Biology subjects

Hagiwara, M.

Publications and source records attributed to Hagiwara, M..

10 recordsLinked to original sources

bioRxiv2026

Engineering developmental transitions during human neurulation through modular integration of multimodal spatial cues

Reconstituting organ development in vitro requires reproducing dynamic developmental transitions rather than only terminal tissue structures. Here, we developed MoSAIC (Modular Spatial Assembly and Integration of Cues), a modular platform that decomposes complex culture operations and subsequently integrates them to reconstruct multimodal mechano-chemical spatial cues. Using human induced pluripotent stem cells, MoSAIC enabled independent control of tissue geometry, extracellular matrix localization, physical boundaries, and morphogen gradients, while also permitting defined mechanical and chemical perturbations during development. Their integration guided a continuous ectodermal sheet through neural induction, tissue bending, and bifurcation into neural and epidermal tissues, followed by formation of a polarized neural tube structure. Spatially controlled morphogen exposure further established dorsoventral patterning, while the resulting tissue exhibited posterior neural tube identity. Using the developed model recapitulating the transition of human neurulation, MoSAIC further enables non-genetic perturbation of mechanical and chemical cues to interrogate the mechanisms governing neural tube closure.

bioengineering↗

bioRxiv2025

Resilin, the gene for the molecular spring: its roles in the flight and jumpingof Drosophila

Resilin is a member of the chitin-binding protein family that was originally found as a major component of an elastic matrix present in insects. The knockdown of the gene for this protein in Drosophila melanogaster causes the characteristic downturned wing posture, and the knockdown flies cannot move their wings. The jump distance of the knockdown flies is around 50% shorter than that of the controls. Null mutant flies show the same phenotypes, which can be rescued by introducing a 4.7 kb genomic fragment harboring the whole coding region of Resilin. In vitro experiments have shown that the elasticity of the matrix made of Resilin is due to the dityrosine-mediated polymerization of Resilin molecules. Dual oxidase (Duox) is the most likely candidate for the in vivo polymerization of Resilin molecules. Duox knockdown induces phenotypes similar to those observed in wing posture and jump performance of the Resilin knockdown and knockout flies, which can be rescued by the overexpression of Duox gene from a beetle. These findings suggest that Duox is an essential factor for the proper function of Resilin as the major component of the resilin matrix.

genetics↗

bioRxiv2025

Modeling the human limb skeletal development using human pluripotent stem cell-derived skeletal assembloid

Despite recent advances in pluripotent stem cell-based approaches to induce skeletal cells, recapitulating human limb skeletal development in terms of structure and longitudinally oriented growth remains an unresolved challenge. Here, we report a method to differentiate human pluripotent stem cells into region-specific skeletal organoids harboring GDF5+PRG4+ interzone/articular chondrocyte progenitors (IZ/ACPs) and SP7+ growth plate chondrocytes (GPCs) via PRRX1+ limb-bud mesenchymal cells. Comparative analysis demonstrated marked similarities of IZ/ACP and GPC organoids to the human embryonic limb, and graft fate and regenerative capacity in vivo were further characterized. We also mimicked the limb skeletal developmental process in a spatially structured manner by vertically positioning two IZ/ACP organoids at both ends of a GPC organoid to generate a human skeletal assembloid. Notably, this human skeletal assembloid recapitulated endochondral ossification with longitudinal skeletal growth upon transplantation. In summary, our study provides a novel research platform for human limb skeletal development and disease.

