Tadahiro Shinozawa

Safety attrition due to drug-induced inotropic changes remains a significant risk factor for drug development. Mitigating these events during early screening remains challenging. Several in vitro predictive models have been developed to address these issues, with varying success in detecting drug-induced inotropic changes. In this study, we compared traditional two-dimensional human-induced pluripotent stem cell-derived cardiomyocytes (2D hiPSC-CMs) with three-dimensional engineered cardiac tissues (3D ECTs) to assess their ability to detect drug-induced inotropic changes in 17 drugs with known mechanisms of action. The models were exposed to various test compounds, and their responses were evaluated by measuring either the active force or maximum contraction speed. The 3D ECTs successfully detected all the tested positive inotropes, whereas the 2D hiPSC-CMs failed to detect the 2 compounds. Both models demonstrated high predictability for negative inotropy and showed similar results for detecting non-active compounds, except for higher concentrations of phentolamine, zimelidine, and tamsulosin. Irregular beating was less likely to occur in the 3D ECTs, suggesting that 3D ECTs provided superior detection of contractility compared to 2D hiPSC-CMs. Genetic analysis revealed a more mature phenotype for the 3D ECTs compared to the 2D hiPSC-CMs, and the compound-related target expression was comparable to that in the adult human heart tissues. The 3D ECTs captured inotropic changes more accurately and thus represented a more translatable model than the 2D hiPSC-CMs. Overall, contractility assessment using the 3D ECTs could be advantageous for profiling candidate compounds and mechanistic investigations of hemodynamic changes during in vivo or clinical studies.

Developing a reliable method to predict thrombocytopenia is imperative in drug discovery. Here, we establish an assay using a microphysiological system (MPS) to recapitulate the in-vivo mechanisms of platelet aggregation and adhesion. This assay highlights the role of shear stress on platelet aggregation and their interactions with vascular endothelial cells. Platelet aggregation induced by soluble collagen was detected under agitated, but not static, conditions using a plate shaker and gravity-driven flow using MPS. Notably, aggregates adhered on vascular endothelial cells under gravity-driven flow in the MPS, and this incident increased in a concentration-dependent manner. Upon comparing the soluble collagen-induced aggregation activity in platelet-rich plasma (PRP) and whole blood, remarkable platelet aggregate formation was observed at concentrations of 30 µg/mL and 3 µg/mL in PRP and whole blood, respectively. Moreover, ODN2395, an oligonucleotide, induced platelet aggregation and adhesion to vascular endothelial cells. SYK inhibition, which mediated thrombogenic activity via glycoprotein VI on platelets, ameliorated platelet aggregation in the system, demonstrating that the mechanism of platelet aggregation was induced by soluble collagen and oligonucleotide. Our evaluation system partially recapitulated the aggregation mechanisms in blood vessels and can contribute to the discovery of safe drugs to mitigate the risk of thrombocytopenia.

Reliable fluid biomarkers for evaluating neurotoxicity have yet to be established. However, recent studies have reported neurofilament light chain as a fluid biomarker of several neurodegenerative disorders. In this study, we investigated changes in the cerebrospinal fluid and plasma levels of neurofilament light chain in mice treated with trimethyltin as a neurotoxicant. Trimethyltin diluted with saline was administered by intraperitoneal injection to mice at dose levels of 0 (vehicle control), 1.0, and 2.6 mg/kg body weight (dosage volume: 10 mL/kg). At 3 or 7 days after administration, animals were euthanized by exsanguination under 2–3% isoflurane inhalation anesthesia. Increased neurofilament light chain levels in both the cerebrospinal fluid and plasma were observed in animals from the trimethyltin 2.6 mg/kg body weight group, which indicated the brain lesions including neuronal cell death. Animals from the trimethyltin 1.0 mg/kg body weight group exhibited changes neither in neurofilament light chain levels in the cerebrospinal fluid and plasma nor in the histopathology of the brain at any time point. These data indicate that plasma neurofilament light chain can serve as a useful peripheral biomarker for detecting brain lesions such as neuronal necrosis in mice.

