A murine small intestinal two-dimensional monolayer platform that induces fate commitment toward a revival stem cell...

Yes-associated protein (YAP) signaling is a key regulator of intestinal epithelial regeneration and drives fetal-like reprogramming that generates revival stem cells (revSC). Because YAP activity is sensitive to extracellular matrix properties and substrate stiffness, we asked whether a stiff plastic substrate could provide a mechanically defined platform to induce a revSC-like state in vitro. We established Matrigel-derived murine small intestinal organoids cultured in Wnt-containing medium, dissociated them into single cells, and plated them onto collagen I/IV-coated plastic plates to generate a two-dimensional (2D) monolayer. The resulting monolayer formed a continuous, proliferative epithelial sheet with abundant Ki67-positive cells. Immunostaining revealed ubiquitous nuclear retention of YAP. RT-qPCR showed induction of canonical YAP target genes (Ctgf, Cyr61, Ankrd1) and upregulation of revSC markers (Ly6a, Clu), accompanied by marked suppression of crypt basal columnar (CBC) markers (Lgr5, Ascl2, Olfm4), compared with a homeostatic Matrigel organoid control. Notably, after withdrawal of the Wnt3a alternative peptide PG-008 from day 3 onward, the 2D monolayer remained viable through day 8 and retained elevated Ly6a and Clu expression, while CBC and all other differentiated lineage markers (Alpi, Defa6, Muc2, Chga) remained strongly repressed. These findings establish a plastic-based 2D monolayer as a complementary in vitro revSC model characterized by high YAP activity and sustained revSC-like properties with reduced dependence on exogenous Wnt stimulation.