Bioengineering human mini-intestines with in vivo-like complexity and function

Biological research heavily relies on the use of animal models, which has made it difficult to answer specific questions about human biology and disease. However, with the advent of human organoids - miniature versions of tissues generated in 3D human stem cell cultures - it has become possible to recreate important aspects of the architecture and cell type diversity of human organs in remarkable detail. Although stem cell-derived organoids already mimic the corresponding natural tissues quite realistically, they have not yet succeeded in adequately replicating the physiological function and complexity of organs, and they suffer from several shortcomings related to the methods by which they are generated. For example, gastrointestinal organoids have proven to be powerful tools for modeling gut biology and disease, but their application as functional assays is limited due to their large heterogeneity, poor diversity of cell types and short lifespan. To address these challenges, I combine tissue engineering and stem cell-intrinsic self-organization to transform normally stochastic organoid development into a robust and physiologically relevant model of the human small and large intestine through scaffold-guided morphogenesis. I first focus on the derivation and propagation of 'classical' human small and large intestinal organoids and characterize them in terms of cell type composition. I then investigate approaches to microfabricate hydrogels to generate human intestinal epithelial microtissues with defined geometry. Using these arrays of shape-controlled microengineered organoids, I describe a mechanobiological role of YAP signaling in epithelial tissue morphogenesis.Next, I fabricate human intestinal epithelia with an in vivo-like architecture and cell type composition. These perfusable human mini-colons exhibit remarkable tissue longevity and maintain homeostatic cell turnover and epithelial barrier integrity. A gradient of growth factors along the crypt-villus axis results in a characteristic spatial compartmentalization of absorptive and secretory cell types. Single-cell RNA sequencing shows that human mini-colons remarkably replicate the cellular diversity of the native epithelium and outperform classical human colon organoids. Next, I show that the described approach can be applied to the generation of human miniature small-intestines and offers unprecedented opportunities for modeling complex multicellular interac-tions that are difficult to achieve with other bioengineered systems and classical organoids. To this end, I establish a co-culture system of human mini-colons with primary intestinal fibroblasts that is stable over many weeks in culture and allowed to systematically uncover the role of factors secreted by fibroblasts in epithelial regeneration, fibrosis, and inflammation. Overall, this work introduced several tissue engineering approaches to improve the complexity and function of human intestinal organoids. The successful implementation of the newly developed human mini-gut models opens exciting perspectives for basic research and greatly expands the translational value of organoids for drug development and personalized medicine. In particular, the models developed here provide a unique approach to shed light on the complex epithelial-mesenchymal crosstalk in the human intestine that is difficult to capture with animal and other more reductionist in vitro models.