Biology subjects

Toyoda, M.

Publications and source records attributed to Toyoda, M..

15 recordsLinked to original sources

bioRxiv2024

Comparative genomic analysis of multidrug-resistant clinical isolates of Mycobacterium abscessus revealed worldwide horizontal plasmid transfer

Genetic epidemiological analysis of mobile genetic elements, such as plasmids, has rarely been carried out in Mycobacterium abscessus, regardless of its usefulness in speculating the past contact between bacteria. In this study, through whole genome sequencing analysis of clinical isolates of M. abscessus sequentially collected from the same patient, we identified a single nucleotide variant that could cause antibiotic resistance, and three plasmids (pMAB625-1, pMAB625-2 and pMAB625-3) not found in the type strain. We investigated the distribution of plasmids previously identified in M. abscessus, including pMAB625 plasmids, in 462 clinical isolates worldwide in silico. pMAB625-3 was detected in the largest number of isolates. Furthermore, phylogenetic tree analysis revealed that these plasmids transferred beyond regions and subspecies and acquired unique mutations. These results indicate that transmission of plasmids increases the genomic diversity in M. abscessus, and plasmid epidemiology is useful for estimating the past contact between bacteria.

genomics↗

bioRxiv2023

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 2^20-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

cell biology↗

bioRxiv2022

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75

SARS-CoV-2 Omicron BA.2.75 emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically different from BA.5, the currently predominant BA.2 descendant. Here, we showed that the effective reproduction number of BA.2.75 is greater than that of BA.5. While the sensitivity of BA.2.75 to vaccination- and BA.1/2 breakthrough infection-induced humoral immunity was comparable to that of BA.2, the immunogenicity of BA.2.75 was different from that of BA.2 and BA.5. Three clinically-available antiviral drugs were effective against BA.2.75. BA.2.75 spike exhibited a profound higher affinity to human ACE2 than BA.2 and BA.5 spikes. The fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were comparable to those of BA.5 but were greater than those of BA.2. Our multiscale investigations suggest that BA.2.75 acquired virological properties independently of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

microbiology↗

bioRxiv2022

The SARS-CoV-2 Omicron BA.1 spike G446S potentiates HLA-A*24:02-restricted T cell immunity

Although the Omicron variant of the SARS-CoV-2 virus is resistant to neutralizing antibodies, it retains susceptibility to cellular immunity. Here, we characterized vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppressed Omicron BA.1 replication. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication was observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation was lost when target cells were treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell immunity towards emerging variants.

immunology↗

bioRxiv2022

SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant

Recent studies have revealed the unique virological characteristics of Omicron, the newest SARS-CoV-2 variant of concern, such as pronounced resistance to vaccine-induced neutralizing antibodies, less efficient cleavage of the spike protein, and poor fusogenicity. However, it remains unclear which mutation(s) in the spike protein determine the virological characteristics of Omicron. Here, we show that the representative characteristics of the Omicron spike are determined by its receptor-binding domain. Interestingly, the molecular phylogenetic analysis revealed that the acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidate that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue in another protomer in the spike trimer, which confers the attenuated spike cleavage efficiency and fusogenicity of Omicron. Our data shed light on the evolutionary events underlying Omicron emergence at the molecular level. HighlightsO_LIOmicron spike receptor binding domain determines virological characteristics C_LIO_LISpike S375F mutation results in the poor spike cleavage and fusogenicity in Omicron C_LIO_LIAcquisition of the spike S375F mutation triggered the explosive spread of Omicron C_LIO_LIF375-H505-mediated {pi}-{pi} interaction in the spike determines the phenotype of Omicron C_LI

microbiology↗

bioRxiv2022

Virological characteristics of SARS-CoV-2 BA.2 variant

Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref1, 2), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.

microbiology↗

bioRxiv2021

Hepatocytes with a phenotype of substantial CYP3A4 induction generated from drug-associated fulminant hepatitis-derived induced pluripotent stem cells

Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.

cell biology↗

bioRxiv2021

Drug metabolism activity is a Critical Intrinsic Selector for Hepatocytes to elongate cell lifespan by using a cell-killing antibiotic

