A high-fidelity in vitro model of the human intestinal epithelium

Primary intestinal epithelial stem cell culture methods have significantly advanced understanding of mammalian intestinal development and disease. However, progress has been hampered by inconsistent methodological reporting and challenges comparing in vitro systems with in vivo observations. We previously established a unique method for long-term 2{-}dimensional (2D) cultivation of mouse lECs using an air-liquid interface (ALI) technique which appears to faithfully recapitulate homeostatic and regenerative features in vitro. Here, we further refined these methods and optimized protocols for long-term, self-organizing 2D cultivation of human lECs. During the culture, we observed that epithelial cells undergo a dynamic morphological transition from squamous to columnar shape. Using single cell transcriptomics, we identified both major lineages and minor populations, including enteroendocrine cells, tuft cells, and BEST4/CA7+ cells. Leveraging the power and scalability of a biomedical foundation model (BMFM) trained on single cell RNA sequencing data, we performed classification tasks to identify cell types across sample sources and to quantitatively benchmark our in vitro differentiated cells against cells collected from patient biopsies. We observed a striking degree of similarity between our in vitro differentiated cells and the corresponding cell types in vivo for multiple differentiated lineages. This novel approach using BMFM holds promise to expand our understanding of the regulatory mechanisms including gene-gene regulation underlying homeostasis and regeneration as well as the functions of rare and poorly understood lineages within the human intestinal epithelia. Moreover, these methods are generalizable to other organs and can be used to assess the correspondence of cells across experimental modalities. SignificanceThis manuscript addresses challenges in quantitative comparisons of cell types grown in vitro vs their in vivo counterparts. Here we use the intestinal epithelium as a model system to address this challenge. We devised an in vitro culture platform that supports multipotent intestinal epithelial stem cells and their numerous differentiated progeny. This system gives rise to all known rare and abundant lineages in correct proportions. Novel use of biomedical foundation models pre-trained on publicly available data and then fine-tuned to data from this platform enabled demonstration of high concordance of multiple in vitro differentiated lineages with the corresponding cells in vivo.