HCC patients with hepatitis B infection. Integrative analyses of genetic and transcriptomic changes were performed to characterize the genomic alterations during hepatocarcinogenesis. RESULTS: We report that changes in transcriptomes of early lesions including eHCC were modest and surprisingly homogenous. Extensive genetic alterations and subsequent activation of prognostic adverse signaling pathways occurred only late during hepatocarcinogenesis and were centered on TGFß, WNT, NOTCH, and EMT-related genes highlighting the molecular diversity of pHCC. We further identify IGFALS as a key genetic determinant preferentially down-regulated in pHCC. CONCLUSIONS: Our results define new hallmarks in molecular stratification and therapy options for patients at risk for HCC, and merit larger prospective investigations to develop a modified clinical-decision making algorithm based on the individualized next-generation sequencing analyses.

Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types.

BACKGROUND & AIMS: Human primary liver cancer is classified into biologically distinct subgroups based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of primary liver cancer. METHODS: We transduced mouse primary hepatic progenitor cells, lineage-committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic H-Ras and SV40LT. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling. RESULTS: Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and self-renewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells coexpressed hepatocyte (hepatocyte nuclear factor 4α), progenitor/biliary (keratin 19, epithelial cell adhesion molecule, A6), and mesenchymal (vimentin) markers and showed dysregulation of genes that control the epithelial-mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage stage-dependent genetic changes to malignant transformation. Activation of c-Myc and its target genes was required to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of c-Myc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice. CONCLUSIONS: Any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC by activating different cell type-specific pathways. Identification of common and cell of origin-specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer.

Loss of c-Met accelerates development of liver fibrosis in response to CCl(4) exposure through deregulation of multiple molecular pathways.

HGF/c-Met signaling plays a pivotal role in hepatocyte survival and tissue remodeling during liver regeneration. HGF treatment accelerates resolution of fibrosis in experimental animal models. Here, we utilized Met(fl/fl);Alb-Cre(+/-) conditional knockout mice and a carbon tetrachloride(CCl(4))-induced liver fibrosis model to formally address the role of c-Met signaling in hepatocytes in the context of chronic tissue injury. Histological changes during injury (4weeks) and healing phase (4weeks) were monitored by immunohistochemistry; expression levels of selected key fibrotic molecules were evaluated by western blotting, and time-dependent global transcriptomic changes were examined using a microarray platform. Loss of hepatocyte c-Met signaling altered hepatic microenvironment and aggravated hepatic fibrogenesis. Greater liver damage was associated with decreased hepatocyte proliferation, excessive stellate cell activation and rapid dystrophic calcification of necrotic areas. Global transcriptome analysis revealed a broad impact of c-Met on critical signaling pathways associated with fibrosis. Loss of hepatocyte c-Met caused a strong deregulation of chemotactic and inflammatory signaling (MCP-1, RANTES, Cxcl10) in addition to modulation of genes involved in reorganization of the cytoskeletal network (Actb, Tuba1a, Tuba8), intercellular communications and adhesion (Adam8, Icam1, Itgb2), control of cell proliferation (Ccng2, Csnk2a, Cdc6, cdk10), DNA damage and stress response (Rad9, Rad52, Ercc4, Gsta1 and 2, Jun). Our study demonstrates that deletion of c-Met receptor in hepatocytes results in pronounced changes in hepatic metabolism and microenvironment, and establishes an essential role for c-Met in maintaining the structural integrity and adaptive plasticity of the liver under adverse conditions.

Hepatocyte growth factor/c-met signaling is required for stem-cell-mediated liver regeneration in mice.

UNLABELLED: Hepatocyte growth factor (HGF)/c-Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c-Met in stem-cell-mediated liver regeneration by utilizing mice harboring c-met floxed alleles and Alb-Cre or Mx1-Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3,5-diethocarbonyl-1,4-dihydrocollidine (DDC). Deletion of c-met in oval cells was confirmed in both models by polymerase chain reaction analysis of fluorescence-activated cell-sorted epithelial cell adhesion molecule (EpCam)-positive cells. Loss of c-Met receptor decreased the sphere-forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration, and decreased hepatocytic differentiation in vivo, as demonstrated by double immunofluorescence using oval- (A6 and EpCam) and hepatocyte-specific (i.e. hepatocyte nuclear factor 4-alpha) antibodies. Furthermore, lack of c-Met had a profound effect on tissue remodeling and overall composition of HSC niche, which was associated with greatly reduced matrix metalloproteinase (MMP)9 activity and decreased expression of stromal-cell-derived factor 1. Using a combination of double immunofluorescence of cell-type-specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC-treated livers. The Mx1-Cre-driven c-met deletion caused the greatest phenotypic impact on HSCs response, as compared to the selective inactivation in the epithelial cell lineages achieved in c-Met(fl/fl); Alb-Cre(+/-) mice. However, in both models, genetic loss of c-met triggered a similar cascade of events, leading to the failure of HSC mobilization and death of the mice. CONCLUSION: These results establish a direct contribution of c-Met in the regulation of HSC response and support a unique role for HGF/c-Met as an essential growth-factor-signaling pathway for regeneration of diseased liver.

Contribution of hepatic lineage stage-specific donor memory to the differential potential of induced mouse pluripotent stem cells.

