Deriving Human Intestinal Organoids with Functional Tissue-Resident Macrophages All From Pluripotent Stem Cells.

Background & Aims: Organs of the gastrointestinal tract contain tissue-resident immune cells that function during tissue development, homeostasis, and disease. However, most published human organoid model systems lack resident immune cells, thus limiting their potential as disease avatars. For example, human intestinal organoids (HIOs) derived from pluripotent stem cells contain epithelial and various mesenchymal cell types but lack immune cells.

Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells.

The fetal liver is a hematopoietic organ, hosting a diverse and evolving progenitor population. While human liver organoids derived from pluripotent stem cells (PSCs) mimic aspects of embryonic and fetal development, they typically lack the complex hematopoietic niche and the interaction between hepatic and hematopoietic development. We describe the generation of human Fetal Liver-like Organoids (FLOs), that model human hepato-hematopoietic interactions previously characterized in mouse models.

Self-Assembled Generation of Multi-zonal Liver Organoids from Human Pluripotent Stem Cells.

Distinct hepatocyte subpopulations are spatially segregated along the portal-central axis and critical to understanding metabolic homeostasis and liver injury. While several bioactive molecules have been described to play a role in directing zonal fates, including ascorbate and bilirubin, replication of zonal liver architecture has not been achieved to date. In order to evaluate hepatic zonal polarity, we developed a self-assembling zone-specific liver organoid culture by co-culturing ascorbate and bilirubin enriched hepatic progenitors derived from human induced pluripotent stem cells.

Design and Mechanistic Insights into α-Helical -Terphenyl Guanidines as Potent Small-Molecule Antifreeze Agents.

Ice formation is a critical challenge across multiple fields, from industrial applications to biological preservation. Inspired by natural antifreeze proteins, we designed and synthesized a new class of small-molecule antifreezes based on α-helical -terphenyl scaffolds with guanidine side chains. These -terphenyl guanidines , among the smallest molecules that mimic α-helical structures, exhibit potent ice recrystallization inhibition (IRI) activity, similar to that of existing large α-helical antifreeze compounds.

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn's Disease.

Background And Aims: We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA).

Methods: Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs.

Self-Organization of Sinusoidal Vessels in Pluripotent Stem Cell-derived Human Liver Bud Organoids.

The induction of tissue-specific vessels in living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b putative liver sinusoidal progenitors (iLSEP) by dictating developmental pathways. By devising an inverted multilayered air-liquid interface (IMALI) culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organized to generate and sustain hepatocyte-like cells neighbored by divergent endothelial subsets composed of CD32bCD31, LYVE1STAB1CD32bCD31THBDvWF, and LYVE1THBDvWF cells.

Deciphering Endothelial and Mesenchymal Organ Specification in Vascularized Lung and Intestinal Organoids.

Unlabelled: To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn's Disease.

Background And Aims: We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA).

Methods: Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs.

Synthetic augmentation of bilirubin metabolism in human pluripotent stem cell-derived liver organoids.

UGT1A1 (UDP glucuronosyltransferase family 1 member A1) is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic anion transporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype.

Detection of stellar light from quasar host galaxies at redshifts above 6.

The detection of starlight from the host galaxies of quasars during the reionization epoch (z > 6) has been elusive, even with deep Hubble Space Telescope observations. The current highest redshift quasar host detected, at z = 4.5, required the magnifying effect of a foreground lensing galaxy.

Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics.

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized.

Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology.

The spatial resolution of fluorescence microscopy has recently been greatly enhanced. However, improvements in temporal resolution have been limited, despite their importance for examining living cells. Here, we developed an ultrafast camera system that enables the highest time resolutions in single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore we identified.

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T.

Directed differentiation of human pluripotent stem cells into diverse organ-specific mesenchyme of the digestive and respiratory systems.

Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. Although the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue.

Eicosatetraynoic Acid and Butyrate Regulate Human Intestinal Organoid Mitochondrial and Extracellular Matrix Pathways Implicated in Crohn's Disease Strictures.

