Molecular Heterogeneity of Breast Milk in Intestinal Development: A Multi-Omics Approach Using Human Organoids and Caco-2 Cells

<title>Abstract</title> <p> Breast milk is recognized as the optimal nutritional source for infants, yet the molecular mechanisms underlying its influence on gastrointestinal development remain incompletely understood. We employed human stem cell-derived small intestinal organoids (SIOs) to analyze BM heterogeneity from seven donors, offering a more accurate model than the traditional Caco-2 cell line. In this study, we used multi-omics approach, including Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), to dive deep into the pathways related to differentially expressed genes (DEGs) to explore the similarities and differences among these pathways. We found that the DEGs were considerably linked to pathways involved in immune function, cellular development, and differentiation. SIOs captured more enriched pathways compared with Caco-2(FDR &lt; 0.05), including developmentally critical Wnt signaling (LEF1, NOTCH1; p &lt; 0.01). We also found that donor-specific molecular signatures drove functional divergence, including ribosomal biogenesis, immune regulation. Overall, our findings suggest that small intestinal organoids are a reliable <italic>in vitro</italic> model for studying absorption and digestion processes. Besides, it offers new understanding of the molecular mechanisms that contribute to the advantages of breast milk, potentially guiding the formulation of infant formulas that more closely mimic human milk. </p>