Biology subjects

Yoshimoto, K.

Publications and source records attributed to Yoshimoto, K..

11 recordsLinked to original sources

bioRxiv2026

Growth-driven mechanical buckling as a mechanism of age-related tortuosity in human cerebral arteries

Human cerebral arteries become increasingly, sometimes abnormally, winding with age, but the mechanical basis of this change remains incompletely understood. To examine how arterial bends form and what sets their spacing, we modeled a cerebral artery as a slender elastic vessel supported by surrounding tissue. The distance between its ends remains constant, and the vessel can bend only in a plane tangent to the brain surface. As its natural length increases, axial compression builds until the vessel buckles. Bending partly relieves the compression, and the resulting waves grow in amplitude as elongation continues. Arterial bending stiffness and tissue support determine the wavelength, which is much larger than and proportional to vessel diameter, with comparatively weak dependence on other parameters. Numerical simulations captured a pre-buckling phase, rapid amplification of waves, and slow post-buckling growth during continued elongation. We evaluated these predictions using three-dimensional centerlines of cerebral arteries reconstructed from magnetic resonance angiography in healthy adults. Older age was associated with greater arterial length and excess-length ratio. For arterial waves oriented mostly parallel to the brain surface, wavelength increased with vessel diameter. Both this relationship and the observed wavelength-to-diameter ratio were consistent with model predictions. However, wave amplitude was not significantly associated with age. Overall, the modeling and imaging results support growth-driven buckling and post-buckling deformation under spatial constraint as a plausible mechanical explanation for age-related cerebral arterial tortuosity. This framework links vascular aging to growth-driven instability and pattern formation, with potential applications to diseased arteries beyond the brain.

biophysics↗

bioRxiv2026

Pharmacological induction of mitochondrial stress counteracts therapy resistance in glioblastoma stem-like cells

Glioblastoma (GBM) stem-like cells (GSCs) contribute to therapeutic resistance and recurrence. We sought to define cellular processes underlying GSC resilience. We discovered that GSCs, unlike differentiated GBM cells (DGCs) or non-malignant neural cells, depend on mitochondrial function for survival. To target this vulnerability, we exploited doxycycline (DOXY), an antibiotic used in humans, to interfere with mitochondrial protein translation. DOXY induced cell death and inhibited sphere formation in GSCs, but not in DGCs or non-malignant cells, indicating a differentiation state-selective effect. Mechanistically, DOXY induced mitochondrial dysfunction and activated a stress-responsive apoptotic program involving HRI-mediated signaling. DOXY displayed antitumor efficacy in patient-derived GBM organoid and orthotopic xenograft models. Our study reveals that DOXY can selectively target undifferentiated glioma cells, informing a drug repurposing-based strategy.

cancer biology↗

bioRxiv2025

High-Purity Production of Endothelial Cells from Human Pluripotent Stem Cells

Endothelial cells (ECs) are essential for vascular network formation and tissue homeostasis, yet the fields of tissue engineering and vascularized organoid generation still relies heavily on human umbilical vein ECs (HUVECs), which are venous, allogeneic, and difficult to mature fully. Human pluripotent stem cells (hPSCs) offer an autologous, developmentally flexible alternative, but most differentiation protocols require fluorescence-activated cell sorting, limiting scalability. Here we present a streamlined method that produces highly pure ECs directly from human embryonic stem cells (hESCs) without cell sorting. Extending Wnt activation with CHIR99021 to three days maximizes mesoderm induction, and brief Notch blockade with DAPT during specification suppresses smooth-muscle commitment. The result is over 90 % CD31+ CD144+ ECs that display classic cobblestone morphology, robust DiI-acetylated LDL uptake, and capillary-like sprouting comparable to HUVECs. Bulk RNA barcoding and sequencing segregates the hESC-derived ECs from both HUVECs and undifferentiated hESCs and uncovers an arterial-skewed transcriptome: NOTCH1, DLL4, and CXCR4 are up-regulated, whereas venous markers (EPHB4, NRP2) are reduced. Enrichment of Notch-responsive pathways further supports an arterial-like identity. Although several adult functional genes (e.g., vWF, NOS3) are expressed at lower levels than in HUVECs, the protocol delivers a scalable source of developmentally relevant ECs ideal for vascularizing organoids derived from the same hPSCs and for future applications in drug screening, disease modeling, and cell-based vascular therapies.

bioengineering↗

bioRxiv2024

Quantifying small GTPase activation status using a novel fluorescence HPLC-based assay