bioengineering↗

bioRxiv2024

Nitric oxide regulates phagocytosis through S-nitrosylation of Rab5

Phagocytosis is mediated mainly by immune cells, such as macrophages, monocytes and neutrophils, that function to clear large pathogens including bacteria. The small GTP-binding protein Rab5 is crucial for clathrin-dependent endocytosis as well as phagocytosis. However, the role and mechanism of Rab5 activation during phagocytosis are poorly understood. Here we report that nitric oxide (NO), a novel regulator of Rab5, regulates phagocytosis through S-nitrosylation of Rab5. NO can promote phagocytosis by activating Rab5 in cultured cells, and it potently S-nitrosylates active Rab5 compared to inactive Rab5. Moreover, we demonstrate that two cysteine residues in the C terminus of Rab5 are S-nitrosylated and are important for phagocytosis. Experiments involving mice also showed that NO activates Rab5 and increases levels of S-nitrosylated Rab5 and that NO is involved in phagocytic bacterial clearance mediated by peritoneal macrophages. These data suggest that NO promotes S-nitrosylation of Rab5 to act as a novel Rab5 activator and a key regulator of phagocytosis. HighlightsO_LINO promotes phagocytosis C_LIO_LINO directly activates Rab5 and increases Rab5 S-nitrosylation C_LIO_LIS-nitrosylation C-terminal Rab5 cysteines residues is involved in phagocytosis C_LIO_LINO increases Rab5 S-nitrosylation and activity to promote bacterial clearance in vivo C_LI

biochemistry↗

bioRxiv2024

Transcriptional landscape of the dorsal raphe serotonin neurons rendering stress resiliency

Major depressive disorder (MDD) is a serious and large social problem, yet the pathophysiology of MDD and the action mechanism of antidepressants are still poorly understood. A number of studies have reported that activation and inactivation of serotonin neurons in the dorsal raphe nucleus (DRN) cause antidepressant-like effects and depressive-like behaviors, respectively. Also, their physiological neural activities are increased when mice were chronically administered an SSRI and decreased in mice exposed to chronic social defeat stress (CSDS), a mouse model of depression. However, the molecular mechanism underlying these neural activity changes in DRN serotonin neurons remains unclear. In this study, we performed a DRN serotonin neuron-specific comprehensive gene expression analysis by using Translating Ribosome Affinity Purification (TRAP) technology in both chronic SSRI-treated mice as a model of antidepressant treatment and CSDS mice as a model of depression. It revealed that many gene expression changes were the opposite between SSRI-treated mice and CSDS-susceptible mice. Among these, we identified S100a10 as a prodepressive gene in DRN serotonin neurons, and we found that Interleukin-4 (IL-4) - Signal Transducer and Activator of Transcription 6 (STAT6) pathway and 5-HT1B receptor were the upstream and downstream molecules of S100a10, respectively. Our findings provide insights into molecular mechanisms underlying the action of antidepressants and stress resiliency.

pharmacology and toxicology↗

bioRxiv2023

Modular Tissue-in-a-CUBE Platform to Model Blood-brain-Barrier (BBB) and Brain Interaction

With the advent of increasingly sophisticated organoids, there is growing demand for technology to replicate the interactions between multiple tissues or organs. This is challenging to achieve, however, due to the varying culture conditions of the different cell types that make up each tissue. Current methods often require complicated microfluidic setups, but fragile tissue samples tend not to fare well with rough handling. Furthermore, the more complicated the human system to be replicated, the more difficult the model becomes to operate. Here, we present the development of a multi-tissue chip platform that takes advantage of the modularity and convenient handling ability of the CUBE device. We first developed a blood-brain barrier (BBB)-in-a-CUBE by layering astrocytes, pericytes, and brain microvascular endothelial cells in the CUBE, and confirmed the expression and function of important tight junction and transporter proteins in the BBB model. Then, we demonstrated the application of integrating Tissue-in-a-CUBE with a chip in simulating the testing of the permeability of a drug through the blood-brain barrier (BBB) to the brain and its effect on treating brain cancer. We anticipate that this platform can be adapted for use with organoids to build complex human systems in vitro by the combination of multiple simple CUBE units.

bioengineering↗

bioRxiv2022

Localization of multiple hydrogels with MultiCUBE platform spatially guides 3D tissue morphogenesis in vitro

