Polyoxyethylene tallow amine and glyphosate exert different developmental toxicities on human pluripotent stem cells-derived heart organoid model.

The early stage of heart development is highly susceptible to various environmental factors. While the use of animal models has aided in identifying numerous environmental risk factors, the variability between species and the low throughput limit their translational potential. Recently, a type of self-assembling cardiac structures, known as human heart organoids (hHOs), exhibits a remarkable biological consistency with human heart. However, the feasibility of hHOs for assessing cardiac developmental risk factors remains unexplored. Here, we focused on the cardiac developmental effects of core components of Glyphosate-based herbicides (GBHs), the most widely used herbicides, to evaluate the reliability of hHOs for the prediction of possible cardiogenesis toxicity. GBHs have been proven toxic to cardiac development based on multiple animal models, with the mechanism remaining unknown. We found that polyoxyethylene tallow amine (POEA), the most common surfactant in GBHs formulations, played a dominant role in GBHs' heart developmental toxicity. Though there were a few differences in transcriptive features, hHOs exposed to sole POEA and combined POEA and Glyphosate would suffer from both disruption of heart contraction and disturbance of commitment in cardiomyocyte isoforms. By contrast, Glyphosate only caused mild epicardial hyperplasia. This study not only sheds light on the toxic mechanism of GBHs, but also serves as a methodological demonstration, showcasing its effectiveness in recognizing and evaluating environmental risk factors, and deciphering toxic mechanisms.

Generation of a human embryonic stem cell line (SKLRMe004-A) carrying NKX2.5-EGFP and TBX5-Tdtomato dual fluorescent reporters.

TBX5 and NKX2-5 are two important transcription factors regulating cardiomyocytes commitment. In this study, we generated a dual fluorescent reporter cell line based on H9 human embryonic stem cells (hESCs) and called it H9-NKX2.5GFP/TBX5Td-T2A-18, which could be short for T2A-18. When T2A-18 was induced towards a cardiomyocyte fate, EGFP and Tdtomato fluorescences were observed, indicating the expression of NKX2.5 and TBX5, respectively. Meanwhile, T2A had normal karyotype and could maintain main characteristics of wildtype H9. Therefore, T2A-18 could be used as a tool to help us study the mechanism of cardiomyocytes specification from hESCs.

Generation of a human extended pluripotent stem cell line (SKLRMe002-A) carrying a doxycycline-inducible Cas9 expression cassette.

Human extended pluripotent stem cell (hEPS) is a novel type of pluripotent stem cell, which possesses bi-potency towards both embryonic and extraembryonic lineages. Here, we generated a hEPS cell line (hEPS1-iCas9-B) from the cell line named hEPS1, carrying a doxycycline-inducible Cas9 expression cassette along with a constitutive reverse tetracycline transactivator (M2rtTA) expression cassette at the AAVS1 locus, thus we could efficiently generate genetically modified hEPS for studies. This cell lined remained self-renewal, differentiation potential and normal karyotype. Meanwhile, it showed robust transcriptional expression of Cas9 with doxycycline induction and could target the site where the sgRNA guided.