Recent studies suggested that induced pluripotent stem cells (iPSCs) retain a residual donor cell gene expression, which may impact their capacity to differentiate into cell of origin. Here, we addressed a contribution of a lineage stage-specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast-derived, HB-iPSCs) and end stage (adult hepatocyte, AH-iPSCs) of hepatocyte differentiation as well as from mouse embryonic fibroblasts (MEFs-iPSCs) using a lentiviral vector encoding four pluripotency-inducing factors Oct4, Sox2, Klf4, and c-Myc. All resulting iPSC lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay, and normal diploid karyotype. However, HB-iPSCs were more efficient in directed differentiation toward hepatocytic lineage as compared to AH-iPSCs, MEF-iPSCs, or mouse embryonic stem cells (mESCs). Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells, and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB-iPSCs retained a transcriptional memory (seven upregulated and 17 downregulated genes) typical of the original cells. Continuous passaging of HB-iPSCs erased most of these differences including a superior capacity for hepatic redifferentiation. These results suggest that retention of lineage stage-specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set may help to improve hepatic differentiation for therapeutic applications and contribute to the better understanding of liver development.

RPL27 contributes to colorectal cancer proliferation and stemness via PLK1 signaling.

Although expression of ribosomal protein L27 (RPL27) is upregulated in clinical colorectal cancer (CRC) tissue, to the best of our knowledge, the oncogenic role of RPL27 has not yet been defined. The present study aimed to investigate whether targeting RPL27 could alter CRC progression and determine whether RPL27 gains an extra­ribosomal function during CRC development. Human CRC cell lines HCT116 and HT29 were transfected with RPL27­specific small interfering RNA and proliferation was assessed in vitro and in vivo using proliferation assays, fluorescence­activated cell sorting (FACS) and a xenograft mouse model. Furthermore, RNA sequencing, bioinformatic analysis and western blotting were conducted to explore the underlying mechanisms responsible for RPL27 silencing­induced CRC phenotypical changes. Inhibiting RPL27 expression suppressed CRC cell proliferation and cell cycle progression and induced apoptotic cell death. Targeting RPL27 significantly inhibited growth of human CRC xenografts in nude mice. Notably, polo­like kinase 1 (PLK1), which serves an important role in mitotic cell cycle progression and stemness, was downregulated in both HCT116 and HT29 cells following RPL27 silencing. RPL27 silencing reduced the levels of PLK1 protein and G2/M­associated regulators such as phosphorylated cell division cycle 25C, CDK1 and cyclin B1. Silencing of RPL27 reduced the migration and invasion abilities and sphere­forming capacity of the parental CRC cell population. In terms of phenotypical changes in cancer stem cells (CSCs), RPL27 silencing suppressed the sphere­forming capacity of the isolated CD133+ CSC population, which was accompanied by decreased CD133 and PLK1 levels. Taken together, these findings indicated that RPL27 contributed to the promotion of CRC proliferation and stemness via PLK1 signaling and RPL27 may be a useful target in a next­generation therapeutic strategy for both primary CRC treatment and metastasis prevention.

Human chemically-derived hepatic progenitors (hCdHs) as a source of liver organoid generation: Application in regenerative medicine, disease modeling, and toxicology testing.

BACKGROUND & AIMS: Several types of human stem cells from embryonic (ESCs) and induced pluripotent (iPSCs) to adult tissue-specific stem cells are commonly used to generate 3D liver organoids for modeling tissue physiology and disease. We have recently established a protocol for direct conversion of primary human hepatocytes (hPHs) from healthy donor livers into bipotent progenitor cells (hCdHs). Here we extended this culture system to generate hCdH-derived liver organoids for diverse biomedical applications. METHODS: To obtain hCdHs, hPHs were cultured in reprogramming medium containing A83-01 and CHIR99021 for 7 days. Liver organoids were established from hCdHs (hCdHOs) and human liver cells (hLOs) using the same donor livers for direct comparison, as well as from hiPSCs. Organoid properties were analyzed by standard in vitro assays. Molecular changes were determined by RT-qPCR and RNA-seq. Clinical relevance was evaluated by transplantation into FRG mice, modeling of alcohol-related liver disease (ARLD), and in vitro drug-toxicity tests. RESULTS: hCdHs were clonally expanded as organoid cultures with low variability between starting hCdH lines. Similar to the hLOs, hCdHOs stably maintained stem cell phenotype based on accepted criteria. However, hCdHOs had an advantage over hLOs in terms of EpCAM expression, efficiency of organoid generation and capacity for directed hepatic differentiation as judged by molecular profiling, albumin secretion, glycogen accumulation, and CYP450 activities. Accordingly, FRG mice transplanted with hCdHOs survived longer than mice injected with hLOs. When exposed to ethanol, hCdHOs developed stronger ARLD phenotype than hLOs as evidenced by transcriptional profiling, lipid accumulation and mitochondrial dysfunction. In drug-induced injury assays in vitro, hCdHOs showed a similar or higher sensitivity response than hPHs. CONCLUSION: hCdHOs provide a novel patient-specific stem cell-based platform for regenerative medicine, toxicology testing and modeling liver diseases.

Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways.

UNLABELLED: Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase-1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal, superior tumor-initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. CONCLUSION: Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs.