Effects of n-3 Long-Chain Polyunsaturated Fatty Acid and Vitamin D Supplementation on Transcriptional Profiles of Human Lung Organoids

<b>Background/Objectives:</b> Randomized clinical trials (RCTs) suggest that n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) and high-dose vitamin D supplementation during pregnancy may protect against childhood asthma. However, the underlying mechanisms remain unclear. <b>Methods:</b> To explore the transcriptional effects of various concentrations of n-3 LCPUFA and vitamin D supplementation on in utero lung development, we cultured human lung organoids derived from BILX and SEHP human-induced pluripotent stem cell lines at the Sanger Institute (Cambridge, UK). The organoids were treated with either no supplementation, or low (0.01 µL/mL) or high (0.1 µL/mL) concentrations of n-3 LCPUFA, as well as no supplementation, or low (5 pM) or high (50 pM) concentrations of vitamin D. Organoids were matured for 50 days, with foregut spheroids embedded in Matrigel and later re-embedded individually to ensure robust growth. We then assessed the impact of these supplementations using RNA sequencing. <b>Results:</b> RNA sequencing of four replicates per condition (36 total samples) revealed that n-3 LCPUFA supplementation had a more substantial impact on gene regulation than vitamin D (differentially expressed genes, <i>n</i> = 907 vs. <i>n</i> = 23). <i>CPT1A</i> and <i>ANGPTL4</i> genes were highly expressed in media cultured with a high concentration of n-3 LCPUFA, while <i>CYP24A1</i> was among the highly expressed genes in media cultured with a high concentration of vitamin D. Enrichment analysis showed activation of PPAR pathways, suggesting that n-3 LCPUFA supplementation may protect against asthma by regulating lipid metabolism and inflammation. <b>Conclusions:</b> We identified several genes and pathways that may provide insights into the biological effects of n-3 LCPUFA and vitamin D supplementation on asthma pathophysiology.