developmental-toxicity-assay-based-on-real-time-monitoring-fibroblast

Developmental Toxicity Assay Based on Real-Time Monitoring of Fibroblast Growth Factor Signal Disruption in Human Induced Pluripotent Stem Cells

Developmental toxicity (Devtox) is typically evaluated in animal models. However, there are challenges in ensuring accuracy, such as false negatives due ...

National Institute of Health Sciences

In this study, a highly transformable electrocardiograph that can considerably deform the position of stretchable electrodes based on the lead method for diagnosing heart disease was developed; these electrodes exhibited high resistance stability against considerable stretching and multiple stretching. To realize the large deformable functionality of the electrodes of a system, liquid metal electrodes and a heteroconnector composed of a liquid metal paste and carbon-based conductive rubber were employed. The developed device can achieve a 200% strain with only 6% resistance change and a high stability of resistances after the 100-time stretching test. In addition, the study demonstrated electrocardiograms in different lead methods of adult and child using the same device. The proposed combination of large deformable electrodes with high electric stability and a robust heteroconnector is an important technology, and it presents a considerable advancement in the application of stretchable electronic systems.

Transformable Electrocardiograph Using Robust Liquid-Solid Heteroconnector.

Neonatal jaundice occurs in >80% of newborns in the first week of life owing to physiological hyperbilirubinemia. Severe hyperbilirubinemia could cause brain damage owing to its neurotoxicity, a state commonly known as kernicterus. Therefore, periodic bilirubin monitoring is essential to identify infants at-risk and to initiate treatment including phototherapy. However, devices for continuous measurements of bilirubin have not been developed yet. Here, we established a wearable transcutaneous bilirubinometer that also has oxygen saturation (SpO) and heart rate (HR) sensing functionalities. Clinical experiments with neonates demonstrated the possibility of simultaneous detection of bilirubin, SpO, and HR. Moreover, our device could consistently measure bilirubin during phototherapy. These results demonstrate the potential for development of a combined treatment approach with an automatic link via the wearable bilirubinometer and phototherapy device for optimization of the treatment of neonatal jaundice.

Neonatal wearable device for colorimetry-based real-time detection of jaundice with simultaneous sensing of vitals.

Notch-Delta signaling regulates many developmental processes, including tissue homeostasis and maintenance of stem cells. Upon interaction of juxtaposed cells via Notch and Delta proteins, intracellular domains of both transmembrane proteins are cleaved and translocate to the nucleus. Notch intracellular domain activates target gene expression; however, the role of the Delta intracellular domain remains elusive. Here, we show the biological function of Delta like 1 intracellular domain (D1ICD) by modulating its production. We find that the sustained production of D1ICD abrogates cell proliferation but enhances neurogenesis in the developing dorsal root ganglia (DRG), whereas inhibition of D1ICD production promotes cell proliferation and gliogenesis. D1ICD acts as an integral component of lateral inhibition mechanism by inhibiting Notch activity. In addition, D1ICD promotes neurogenesis in a Notch signaling-independent manner. We show that D1ICD binds to Erk1/2 in neural crest stem cells and inhibits the phosphorylation of Erk1/2. In summary, our results indicate that D1ICD regulates DRG development by modulating not only Notch signaling but also the MAP kinase pathway.

Cleaved Delta like 1 intracellular domain regulates neural development via Notch signal-dependent and -independent pathways.

The number of man-made chemicals has increased rapidly in recent decades, with certain chemicals potentially causing malformations in fetuses. Although the toxicities of chemicals have been tested in animals, chemicals that are not teratogenic in rodents can cause severe malformations in humans, owing to the differences in the susceptibility to the teratogenicity of chemicals among species. One possible cause of such species differences, other than pharmacokinetics, could be the difference in sensitivity to such chemicals at the cellular level. Therefore, a human cell-based high-throughput assay system is needed for detecting potential teratogenic chemicals. In this study, we proposed a signal reporter assay using human induced pluripotent stem cells (iPSCs). Because developmental processes are governed by highly intricate and precisely programmed signaling pathways, external chemical-induced disruption of these pathways often triggers developmental toxicities. The reporter assay using hiPSCs was used to detect changes in the fibroblast growth factor (FGF) signaling pathway, a pathway essential for limb morphogenesis. The method was based on monitoring and time-accumulation of the signal disruption over time, rather than the classical endpoint detection of the signal disruption. This approach was useful for detecting signal disruptions caused by the malformation chemicals listed in the ICH S5 guideline, including thalidomide. The human iPSC-based signal disruption assay could be a promising tool for the initial screening of developmental toxicants.

