Intestine organotypic culture recapitulates human embryonic villus morphogenesis at week 8.

The human small intestine depends on villi to generate the large absorptive surface area required for nutrient uptake and barrier integrity. Existing human in vitro models fail to reproduce early villus morphogenesis, as they lack developmental signaling and epithelial-mesenchymal interactions that drive villus initiation. Such models are crucial for understanding the mechanisms of human villus formation to study intestinal development and disease. This study aimed to establish a human organotypic intestine model that recapitulates early villus development and to compare its features with human embryos. Primary organoid-derived epithelial cells containing intestinal stem and progenitor populations, and Caco-2/HT29 cocultures (as reference) were both cultured on a hydrogel containing primary small-intestinal stromal cells in a Transwell system. Models were assessed by histology, electron microscopy, transepithelial electrical resistance (TEER), permeability assays, gene expression analysis, growth factor secretion, and comparison with human embryonic duodenum (Carnegie stages 20-23). Both models formed polarized epithelial monolayers with sparse microvilli compared with mature in vivo intestine. Strikingly, only the organoid-derived model generated stromal cell and extracellular matrix (ECM)-filled epithelial protrusions with localized laminin deposition. This model activated key developmental pathways (SHH, PDGF-AA, BMP4, and WNT5A), upregulated villus-associated transcription factors (FOXF1, FOXF2, and FOXL1), and expressed laminin isoforms linked to villus initiation. The morphology and height of the protrusions (approx. 28 µm) closely resembled early villi in Carnegie stage 21 embryos.