Infant and Adult Human Intestinal Enteroids are Morphologically and Functionally Distinct

Background & Aims Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. Methods We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. Results RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. Conclusions HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population. Importance Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begins during infancy, tools that appropriately model intestinal development and diseases are critical. Infant enteroids model key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases and responses to pathogens. ### Competing Interest Statement The authors have declared no competing interest. * ( AFP ) : Alpha-fetoprotein (ALPI) : alkaline phosphatase (APOB) : Apolipoprotein B-48 ( CAECAM7 ) : carcinoembryonic antigen-related cell adhesion molecule 7 ( CHGA ) : chromogranin A (CHGB) : chromogranin B (CLCA1) : chloride channel accessory 1 (+/−) : complete media with/without growth factors CMGF (DEFA6) : defensin alpha 6 (DEG) : differentially expressed gene (EdU) : 5-Ethynyl-2’deoxyuridine (EECs) : enteroendocrine cells (EGTA) : ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetra acetic acid ( FABP6 ) : fatty acid binding protein 6 (FDR) : false discovery rate (FITC) : fluorescein isothiocyanate (GSEA) : gene set enrichment analysis (GOBP) : gene ontology biological processes compendium (HIEs) : human intestinal enteroids (IFNλ) : interferon lambda (IFI44L) : IFN-induced protein 44-like (IP-10) : gamma-induced protein 10 (LCT) : lactase ( LIFR ) : leukemia inhibitory factor receptor (LYZ) : lysozyme (mOPV1) : monovalent type 1 oral poliovirus vaccine (ORA) : over-representation analysis (MOI) : multiplicity of infection (MTTP) : microsomal triglyceride transfer protein ( MUC2 ) : mucin 2 (NEC) : necrotizing enterocolitis (PCA) : principal component analysis (POU2F3) : POU class 2 homeobox 3 (REG3A) : regenerating family member 3 alpha ( SLC10A2 ) : solute carrier family 10 member 2 ( SI ) : sucrase isomaltase (SST) : somatostatin (TLR) : toll-like receptor (TEER) : transepithelial electrical resistance (TFP) : tight junction protein (TFF3) : trefoil factor 3