Background: Perturbagen analysis of Crohn's disease (CD) ileal gene expression data identified small molecules including eicosatetraynoic acid (ETYA), which may exert an antifibrotic effect. We developed a patient-specific human intestinal organoid (HIO) model system to test small molecule regulation of mitochondrial and wound-healing functions implicated in stricturing behavior.

Methods: HIOs were made from CD induced pluripotent stem cells with and without a loss-of-function haplotype in the DUOX2 gene implicated in ileal homeostasis and characterized under basal conditions and following exposure to butyrate and ETYA using RNA sequencing, flow cytometry, and immunofluorescent and polarized light microscopy.

Correction of a Factor VIII genomic inversion with designer-recombinases.

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells.

Synthesis and application of POLYseq for profiling human liver organoids.

Detailed herein is the protocol for synthesis, characterization, and application of POLYseq for cell pooling in single-cell sequencing runs. POLYseq is synthesized through commercially available reagents and is highly tailorable. Synthesis is easily performed in a two-step protocol, utilizing Michael addition only requiring a temperature-stable hot bath capable of holding 90°C.

Interpretations of SARS-CoV-2 IgM and IgG antibody titers in the seroepidemiological study of asymptomatic healthy volunteers.

Introduction: The usefulness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests in asymptomatic individuals has not been well validated, although they have satisfied sensitivity and specificity in symptomatic patients. In this study, we investigated the significance of IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects.

Methods: From June 2020, we recruited 10,039 participants to the project named the University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS), and measured iFlash-SARS-CoV-2 IgM and IgG (YHLO IgM and IgG) titers in the collected serum.

POLYseq: A poly(β-amino ester)-based vector for multifunctional cellular barcoding.

Despite evolving biological application of next-generation sequencing (NGS) at single-cell level, current techniques in NGS library preparation restrict multiplexing, necessitating the costly preparation of distinct libraries for each sample. Here, we report the development of a novel poly(β-amino) ester labeling system synthesized with inexpensive, common reagents, termed POLYseq, capable of efficiently delivering fluorescent molecules or sample-distinguishing DNA barcodes through non-covalent binding enabling rapid creation of custom sample pools. Chemical formulation was found to determine cellular labeling propensity.

Organogenesis in vitro.

Organoids are three-dimensional structures that self-organize from human pluripotent stem cells or primary tissue, potentially serving as a traceable and manipulatable platform to facilitate our understanding of organogenesis. Despite the ongoing advancement in generating organoids of diverse systems, biological applications of in vitro generated organoids remain as a major challenge in part due to a substantial lack of intricate complexity. The studies of development and regeneration enumerate the essential roles of highly diversified nonepithelial populations such as mesenchyme and endothelium in directing fate specification, morphogenesis, and maturation.

Common Genetic Variation in Humans Impacts In Vitro Susceptibility to SARS-CoV-2 Infection.

The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant interindividual variability. In addition to showing more disease in males, the elderly, and individuals with underlying comorbidities, SARS-CoV-2 can seemingly afflict healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants influence vulnerability to infection.

Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells.

Human organoids are emerging as a valuable resource to investigate human organ development and disease. The applicability of human organoids has been limited, partly due to the oversimplified architecture of the current technology, which generates single-tissue organoids that lack inter-organ structural connections. Thus, engineering organoid systems that incorporate connectivity between neighboring organs is a critical unmet challenge in an evolving organoid field.

Common genetic variation in humans impacts susceptibility to SARS-CoV-2 infection.

The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant inter-individual variability. In addition to showing more disease in males, the elderly, and individuals with underlying comorbidities, SARS-CoV-2 can seemingly render healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants also impact vulnerability to infection.

Single cell transcriptomics identifies a signaling network coordinating endoderm and mesoderm diversification during foregut organogenesis.

Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut.

The β-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts.

β-catenin is a multifunctional protein that plays crucial roles in embryonic development, physiological homeostasis, and a wide variety of human cancers. Previously, we showed that in vivo targeted ablation of β-catenin in melanoma-associated fibroblasts after melanoma formation significantly suppressed tumor growth. However, when the expression of β-catenin was ablated in melanoma-associated fibroblasts before tumor initiation, melanoma development was surprisingly accelerated.