Biliary organoids uncover delayed epithelial development and barrier function in biliary atresia.

BACKGROUND AND AIMS: Biliary atresia is a severe inflammatory and fibrosing cholangiopathy of neonates of unknown etiology. The onset of cholestasis at birth implies a prenatal onset of liver dysfunction. Our aim was to investigate the mechanisms linked to abnormal cholangiocyte development. APPROACH AND RESULTS: We generated biliary organoids from liver biopsies of infants with biliary atresia and normal and diseased controls. Organoids emerged from biliary atresia livers and controls and grew as lumen-containing spheres with an epithelial lining of cytokeratin-19pos albuminneg SOX17neg cholangiocyte-like cells. Spheres had similar gross morphology in all three groups and expressed cholangiocyte-enriched genes. In biliary atresia, cholangiocyte-like cells lacked a basal positioning of the nucleus, expressed fewer developmental and functional markers, and displayed misorientation of cilia. They aberrantly expressed F-actin, ß-catenin, and Ezrin, had low signals for the tight junction protein zonula occludens-1 (ZO-1), and displayed increased permeability as evidenced by a higher Rhodamine-123 (R123) signal inside organoids after verapamil treatment. Biliary atresia organoids had decreased expression of genes related to EGF signaling and FGF2 signaling. When treated with EGF+FGF2, biliary atresia organoids expressed differentiation (cytokeratin 7 and hepatocyte nuclear factor 1 homeobox B) and functional (somatostatin receptor 2, cystic fibrosis transmembrane conductance regulator [CFTR], aquaporin 1) markers, restored polarity with improved localization of F-actin, ß-catenin and ZO-1, increased CFTR function, and decreased uptake of R123. CONCLUSIONS: Organoids from biliary atresia are viable and have evidence of halted epithelial development. The induction of developmental markers, improved cell-cell junction, and decreased epithelial permeability by EGF and FGF2 identifies potential strategies to promote epithelial maturation and function.

Multilineage communication regulates human liver bud development from pluripotency.

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

Problem-appropriate diagram instruction for improving mathematical word problem solving.

The use of diagrams can be effective in solving mathematical word problems solving. However, students worldwide do not construct diagrams unprompted or have trouble using them. In the present study, the effects of problem-appropriate diagram use instruction were investigated with an adaptation of the multiple baseline design method. The instruction for using line diagrams, tables, and graphs was provided to 67 junior high school students in a staggered manner and the effects on problem solving of three different types of problems was examined. The results showed that use of problem-appropriate diagrams increased and persisted over time. More importantly, the instruction led to increases in problem solving performance and to decreases in perceived cognitive load. These findings support the argument that effective diagram use depends on the acquisition not only of declarative knowledge, but also sufficient procedural and conditional knowledge.

Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling.

Timely controlled oxygen (O2) delivery is crucial for the developing liver. However, the influence of O2 on intercellular communication during hepatogenesis is unclear. Using a human induced pluripotent stem cell-derived liver bud (hiPSC-LB) model, we found hypoxia induced with an O2-permeable plate promoted hepatic differentiation accompanied by TGFB1 and TGFB3 suppression. Conversely, extensive hypoxia generated with an O2-non-permeable plate elevated TGFBs and cholangiocyte marker expression. Single-cell RNA sequencing revealed that TGFB1 and TGFB3 are primarily expressed in the human liver mesenchyme and endothelium similar to in the hiPSC-LBs. Stromal cell-specific RNA interferences indicated the importance of TGFB signaling for hepatocytic differentiation in hiPSC-LB. Consistently, during mouse liver development, the Hif1a-mediated developmental hypoxic response is positively correlated with TGFB1 expression. These data provide insights into the mechanism that hypoxia-stimulated signals in mesenchyme and endothelium, likely through TGFB1, promote hepatoblast differentiation prior to fetal circulation establishment.

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.

Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.

Sequencing analysis of a putative human O-sialoglycoprotein endopeptidase gene (OSGEP) and analysis of a bidirectional promoter between the OSGEP and APEX genes.

We performed cDNA and genomic cloning, sequencing and promoter analysis of the putative human O-sialoglycoprotein endopeptidase gene OSGEP (a homologue of gcp, a Pasteurella haemolytica A1 glycoprotease). The cloned OSGEP cDNA is 1311 nucleotides long, and encodes a protein consisting of 335 amino acids with predicted molecular mass of 36.4 kDa. The amino acid sequence of OSGEP showed 29.7% identity with that of P. haemolytica glycoprotease. The OSGEP gene is 7.75 kb long, consists of 11 exons and 10 introns, and lies immediately adjacent to the APEX gene (which encodes APEX nuclease, a multifunctional DNA repair enzyme) in 5'-to-5' orientation. The promoter region of the OSGEP gene lacks the typical TATA box, but has putative regulatory elements in the CpG island. Northern blot analysis showed ubiquitous expression of the OSGEP gene in several tissues, and we observed similarities in expression patterns between OSGEP and APEX. In order to study the regulation of OSGEP gene expression, we analyzed the OSGEP promoter region by luciferase assay using HeLa cells. A functional region required for full transcription activity was narrowed down to a 23 bp region containing a CCAAT box. It has been reported that this CCAAT box promotes basal transcription in the APEX direction. We thus conclude that a bidirectional promoter containing a CCAAT box regulates transcription of both the OSGEP and APEX genes.

Development of an efficient genotyping method to detect obese mutation in the mouse leptin gene for use in SPF barrier facilities.

We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lep(ob)) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lep(ob)/Lep(ob), Lep(ob)/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.

Identification of the functional elements in the bidirectional promoter of the mouse O-sialoglycoprotein endopeptidase and APEX nuclease genes.

The gene for mammalian O-sialoglycoprotein endopeptidase (Osgep) lies immediately adjacent to the gene for the APEX nuclease (Apex), a multifunctional DNA repair enzyme, in a head-to-head orientation. To clarify the regulation of these divergent genes, we studied their promoter regions with luciferase reporters. Deletion analysis of a fragment containing the entire mouse Apex gene suggested that cis-acting elements driving in the direction of Osgep are widely distributed in the mApex gene, in the antisense orientation. We investigated in detail cis-acting elements near the transcription initiation site of mOsgep. The spacer sequence between mOsgep and mApex was shown to have bidirectional promoter activity and it has been reported that two CCAAT boxes promote basal transcription in the direction of mApex. However, only one of the CCAAT boxes proximal to the transcription initiation site of mOsgep was important for transcription towards mOsgep. An Sp1-binding sequence was found to be involved in bidirectional transcription and a CRE/ATF-like sequence was shown to function as a repressor of mOsgep transcription. Quantitative RT-PCR showed that the mApex and mOsgep genes were expressed in all tissues examined and that expression of mOsgep was low compared with mApex.