Highly reproducible and cost-effective one-pot organoid differentiation using a novel platform based on PF-127 triggered spheroid assembly

Using a novel platform based on the findings that Pluronic F-127 (PF-127) could trigger highly uniform spheroid assembly, we develop a one-pot organoid differentiation strategy. Using our strategy, we successfully generate cortical, nephron, hepatic, and lung organoids with improved reproducibility compared to previous methods while reducing the original costs by 80-95%. In addition, we adapt our platform to microfluidic chips allowing automated culture. We showcase that our platform can be applied to tissue-specific screening, such as drug toxicity and transfection reagents testing. And, we generate NEAT1 knockout tissue-specific organoids and show NEAT1 modulates multiple signaling pathways fine-tuning the differentiation of nephron and hepatic organoids and suppresses immune responses in cortical organoids. In summary, our strategy provides a powerful platform for advancing organoid research and studying human development and diseases. Overall design: We use bulk RNA-seq analyses to suggest functions of PF-127 to hESCs, and prove that our methods are robust for generating multiple types of organoids from all three germ layers. Additionally, we use the lncRNA NEAT1 knockout organoids to perform RNA-seq to study tissue-specific gene regulation functions of NEAT1.