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

Abstract

Summary 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.

Fungal Depsidones Stimulate AKT-Dependent Glucose Uptake in 3T3-L1 Adipocytes

“…Cell viability was determined using a cell viability assay kit (Fluorometric-green, Abcam) according to the manufacturer’s instructions. Viable cells were stained with a hydrophobic green fluorescent dye . The fluorescence intensity was determined at Ex/Em ≈ 490 nm/520 nm using a SPARK multimode microplate reader (TECAN).…”

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

“…The two key reagents required are a construct expressing the Nano-luciferase ( Nluc ) gene downstream of the serum response element (SRE) and a vector backbone with adeno-associated virus integration site 1 ( AAVS1 ) homology arms of about 800 bp at both ends of the donor plasmid for efficient knock-in ( Figure 1 A). The following protocol has been used to successfully knock-in target DNA in the AAVS1 region using CRISPR-Cas9 genome editing technology after introducing the SRE-Nluc plasmid into human iPS cells by electroporation ( Kanno et al., 2022a , Kanno et al, 2022b ).

Figure 1 Generation of FGF signal reporter cells from human iPSCs (A) Schematic illustration for plasmid vector construction.

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“…To assess the magnitude of signal disruption-based on temporal changes in FGF signal reporter activity, we developed the R software outlined in Kanno et al (2022a) . The steps below identify the prerequisites necessary for running the software.…”

“…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) .…”

“…For complete details on the use and execution of this protocol, please refer to Kanno et al (2022a) .…”