Gastrointestinal Organoid Technology Advances Studies of Enteric Virus Biology

The historical lack of models recapitulating the complexity of the human intestinal epithelium has hindered studies into many aspects of human enteric virus biology. Immortalized and transformed cell lines are typically limited by the presence of only one cell type, whereas susceptibility in animal models often requires infection routes that differ from humans or necessitates modification of immune system components [1, 2]. In addition, comparing animals from different species remains a confounding factor when trying to infer how findings may apply to human health. Thus, the development of human gastrointestinal organoids to study virus–host interactions marks a significant advance. They provide a physiologically relevant ex vivo platform in which human enteric microorganisms can be studied interacting with the human intestinal epithelium. Furthermore, their intermediate complexity, falling between cell lines and animal models, adds an additional tool to better understand these viruses. Here, we highlight how gastrointestinal organoids have provided new insights into the biology of human enteric viruses and the potential of this technology for advancing the field. The different flavors of gastrointestinal organoids Gastrointestinal organoids are three-dimensional (3D) structures derived from primary tissues (i.e., patient biopsy) containing intestinal stem/progenitor cells or from human pluripotent stem cells (hPSCs) [3]. They contain multiple intestinal epithelial cell types that perform critical functions that are also observed in the human intestine (e.g., absorption, barrier function, differentiation). Induced pluripotent stem cell–derived human intestinal organoids (HIOs) most closely resemble the human fetal intestine [4] and may encompass an epithelium alone [5] or also contain a mesenchyme [6]. However, most infectious disease laboratories work with human intestinal enteroids (HIEs), patient-derived, 3D epithelium-only structures that can be transitioned to a 2D monolayer on plates or transwells. HIEs maintain the physiological and genetic characteristics of their sources for long periods [7–10]. They can be differentiated from the crypt-like state into villus-like state by withdrawing growth factors required to maintain stem cells (i.e., WNT3A) from the culture media [11–13]. Cellular differentiation of specific cell lineages can be further achieved by pharmacologic or genetic means. For example, secretory cells are enriched following dibenzazepine (DBZ) treatment to block NOTCH signaling [12] and enteroendocrine cells by overexpression of NEUROGENIN-3 [14]. In addition, receptor activator of NF-κB ligand (RANKL) treatment of HIE with and without tumor necrosis factor alpha (TNF-α) addition has been used to drive microfold cell development [15, 16]. However, HIE and HIO do not completely mimic the intestinal epithelium in vivo. For example, they do not entirely reproduce the cellularity observed in vivo as few Paneth cells and no Tuft cells are present [17]. Furthermore, the lack of villi and genetic or pharmacologic skewing of differentiation may also affect the proportionality of individual cell lineages.