Small GTPases play crucial roles in cellular signaling pathways, with their activation states tightly regulated between GDP-bound inactive and GTP-bound active forms. Dysregulation of these nucleotide-binding states, such as in oncogenic RAS, is implicated in diseases like cancer. Accurately quantifying these states in cells is thus crucial for deciphering their functional roles and regulatory mechanisms. However, current methods do not fully meet the necessary sensitivity and versatility, limiting their effectiveness in small GTPase analysis. Here, we present a highly sensitive HPLC-based assay with fluorescence detection (Fluor-HPLC), enabling precise quantification of guanine nucleotide-binding states in small GTPases. Applying this technique, we successfully quantified the guanine nucleotide-binding states of RHEB and KRAS at their endogenous expression levels. We demonstrated the utility of Fluor-HPLC by elucidating RHEB activation dynamics in response to insulin stimulation and amino acid availability. Furthermore, integration of Fluor-HPLC with syngeneic mouse models provided insights into KRAS activation dynamics in tumor tissues and evaluated the effectiveness of targeted therapeutics. Overall, this versatile method paves the way for investigating activation states and regulatory mechanisms of various small GTPases, potentially accelerating our understanding of their roles in cellular processes and disease pathogenesis.

cell biology↗

bioRxiv2023

NMFClustering: Accessible NMF-based clustering utilizing GPU acceleration

SummaryNon-negative Matrix Factorization (NMF) is an algorithm that can reduce high dimensional datasets of tens of thousands of genes to a handful of metagenes which are biologically easier to interpret. Application of NMF on gene expression data has been limited by its computationally intensive nature, which hinders its use on large datasets such as single-cell RNA sequencing (scRNA-seq) count matrices. We have implemented NMF based clustering to run on high performance GPU compute nodes using CuPy, a GPU backed python library, and the Message Passing Interface (MPI). This reduces the computation time by up to three orders of magnitude and makes the NMF Clustering analysis of large RNA-Seq and scRNA-seq datasets practical. We have made the method freely available through the GenePattern gateway, which provides free public access to hundreds of tools for the analysis and visualization of multiple omic data types. Its web-based interface gives easy access to these tools and allows the creation of multi-step analysis pipelines on high performance computing (HPC) clusters that enable reproducible in silico research for non-programmers. Availability and ImplementationNMFClustering is freely available on the public GenePattern server at https://genepattern.ucsd.edu. Code for the NMFClustering is available under a BSD style license on github at https://github.com/genepattern/nmf-gpu. ContactTed Liefeld, jliefeld@cloud.ucsd.edu Supplementary InformationSupplementary data are available at Bioinformatics online and at https://datasets.genepattern.org/?prefix=data/test_data/NMFClustering/.

bioinformatics↗

bioRxiv2023

Cyclic mechanical stretching stimuli promotes angiocrine signals during in vitro liver bud formation from human pluripotent stem cells

Liver organoids derived from human pluripotent stem cells (hPSCs) allow elucidation of liver development and have great potential for drug discovery. However, current methods for generating liver organoids using biochemical substances do not realize the vascular network of the liver lobule, due to the lack of knowledge of the role of in vivo mechanical environments during liver development. Here, we investigate the role of cyclic mechanical stretch (cMS) to angiocrine signals of hepatoblasts (HBs) and endothelial progenitor cells (EPCs) using an organ-on-a-chip platform to emulate in vivo-like mechanical environments and hPSCs to recapitulate hepatic differentiation. RNA sequencing revealed that the expression of angiocrine signal genes, such as HGF and matrix metallopeptidase 9 (MMP9), was increased by cMS in co-cultured HBs and EPCs. The secretion of HGF and MMP9 increased by 3.23-folds and 3.72-folds with cMS in the co-cultured HBs and EPCs but was not increased by cMS in the mono-cultured HBs and EPCs. Immunofluorescence micrographs with anti-KRT19, HGF, and MMP9 antibodies also revealed that cMS increased HGF and MMP9 expression when HBs and EPCs were co-cultured. cMS increased HGF and MMP9 expression and secretion when HBs and EPCs were co-cultured. Our findings provide new insights into the mechanical factors involved in the vascular network of human liver bud formation and liver organoid generation.

bioengineering↗

bioRxiv2022

Human blastoid from primed human embryonic stem cells

Artificial human blastoids are used to investigate early embryo development, pregnancy failures, and birth deficiencies, previously impossible without human embryos. Recent methods generating blastoids used human naive pluripotent stem cells, which are prone to genomic instability during in vitro culturing. Here, we introduce a simple, robust, and scalable method for generating human blastoids from more stable, primed human pluripotent stem cells (hPSC). Using a non-cell-adhesive hydrogel, hPSC aggregates formed an asymmetric blastoid structure with a cellular distribution similar to that of a human blastocyst, with in vitro implantation capability. Single-cell RNA-seq followed by cellular trajectory analysis revealed that hydrogel promoted differentiation to tri-lineage cells associated with a blastocyst. This model will allow studies on the underlying mechanisms of human pre- and postimplantation processes, consider elaborating on the potential implications of the model on assistive reproductive technology. One Sentence SummaryNo more than 125 characters and spaces.

