Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes.

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

Generation of functional ciliated cholangiocytes from human pluripotent stem cells.

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

Archival bone marrow smears are useful in targeted next-generation sequencing for diagnosing myeloid neoplasms.

Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.

Potential prognostic impact of EBV RNA-seq reads in gastric cancer: a reanalysis of The Cancer Genome Atlas cohort.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC), whose prognosis remains controversial, is diagnosed by in situ hybridization of EBV-derived EBER1/2 small RNAs. In The Cancer Genome Atlas (TCGA) Stomach Adenocarcinoma (STAD) project, the EBV molecular subtype was determined through a combination of multiple next-generation sequencing methods, but not by the gold standard in situ hybridization method. This leaves unanswered questions regarding the discordance of EBV positivity detected by different approaches and the threshold of sequencing reads. Therefore, we reanalyzed the TCGA-STAD RNA sequencing (RNA-seq) dataset including 375 tumor and 32 normal samples, using our analysis pipeline. We defined a reliable threshold for EBV-derived next-generation sequencing reads by mapping them to the EBV genome with three different random arbitrary alignments. We analyzed the prognostic impact of EBV status on the histopathological subtypes of gastric cancer. EBV-positive cases identified by reanalysis comprised nearly half of the cases (49.6%) independent from infiltrating lymphocyte signatures, and showed significantly longer overall survival for adenocarcinomas of the 'not-otherwise-specified' type [P = 0.016 (log-rank test); hazard ratios (HR): 0.476; 95% CI: 0.260-0.870, P = 0.016 (Cox univariate analysis)], but shorter overall survival for the tubular adenocarcinoma type [P = 0.005 (log-rank test); HR: 3.329; 95% CI: 1.406-7.885, P = 0.006 (Cox univariate analysis)]. These results demonstrate that the EBV positivity rates were higher when determined by RNA-seq than when determined by EBER1/2 in situ hybridization. The RNA-seq-based EBV positivity demonstrated distinct results for gastric cancer prognosis depending on the histopathological subtype, suggesting its potential to be used in clinical prognoses.

Large-scale Generation of Functional and Transplantable Hepatocytes and Cholangiocytes from Human Endoderm Stem Cells.

The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.

Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.

The liver is the largest solid organ in the body and is critical for metabolic and immune functions. However, little is known about the cells that make up the human liver and its immune microenvironment. Here we report a map of the cellular landscape of the human liver using single-cell RNA sequencing. We provide the transcriptional profiles of 8444 parenchymal and non-parenchymal cells obtained from the fractionation of fresh hepatic tissue from five human livers. Using gene expression patterns, flow cytometry, and immunohistochemical examinations, we identify 20 discrete cell populations of hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, B cells, conventional and non-conventional T cells, NK-like cells, and distinct intrahepatic monocyte/macrophage populations. Together, our study presents a comprehensive view of the human liver at single-cell resolution that outlines the characteristics of resident cells in the liver, and in particular provides a map of the human hepatic immune microenvironment.

Analysis of histone modification around the CpG island region of the p15 gene in acute myeloblastic leukemia.

Seven of 11 patients with acute myeloblastic leukemia (AML) had allele(s) in which more than half of 27 CpG sites in the p15 gene were methylated. The p15 CpG island region was surrounded with both the acetylated histone H3 (AcH3) and dimethylated histone H3-lysine 9 (MeH3K9) in bone marrow cells of AML patients, whereas with AcH3 alone in normal marrow cells. The p15 CpG islands of DNA immunoprecipitated with anti-AcH3 antibody and anti-MeH3K9 antibody were not always unmethylated and methylated, respectively, in the patients. These results suggest perturbed modifications of histone H3 around the p15 CpG island region in AML.

Directed differentiation of cholangiocytes from human pluripotent stem cells.

Although bile duct disorders are well-recognized causes of liver disease, the molecular and cellular events leading to biliary dysfunction are poorly understood. To enable modeling and drug discovery for biliary disease, we describe a protocol that achieves efficient differentiation of biliary epithelial cells (cholangiocytes) from human pluripotent stem cells (hPSCs) through delivery of developmentally relevant cues, including NOTCH signaling. Using three-dimensional culture, the protocol yields cystic and/or ductal structures that express mature biliary markers, including apical sodium-dependent bile acid transporter, secretin receptor, cilia and cystic fibrosis transmembrane conductance regulator (CFTR). We demonstrate that hPSC-derived cholangiocytes possess epithelial functions, including rhodamine efflux and CFTR-mediated fluid secretion. Furthermore, we show that functionally impaired hPSC-derived cholangiocytes from cystic fibrosis patients are rescued by CFTR correctors. These findings demonstrate that mature cholangiocytes can be differentiated from hPSCs and used for studies of biliary development and disease.

Farside gravity field of the moon from four-way Doppler measurements of SELENE (Kaguya).

The farside gravity field of the Moon is improved from the tracking data of the Selenological and Engineering Explorer (SELENE) via a relay subsatellite. The new gravity field model reveals that the farside has negative anomaly rings unlike positive anomalies on the nearside. Several basins have large central gravity highs, likely due to super-isostatic, dynamic uplift of the mantle. Other basins with highs are associated with mare fill, implying basalt eruption facilitated by developed faults. Basin topography and mantle uplift on the farside are supported by a rigid lithosphere, whereas basins on the nearside deformed substantially with eruption. Variable styles of compensation on the near- and farsides suggest that reheating and weakening of the lithosphere on the nearside was more extensive than previously considered.

CD30+ anaplastic large cell lymphoma complicated by pyoderma gangrenosum with increased levels of serum cytokines.

We here present the case of a 5-yr-old girl with pyoderma gangrenosum (PG) in association with underlying CD30+ anaplastic large cell lymphoma with increased serum cytokine levels (interleukin-8, granulocyte colony-stimulating factor). An association between PG and increased cytokine levels was suggested. Even in children, dermatosis of PG should receive prompt careful evaluation for underlying hematological malignancy.

Quinolinate dehydrogenase and 6-hydroxyquinolinate decarboxylase involved in the conversion of quinolinic acid to 6-hydroxypicolinic acid by Alcaligenes sp. strain UK21.

In the conversion of quinolinic acid to 6-hydroxypicolinic acid by whole cells of Alcaligenes sp. strain UK21, the enzyme reactions involved in the hydroxylation and decarboxylation of quinolinic acid were examined. Quinolinate dehydrogenase, which catalyzes the first step, the hydroxylation of quinolinic acid, was solubilized from a membrane fraction, partially purified, and characterized. The enzyme catalyzed the incorporation of oxygen atoms of H(2)O into the hydroxyl group. The dehydrogenase hydroxylated quinolinic acid and pyrazine-2,3-dicarboxylic acid to form 6-hydroxyquinolinic acid and 5-hydroxypyrazine-2,3-dicarboxylic acid, respectively. Phenazine methosulfate was the preferred electron acceptor for quinolinate dehydrogenase. 6-Hydroxyquinolinate decarboxylase, catalyzing the nonoxidative decarboxylation of 6-hydroxyquinolinic acid, was purified to homogeneity and characterized. The purified enzyme had a molecular mass of approximately 221 kDa and consisted of six identical subunits. The decarboxylase specifically catalyzed the decarboxylation of 6-hydroxyquinolinic acid to 6-hydroxypicolinic acid, without any co-factors. The N-terminal amino acid sequence was homologous with those of bacterial 4,5-dihydroxyphthalate decarboxylases.