Intestinal epithelial migration is supposed to occur in a passive manner driven by the mitotic pressure exerted either by the cryptal stem cells, under physiological conditions, or the newly formed epithelium, upon damage. However, whether interactions between different neighboring cell types and with the matrix contribute to epithelial movement remain elusive. Here, we developed a novel three-dimensional in vitro intestinal mucosa model of gap closure, that includes both the epithelium and the basement membrane cellular compartments in a spatially relevant manner, to show that intestinal subepithelial myofibroblasts (ISEMFs) play a crucial role in epithelial restoration. ISEMF-derived biochemical cues boost epithelial proliferation and maintain epithelial barrier integrity. While, at the wounded area, ISEMF actively migrate to the epithelial front where they activate and generate α-SMA contractile stress fibers along the direction of the epithelial cells promoting epithelial migration. Furthermore, ISEMF also deposit collagen paths that act as'guide rails' for directing IEC migration. Thus, the mere physical presence of ISEMFs can greatly accelerate restoration in wound healing, which suggests that ISEMFs should be recognized as new potential therapeutic targets.

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they formed and sensed by the epithelium is still not fully understood. Here we present a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v). Biochemical gradients are formed through the hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, we employ light-sheet fluorescence microscopy and in-silico modelling. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villuslike architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they formed and sensed by the epithelium is still not fully understood. Here we present a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and biomolecular gradients of the ISC niche (v). Biochemical gradients are formed through the hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of precise spatiotemporally controlled gradients, we employ light-sheet fluorescence microscopy and in-silico modelling. The ISC niche biochemical gradients applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.