Long-Term Monolayer Cultivation Captures Homeostatic and Regenerative Features of Human Colonic Epithelial Cells

While organoid culture technique has significantly advanced research on intestinal epithelial cells (IECs), reproducing the homeostatic and regenerative processes of human IECs in vitro remains challenging. We previously established a unique long-term 2-dimensional (2D) cultivation for mouse IECs using the air-liquid interface (ALI) technique, successfully recapitulating homeostasis and regeneration. Here, applying this to human IECs, we developed a long-term, self-organizing 2D cultivation for human IECs. During the culture, we observed a dynamic transition in cellular shape from squamous to columnar, resembling in vivo regeneration. RNA sequencing indicated that intestinal stem cells (SCs) with canonical SC markers like LGR5 may play a main role in both homeostasis and regeneration. Moreover, we identified minor populations, including deep crypt secretory (DCS)-like cells, CA7-positive cells, and non-canonical Goblet cells, previously overlooked in vitro. This novel technique holds promise for not only broadening our understanding of homeostasis, regeneration, and the functions of minor populations as well as SCs in human intestinal mucosa, but also facilitating future studies such as analyzing host-microbe interactions. ### Competing Interest Statement T.S.S. is on the scientific advisory board for Janssen Pharmaceuticals and Amgen. T.S.S. is a founder of Mobius Care, Inc.