Localization of multiple hydrogels is expected to develop the structure of 3D tissue models in a location specific manner. Here, we successfully localize morphogenesis within individual tissues by exposing different hydrogel conditions to different parts of the tissues. We develop a unit-based scaffold with a unique frame design to trap hydrogel solutions inside their designated units. Interestingly, this unit-based scaffold within an optimal range of dimensional size and surface wettability can trap several cubic millimeters of hydrogels. This localization capability enables the spatial organization of hydrogel compositions, growth factors and physical conditions, as well as the position of biological samples (cells, spheroids, reconstituted tissues) relative to each hydrogel compartment. We succeed to localize the branching development of reconstituted human epithelial tissues according to the localized biomolecular and physical cues from hydrogels, regardless of the initial tissue configurations. Unlike 3D-bioprinting or microfluidics, the localization with this unit-based scaffold requires only manual pipetting and handling without any specialized equipment or skills, thus ready to use by researchers from any field. This scaffold-based localization provides a new promising route to spatially control morphogenesis, differentiation, and other developmental processes within organoids or other 3D tissues, resulting in 3D functional models for practical biomedical applications.

bioengineering↗

bioRxiv2022

From Gradient to Sectioning in CUBE: Workflow for Generating and Imaging Organoid with Localized Differentiation

Advancements in organoid culture methods have led to a widespread availability of various in vitro mini-organs that mimic native tissues in many ways. Yet, the bottleneck remains to generate complex organoids with body axis patterning, as well as keeping the orientation of organoid samples during post-experiment analysis processes. In this paper, we present a workflow for culturing organoids with gradient using a previously developed CUBE culture device, then sectioning samples with the CUBE to retain information on the gradient direction of the sample. We show that hiPSC spheroids cultured with separate differentiation media on two opposing ends of the CUBE resulted in localized expressions of the respective differentiation markers, in contrast to homogeneous distribution of markers in controls with the same media on both ends. We also describe the processes for cryo and paraffin sectioning of gradient-cultured spheroids in CUBE to retain gradient orientation information. This workflow from gradient culture to sectioning with CUBE can provide researchers with a convenient tool to generate increasingly complex organoids and study their developmental processes in vitro.

bioengineering↗

bioRxiv2021

Switching of OAS1 splicing isoforms mitigates SARS-CoV-2 infection

BackgroundThe rapidly accumulating disease susceptibility information collected from coronavirus disease (COVID-19) patient genomes must be urgently utilized to develop therapeutic interventions for SARS-CoV-2 infection. Chromosome 12q24.13, which encodes the 2-5-oligoadenylate synthetase (OAS) family of proteins that sense viral genomic RNAs and trigger an antiviral response, is identified as one of the genomic regions that contains SNPs associated with COVID-19 severity. A high-risk SNP identified at the splice acceptor site of OAS1 exon 6 is known to change the proportions of the various splicing isoforms and the activity of the enzyme. MethodsWe employed in-silico motif search and RNA pull-down assay to define a factor responsible for the OAS1 splicing. Next, we rationally selected a candidate for slicing modulator to modulate this splicing. ResultsWe found that inhibition of CDC-like kinase with a small chemical compound induces switching of OAS1 splice isoforms in human lung cells. In this condition, increased resistance to SARS-CoV-2 infection, enhanced RNA degradation, and transcriptional activation of interferon {beta}1, were also observed. ConclusionsThe results indicate the possibility of using chemical splicing modifiers aided by genome-based precision medicine to boost the innate immune response against SARS-CoV-2 infection.

genomics↗

bioRxiv2018

Sub-voxel light-sheet microscopy for high-resolution, high-throughput volumetric imaging of large biomedical specimens

A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). Here, we report a sub-voxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular-resolution. A non-axial, continuous scanning strategy is used to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then by adopting a sub-voxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and iteratively recovers a 3-D image with improved resolution throughout the sample. This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times, with high acquisition speeds of gigavoxels per minute. As demonstrated by quick reconstruction (minutes to hours) of various samples, e.g., 3-D cultured cells, an intact mouse heart, mouse brain, and live zebrafish embryo, the SLSM method presents a high-throughput way to circumvent the tradeoff between intoto mapping of large-scale tissue (>100 mm3) and isotropic imaging of single-cell (~1-m resolution). It also eliminates the need of complicated mechanical stitching or precisely modulated illumination, using a simple light-sheet setup and fast graphics-processing-unit (GPU)-based computation to achieve high-throughput, high-resolution 3-D microscopy, which could be tailored for a wide range of biomedical applications in pathology, histology, neuroscience, etc.

bioengineering↗