Generation and Validation of an Aryl Hydrocarbon Receptor Knockout Human Embryonic Stem Cell Line

Glucose homeostasis is tightly controlled by hormones secreted from pancreatic islets. The most abundant cell type in islets is the β-cell, which secretes insulin in response to nutritional stimuli. We previously reported that the adverse metabolic effects of high-dose dioxin exposure in mice are regulated by the aryl hydrocarbon receptor (AHR) specifically in β-cells. Additionally, fetal exposure to low-dose dioxin reduced β-cell area in female mice at birth; however, the role of AHR in β-cell development has not been explored. To characterize the AHR pathway in developing human β-cells, we differentiated human embryonic stem cells (hESCs) into "islet-like" cell clusters (SC-islets) in vitro and treated cells with vehicle or dioxin for 24 hours at key stages of differentiation. Dioxin exposure robustly upregulated AHR gene targets (CYP1A1, AHRR) at all stages of differentiation but only had modest effects on markers of islet development and maturity. We next generated an AHR knockout (KO) hESC line and found that basal CYP1A1 expression was profoundly suppressed in AHR-KO cells compared to parental cells at all stages of differentiation. Key markers of developing and mature pancreatic islets were largely unaffected by AHR deletion; however, G6PC2 was consistently downregulated in SC-islets from AHR-KO cells compared to parental cells. Interestingly, AHR-KO SC-islets also showed modestly increased insulin secretion relative to the parental line, suggesting a role for AHR in islet development. This novel AHR-KO cell line will allow for deeper investigation into the impact of AHR on the development of human islets and other cell lineages.