Differentiating Intestinal Stem Cells in a 3D Niche

Differentiating Intestinal Stem Cells in a 3D Niche

Cait M. Costello and John C. March

Purpose: In-vitro intestinal models are useful tools for studies into small intestinal function, including cellular growth and proliferation mechanisms, drug absorption capabilities and host-microbial interactions. In the past, these models have typically been limited to simple cultures on 2-D scaffolds or transwell inserts, but we have shown previously that epithelial cells cultured in 3-D environments exhibit different phenotypes that are more reflective of native tissue.   It is widely accepted that different microbial species will preferentially adhere to select locations along the intestinal villi.  Some of this niche determination is based on topography, but some is based on epithelial cell function. Our focus was to develop a porous, synthetic 3-D tissue scaffold with villous features that could support the co-culture of different epithelial cell types derived from a single stem cell precursor that we could co-culture with select bacterial populations. Our end goal was to establish microbial niches along the crypt-villus axis in order to mimic the natural microenvironment of the small intestine, which could potentially provide new insights into microbe-induced intestinal disorders, as well as enabling targeted probiotic therapies.

Methods and Results: We recreated the surface topography of the small intestine by fabricating a biodegradable and biocompatible villous scaffold using poly-lactic-glycolic-acid (PLGA), made porous through particulate leaching and phase separation of the solvent (Fig 1). By initially seeding the scaffolds with a simple co-culture of Caco-2 and HT29-MTX, we demonstrated that our scaffolds enable cellular differentiation along the crypt-villus axis in a similar manner to native intestines, with greater epithelial cell polarization and brush-border enzyme expression near the tips of the villi (Fig 2) compared to the crypt region, as well as statistically significant supporting TEER values. In addition, we are also able to simulate the intestinal mesenchyme by either feeding growth factors directly, or by co-culturing with a basolateral fibroblast feeder layer. This enables us to increase the complexity of our epithelial layer by allowing the growth and differentiation of intestinal stem cells, including the established stem cell line rat IEC-6, and through primary cultures of mouse crypts. Intestinal stem cells can differentiate into secretory epithelial cell types, as well as enterocytes, and these have been shown to affect the spatial location of bacteria along the crypt-villus axis.  Using RT-PCR and immunofluorescence, we have shown expression of markers for goblet, paneth, absorptive and enteroendocrine cell types with similar numbers and location along the crypt-villus axis to real small intestine epithelium (Fig 3). In addition, we have shown through preliminary experimentation with intestinal pathogens that on our scaffolds we are able to mimic the adhesion and invasion profiles of both salmonella and pseudomonas, when co-cultured with intestinal epithelial and probiotics (Fig 4).

Conclusions: We have demonstrated the feasibility of developing an in-vitro artificial intestine from a biocompatible polymer, which can be molded into villous shapes to mimic the topography of the small intestine, providing a platform for the differentiation of intestinal stem cells into epithelial cell types, and the subsequent adhesion/invasion of pathogenic bacteria.