High-throughput organoid screening enables engineering of intestinal epithelial composition

Abstract

Barrier tissue epithelia play an essential role in maintaining organismal homeostasis, and changes in their cellular composition have been observed in multiple human diseases.Within the small intestinal epithelium, adult stem cells integrate diverse signals to regulate regeneration and differentiation, thereby establishing overall cellularity. Accordingly, directing stem cell differentiation could provide a tractable approach to alter the abundance or quality of specialized cells of the small intestinal epithelium, including the secretory Paneth, goblet, and enteroendocrine populations. Yet, to date, there has been a lack of suitable tools and rigorous approaches to identify biological targets and pharmacological agents that can modify epithelial composition to enable causal testing of disease-associated changes with novel therapeutic candidates. To empower the search for epithelia-modifying agents, we establish a first-of-its-kind high-throughput phenotypic organoid screen. We demonstrate the ability to screen thousands of samples and uncover biological targets and associated small molecule inhibitors which translate to in vivo. This approach is enabled by employing a functional, cell-type specific, scalable assay on an organoid model designed to represent the physiological cues of in vivo Paneth cell differentiation from adult intestinal stem cells. Further, we miniaturize and adapt the organoid culture system to enable automated plating and screening, thereby providing the ability to test thousands of samples. Strikingly, in our screen we identify inhibitors of the nuclear exporter Xpo1 modulate stem cell fate commitment by inducing a panepithelial stress response combined with an interruption of mitogen signaling in cycling intestinal progenitors, thereby significantly increasing the abundance of Paneth cells independent of known WNT and Notch differentiation cues. We extend our observation in vivo, demonstrating that oral administration of Xpo1 inhibitor KPT-330 at doses 1,000fold lower than conventionally used in hematologic malignancies increases Paneth cell abundance. In total, we provide a framework to identify novel biological cues and therapeutic leads to rebalance intestinal stem cell differentiation and modulate epithelial tissue composition via high-throughput phenotypic screening in rationally-designed organoid model of differentiation.

Characterization of human nasal organoids from chronic rhinosinusitis patients

“…However, the requirement of porous culture inserts limits the application of ALI culture to smaller-scale experiments. In contrast, nasal organoids bring the possibility to perform high-throughput screening ( Phan et al, 2019 ; Mead et al, 2020 preprint). Furthermore, evaluation of the expression levels of the mesenchymal stem cell markers CD73, CD90, CD105 and the hematopoietic stem cell marker CD45 in HNECs (monolayers) showed that the quality of the cells was maintained up to passage four ( Muhammed et al, 2015 ).…”