Organoids are three-dimensional structures fabricated in vitro from pluripotent stem cells or adult tissue stem cells via a process of self-organization that results in the formation of organ-specific cell types. Human organoids are expected to mimic complex microenvironments and many of the in vivo physiological functions of relevant tissues, thus filling the translational gap between animals and humans and increasing our understanding of the mechanisms underlying disease and developmental processes. In the last decade, organoid research has attracted increasing attention in areas such as disease modeling, drug development, regenerative medicine, toxicology research, and personalized medicine. In particular, in the field of toxicology, where there are various traditional models, human organoids are expected to blaze a new path in future research by overcoming the current limitations, such as those related to differences in drug responses among species. Here, we discuss the potential usefulness, limitations, and future prospects of human liver, heart, kidney, gut, and brain organoids from the viewpoints of predictive toxicology research and drug development, providing cutting edge information on their fabrication methods and functional characteristics.

Background: Sunitinib is known to cause cardiotoxicity in clinical settings. However, among sunitinib-treated patients experiencing adverse cardiac events, decreased cardiac function was reportedly reversible in > 50% of the patients. We previously showed that anti-cancer drugs such as sunitinib cause marked sarcomere disruption in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and the extent of sarcomere disruption can be used to predict drug-induced cardiotoxicity in humans. The aim of this study is to investigate whether the reversibility of sunitinib-induced cardiac events in clinical settings can be mimicked in vitro, and to examine the molecular mechanism responsible for sunitinib-induced cardiotoxicity focusing on the Hippo pathway. Methods: iPSC-CMs were stimulated with sunitinib for 72 h and the morphology of sarcomere structures were analyzed by high-content analysis before and after sunitinib washout. To examine the involvement of the Hippo pathway in the sunitinib-induced sarcomere disruption, the extent of nuclear localization of YAP1 (yes-associated protein 1, a Hippo signaling target) was determined. iPSC-CMs were also stimulated with sunitinib and a small molecule inhibitor of the Hippo pathway, XMU-MP-1 and sarcomere structures were analyzed. Results: We observed a spontaneous recovery in cardiac sarcomeres in iPSC-CMs that were significantly disrupted by sunitinib treatment after a 72 h or 144 h washout of sunitinib. The extent of nuclear localization of YAP1 was significantly reduced after sunitinib stimulation and tended to return to normal levels after drug washout. Simultaneous stimulation of iPSC-CM with sunitinib and XMU-MP-1 suppressed the sunitinib-induced disruption of sarcomeres. Conclusions: These results indicate that iPSC-CMs have the ability to recover from sunitinib-induced sarcomere disruption, and the Hippo pathway plays a role in the process of sunitinib-induced disruption of sarcomere and its recovery. Inhibition of the Hippo pathway may help to develop a co-medication strategy for mitigating the risk of sunitinib-induced adverse cardiac events.

Drug-induced toxicity remains one of the leading causes of discontinuation of the drug candidate and post-marketing withdrawal. Thus, early identification of the drug candidates with the potential for toxicity is crucial in the drug development process. With the recent discovery of human- Induced Pluripotent Stem Cells (iPSC) and the establishment of the differentiation protocol of human iPSC into the cell types of interest, the differentiated cells from human iPSC have garnered much attention because of their potential applicability in toxicity evaluation as well as drug screening, disease modeling and cell therapy. In this review, we expanded on current information regarding the feasibility of human iPSC-derived cells for the evaluation of drug-induced toxicity with a focus on human iPSCderived hepatocyte (iPSC-Hep), cardiomyocyte (iPSC-CMs) and neurons (iPSC-Neurons). Further, we CSAHi, Consortium for Safety Assessment using Human iPS Cells, reported our gene expression profiling data with DNA microarray using commercially available human iPSC-derived cells (iPSC-Hep, iPSC-CMs, iPSC-Neurons), their relevant human tissues and primary cultured human cells to discuss the future direction of the three types of human iPSC-derived cells.