bioengineering↗

bioRxiv2021

Use of the Azure Kinect to measure foot clearance during obstacle crossing

Obstacle crossing is a typical adaptive locomotion known to be related to the risk of falls. Previous conventional studies have used elaborate and costly optical motion capture systems, which not only represent a considerable expense but also require participants to visit a laboratory. To overcome these shortcomings, we aimed to develop a practical and inexpensive solution for measuring obstacle-crossing behavior by using the Microsoft Azure Kinect, one of the most promising markerless motion capture systems. We validated the Azure Kinect as a tool to measure foot clearance and compared its performance to that of an optical motion capture system (Qualisys). We also determined the effect of the Kinect sensor placement on measurement performance. Sixteen healthy young men crossed obstacles of different heights (50, 150, and 250 mm). Kinect sensors were placed in front of and beside the obstacle as well as diagonally between those positions. As indices of measurement quality, we counted the number of measurement failures and calculated the systematic and random errors between the foot clearance measured by the Kinect and Qualisys. We also calculated the Pearson correlation coefficients between the Kinect and Qualisys measurements. The number of measurement failures and the systematic and random error were minimized when the Kinect was placed diagonally in front of the obstacle on the same side as the trail limb. The high correlation coefficient (r > 0.890) observed between the Kinect and Qualisys measurements suggests that the Azure Kinect has excellent potential for measuring foot clearance during obstacle-crossing tasks.

bioengineering↗

bioRxiv2020

Recapitulation of human embryonic heart beating to promote differentiation of hepatic endoderm to hepatoblasts

A microfluidic platform recapitulating human embryonic heart beat improves the functionalization state of hepatocytes derived from hepatic endoderm (HE). Mechanical stretching of mimicked heart beats was applied to HE cells cultured on the microfluidic platform. Stimulated HE-derived hepatoblasts increased cytochrome P450 3A (CYP3A) metabolic activities and hepatoblast functional markers expression, leading for advancement of regenerative medicine and drug screening.

bioengineering↗

bioRxiv2020

39°C-in vitro culture facilitates hepatic functions of hepatocyte-like cells derived from human embryonic stem cells

Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. Although tremendous efforts have been made to facilitate hepatic functions of hPSC-HLCs using growth factors and chemicals, these cells have not yet reached hepatic functions comparable to hepatocytes in vivo. Therefore, there exists a critical need to use an alternative trigger to facilitate hepatic functions in hPSC-HLCs. We noted that human liver temperature (around 39{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), yet hepatocytes are generally cultured at 37{degrees}C in-vitro. Here we showed that hepatic functions of hPSC-HLCs would be facilitated under physiological liver temperatures. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39{degrees}C and 42{degrees}C. 42{degrees}C-treatment caused significantly greater cell death compared to 39{degrees}C. We also confirmed the increases of hepatic functions, such as secretion of albumin, cytochrome P450 3A4 (CYP3A4) activities, and collagen productions, without severe cell damages. To elucidate the underlying mechanisms of heat-induced hepatic functions, RNA-seq was to identify gene expression signatures due to 39{degrees}C-treated hESC-HLCs. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs, and lead to the realization of drug discovery efforts and drug toxicological tests. Significance statementHepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. We noted that human liver temperature (around 39{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), affecting the in-vitro hepatic functions of hPSC-HLCs, such as metabolic activities. Here we showed that hepatic functions of hPSC-HLCs, albumin secretion, CYP3A4 activities, and collagen production would be facilitated under physiological liver temperatures at 39{degrees}C, without severe cell damages. RNA-seq was used to elucidate the underlying mechanisms of heat-induced hepatic functions. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons.

bioengineering↗

bioRxiv2019

Direct synthesis of self-organized blastocyst-like cysts derived from human pluripotent stem cells

We introduce a simple, robust and scalable method to generate self-organized blastocyst-like cysts (soBLCs) from human pluripotent stem cells (hPSCs). We use a copolymer hydrogel of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG). hPSC aggregates with a diameter of approximately 117.2 {+/-} 5.1 {micro}m are cultured in a medium supplemented with a hydrogel and a serum for three days. Molecular signatures in the medium revealed the generation of trophoblasts and inner cell mass at specific positions in the soBLCs.

bioengineering↗