Oral nicotine pouch (ONP) use has increased in recent years due to the perception that these products are a safer alternative to combustible tobacco products. However, little is known about the potential developmental toxicity of ONPs, particularly during bone formation. In this study, we evaluated the effects of ONP extracts from multiple ZYN flavors and nicotine concentrations on osteogenic differentiation using a human embryonic stem cell (hESC) model. H9 hESCs were differentiated toward the osteogenic lineage and continuously exposed to ONP extracts throughout differentiation. Cellular metabolic activity was assessed using the MTT assay, while ATP content, total protein content, alkaline phosphatase (ALP) activity, glutathione peroxidase (GPx) activity, and calcium deposition were evaluated using biochemical assays and Alizarin Red S staining. Gene expression was assessed using RT-qPCR and single-cell RNA sequencing, followed by pseudotime trajectory and transcriptomic point of departure (tPOD) analyses. ONP exposure caused concentration-dependent reductions in MTT activity, ATP content, total protein content, ALP activity, GPx activity, and calcium deposition. Expression of the osteogenic marker RUNX2 was generally decreased following ONP exposure, while TWIST1, SOX10, and TBX5 expression were increased in several treatment groups. Single-cell transcriptomic analysis demonstrated significant alterations in cell cluster distribution and pseudotime trajectory following ONP exposure, suggesting disrupted osteogenic lineage progression. Transcriptomic analysis further identified widespread perturbation of bone development-related genes across treatments. Wintergreen 6 mg had among the greatest transcriptomic potency metrics. Together, these findings reveal that ONP extracts disrupt osteogenic differentiation in hESCs and alter developmental lineage progression. This study provides mechanistic evidence that oral nicotine pouch extracts perturb early osteogenic differentiation in a human stem cell model and supports further evaluation of their developmental toxicity.