Integrated fibroblast growth factor signal disruptions in human iPS cells for prediction of teratogenic toxicity of chemicals.

The number of man-made chemicals has increased exponentially recently, and exposure to some of them can induce fetal malformations. Because complex and precisely programmed signaling pathways play important roles in developmental processes, their disruption by external chemicals often triggers developmental toxicity. However, highly accurate and high-throughput screening assays for potential developmental toxicants are currently lacking. In this study, we propose a reporter assay that utilizes human-induced pluripotent stem cells (iPSCs) to detect changes in fibroblast growth factor signaling, which is essential for limb morphogenesis. The dynamics of this signaling after exposure to a chemical were integrated to estimate the degree of signaling disruption, which afforded a good prediction of the capacity of chemicals listed in the ECVAM International Validation Study that induce limb malformations. This study presents an initial report of a human iPSC-based signaling disruption assay, which could be useful for the screening of potential developmental toxicants.

Establishment of a developmental toxicity assay based on human iPSC reporter to detect FGF signal disruption.

We describe a protocol for a live-cell luciferase assay system for continuously monitoring fibroblast growth factor (FGF) signal disruption in human-induced pluripotent stem cells (iPSCs). Signal disrupting effects of chemicals are used as an indicator to evaluate toxicity. The assay is reliably predictive of the effects of limb malformation chemicals (AUC = 0.93). The current approach is limited to FGF signal disruption, and combinations with other types of signaling will be required to detect the effects of different toxicants. For complete details on the use and execution of this protocol, please refer to Kanno et al. (2022a).

Luciferase assay system to monitor fibroblast growth factor signal disruption in human iPSCs.

During embryonic development, reciprocal interactions between epidermal and mesenchymal layers trigger hair follicle morphogenesis. This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro. A key approach is to modulate spatial distributions of epithelial and mesenchymal cells in their spontaneous organization. The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids, and hair shafts sprouted with near 100% efficiency in vitro. The hair shaft length reached ~3 mm in culture. Typical trichogenic signaling pathways were up-regulated in hair follicloids. Owing to replication of hair follicle morphogenesis in vitro, melanosome production and transportation were also monitored in the hair bulb region. This in vitro hair follicle model might be valuable for better understanding hair follicle induction, evaluating hair growth and inhibition of hair growth by drugs, and modeling gray hairs in a well-defined environment.

Article	Year
Transformable Electrocardiograph Using Robust Liquid-Solid Heteroconnector. ACS sensors\| PubMed ID: 33395271	2021
Neonatal wearable device for colorimetry-based real-time detection of jaundice with simultaneous sensing of vitals. Science advances\| PubMed ID: 33658197	2021
Cleaved Delta like 1 intracellular domain regulates neural development via Notch signal-dependent and -independent pathways. Development (Cambridge, England)\| PubMed ID: 34519339	2021
Integrated fibroblast growth factor signal disruptions in human iPS cells for prediction of teratogenic toxicity of chemicals. Journal of bioscience and bioengineering\| PubMed ID: 35034848	2022
Establishment of a developmental toxicity assay based on human iPSC reporter to detect FGF signal disruption. iScience\| PubMed ID: 35146387	2022
Luciferase assay system to monitor fibroblast growth factor signal disruption in human iPSCs. STAR protocols\| PubMed ID: 35677614	2022
Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction. Science advances\| PubMed ID: 36269827	2022

Yusuke Okubo

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