The human gastrointestinal (GI) epithelium performs major physiologic functions that are critical to survival, health, and homeostatic equilibrium. While model organisms and in vitro cell culture systems have been widely used to study both normal and disease states of the GI tract, these often fail to fully recapitulate critical features of in vivo intestinal tissue. In recent years, investigators have harnessed the ability to perform directed differentiation of human induced pluripotent stem cells (iPSCs) towards cell types originating from all three embryonic germ layers, most notably a wide variety of endodermal lineages, in an attempt to generate in vitro models that better recapitulate human physiology and key developmental milestones. These iPSC-derived cells contain the exact genetic background of a particular donor or patient and are easily amenable to gene-editing, making them particularly advantageous in comparison to non-human model organisms or in vitro cell culture systems often derived from malignant tissue. Here, we report the efficient generation of iPSC-derived mesenchyme-free human intestinal organoids (HIOs) that can be primed towards colonic or proximal intestinal lineages in serum-free defined conditions. By generating a novel CDX2-eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors. We employ these mesenchyme free HIOs to highlight cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction using cystic fibrosis (CF) patient-derived iPSC lines before and after correction of the CFTR mutation. We also demonstrate that these HIOs represent a powerful tool to model pathogen-mediated GI illness, characterizing the intestinal epithelial host response to infection by the coronavirus SARS-CoV-2 as well as two filoviruses, Ebola (EBOV) and Marburg (MARV). Finally, we report the generation of a clinically relevant library of iPSCs derived from patients with Crohn’s Disease (CD), including successful directed differentiation of these lines to a relevant immune cell type, as a proof of concept for their use in CD in vitro modeling. Taken together, our results provide a comprehensive and reductive iPSC-based model to study disease states of the intestinal epithelium, ranging from enteric viral infection to mendelian disorders such as CF and autoimmune conditions such as inflammatory bowel disease (IBD), highlighting the potential of organoids as a powerful tool for disease modeling and therapeutics development.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43769 |
| Date | 03 February 2022 |
| Creators | Mithal, Aditya |
| Contributors | Mostoslavsky, Gustavo |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
Page generated in 0.0019 seconds