Publications

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here 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) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. 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 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.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.

JTD Keywords: 3d architectures, biomolecular gradients, colon, crypt, engineering organoids, hydrogels, identification, in silico modeling, intestinal stem cell niches, light sheet fluorescence microscopy, niche, permeability, photolithography, regeneration, villus, wnt, 3d architectures, Biomolecular gradients, Engineering organoids, In silico modeling, Intestinal stem cell niches, Light sheet fluorescence microscopy, Photolithography, Stem-cell

Engineering dynamic stem cell niche-like environment offers opportunity to obtain better control of the fate of stem cells. We identified, for the first time, that periodic changes in the adhesive environment of human adipose derived mesenchymal stem cells (ADSCs) alters dramatically their asymmetric division but not their ability for symmetric renewal. Hereby, we used smart thermo-responsive polymer (PNIPAM) to create a dynamic adhesive environment for ADSCs by applying periodic temperature cycles to perturb adsorbed adhesive proteins to substratum interaction. Cumulative population doubling time (CPDT) curves showed insignificant decline in the symmetric cell growth studied for up to 13th passages accompanied with small changes in the overall cell morphology and moderately declined fibronectin (FN) matrix deposition probably as a functional consequence of ADSCs ageing. However, a substantial alteration in the differentiation potential of ADSCs from both early and late passages (3rd and 14th, respectively) was found when the cells were switched to osteogenic differentiation conditions. This behavior was evidenced by the significantly altered alkaline phosphatase activity and Ca deposition (Alizarin red) assayed at 3, 14 and 21 day in comparison to the control samples of regular TC polystyrene processed under same temperature settings.

JTD Keywords: Cell ageing, Dynamic adhesive environment, Extracellular matrix, Mesenchymal stem cells, PNIPAM, Stem cell niche, Symmetric and asymmetric cell growth, Thermo-cycling, Thermo-responsive polymer