Hepatocytes can be used to study the pathogenesis of liver diseases and drug discovery research. Human hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. We purified human hepatocytes by selection with cytocidal puromycin, and cultured them for more than 60 population doublings over a span of more than 350 days. These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

cell biology↗

bioRxiv2021

An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity

During the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/438288v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@153428forg.highwire.dtl.DTLVardef@136ca5aorg.highwire.dtl.DTLVardef@1ee490org.highwire.dtl.DTLVardef@2fe478_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Puromycin selection of cells with a high expression of the cytochrome P450 CYP3A4 gene activity from a patient with drug-induced liver injury (DILI) and their lifespan prolongation using a combination of CDK4R24C, cyclin D1 and TERT

Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can accurately evaluate drug-mediated CYP3A4 induction as the gold standard for in vitro hepatic toxicology test, but their lot variation is an issue to be solved. Only a limited number of immortalized hepatocyte cells have been reported. In this study, we generated an immortalized cell expressing CYP3A4 from a patient with drug-induced liver injury (DILI). To generate DILI-derived cells with a high expression of CYP3A4, we employed a three-step approach: 1. Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); 2. Immortalization of the differentiated cells; 3. Selection of the cells with puromycin. We hypothesize that cells with a high expression of cytochrome P450 genes can survive even after exposure to cytotoxic antibiotics because of high drug-metabolism activity. Puromycin, one of the cytotoxic antibiotics, was used in this study because of its rapid cytocidal effect at a low concentration. Phenotypic studies in vitro revealed that the puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at an extremely high level, and continued to proliferate at least up to 34 population doublings for more than 250 days. The expression profiles were independent of population doublings. Drug-mediated induction test revealed that the cells significantly increased CYP3A4 after exposure to rifampicin, suggesting that the immortalized cells would serve as another useful source for in vitro examination of drug metabolism and CYP3A4 induction.

cell biology↗

bioRxiv2020

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammonia-selected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

cell biology↗

bioRxiv2019

Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1

Gorlin syndrome is a rare autosomal dominant hereditary disease with high incidence of tumors such as basal cell carcinoma and medulloblastoma. Disease-specific induced pluripotent stem cells (iPSCs) have now been used as a model to analyze disease pathogenesis as well as an animal model. In this study, we generated iPSCs derived from fibroblasts of four patients with Gorlin syndrome (Gln-iPSCs) with a heterozygous mutation of the PTCH1 gene. Gln-iPSCs from the four patients developed medulloblastoma in 100% (four out of four), a manifestation of Gorlin syndrome, in the teratomas after implantation into immunodeficient mice, but none (0/584) of the other iPSC-teratomas. One of the medulloblastomas had loss of heterozygosity in the PTCH1 gene while benign teratoma, i.e. non-medulloblastoma part, did not, indicating a close clinical correlation between tumorigenesis in Gorlin syndrome patients and Gln-iPSCs.

cell biology↗

bioRxiv2019

Immortalization of human zone I hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

BackgroundHepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia.\n\nMethodsHepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10.\n\nResultsHepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings.\n\nConclusionsHepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

cell biology↗

bioRxiv2018

Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hyper-methylated regions compared with embryonic stem cells. These random aberrant hyper-methylations might be a cause of the differences in the properties of each of the iPSC lines.

cell biology↗

bioRxiv2017

Can Human Embryonic Stem Cell-Derived Stromal Cells Serve A Starting Material For Myoblasts?

A large number of myocytes is necessary to treat intractable muscular disorders such as Duchenne muscular dystrophy with cell-based therapies. However, starting materials for cellular therapy products such as myoblasts, marrow stromal cells, menstrual blood-derived cells and placenta-derived cells have a limited lifespan and cease to proliferate in vitro. From the viewpoints of manufacturing and quality control, cells with a long lifespan are more suitable as a starting material. In this study, we generated stromal cells for future myoblast therapy from a working cell bank of human embryonic stem cells (ESCs). The ESC-derived CD105+ cells with extensive in vitro proliferation capability exhibited myogenesis and genetic stability in vitro. These results imply that ESC-derived CD105+ cells are another cell source for myoblasts in cell-based therapy for patients with genetic muscular disorders. Since ESCs are immortal, mesenchymal stromal cells generated from ESCs can be manufactured at a large scale in one lot for pharmaceutical purposes.

cell biology↗