Recent progress in organoid culture to model intestinal epithelial barrier functions

International Immunology, Vol. 31, No. 1, pp. 13–21 doi:10.1093/intimm/dxy065 Advance Access publication 3 October 2018 © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: Recent progress in organoid culture to model intestinal epithelial barrier functions Tetsuya Nakamura Received 28 June 2018, editorial decision 21 September 2018; accepted 2 October 2018 Abstract The intestinal epithelium not only acts as the physical structure that separates the intestinal lumen from the body but also actively participates in intestinal barrier functions. In the past decade, significant progress has been made in the development of culture technologies to maintain intestinal epithelial cells (IECs) as various forms of intestinal organoids. As these organoids allow for restoration of the physiological composition of IECs, they represent suitable models to study the mechanisms of development and differentiation or the molecular basis of functions in specific types of IECs, such as goblet cells, Paneth cells, tuft cells and M cells. In addition, intestinal organoids are now widely used as model systems to investigate the dynamic processes occurring at the host–microbe interface and the mutual interactions between IECs and the cells involved in the maintenance of local immune homeostasis. In this review article, I showcase recent work that has utilized intestinal organoids to study various aspects of intestinal epithelial barrier functions. Keywords: epithelial–immune cell interaction, host–microbe interaction, intestinal organoids Introduction The intestinal epithelium is composed of a monolayer of simple columnar epithelial cells and folded to generate crypt and villus architecture. It comprises differentiated cells of multiple lineages—absorptive enterocytes, goblet cells, enteroendocrine cells, Paneth cells, tuft cells and M cells—all of which originate from intestinal stem cells (ISCs). As tight junctions connect adjacent epithelial cells and seal the intercellular spaces, the epithelium separates the intestinal lumen from the underlying tissues (1). The epithelium not only acts as a static barrier but also serves as an active participant in dynamic processes of intestinal barrier functions. Goblet cells and Paneth cells secrete protective mucins and anti-microbial peptides, respectively, and contribute to the first line of defense against pathogenic microorganisms (2). Tuft cells initiate immune responses, particularly against parasitic infections in the intestine (3). The intestinal M cells, a subset of intestinal epithelial cells (IECs) covering gut-associated lymphoid tissue (GALT) lymphoid follicles, are specialized to take up intestinal luminal antigens to regulate mucosal immune responses (4). Furthermore, the intestinal epithelium is known to communicate with various types of non-epithelial cells, for example through cytokine production or direct antigen presentation, and control inflammatory and immune responses (2). It is thus apparent that the intestinal epithelium is an integral component of the protective barrier between the intestinal luminal environment and internal milieu of our bodies. In the last decade, there has been significant progress in the development of culture technologies to maintain IECs in vitro. The IECs grown in those novel culture systems recapitulate the physiology, the three-dimensional (3D) architecture and the genetic signature of the original intestinal epithelium and thus are called intestinal organoids (5, 6). Studies have shown that at least three sources of cells—isolated IECs (7–14), IECs generated in vitro through methods such as direct lineage reprogramming of fibroblasts (15) or cells that develop from directed differentiation of pluripotent stem cells (PSCs) (16)—can be grown as epithelium-only organoids or as epithelium plus non-epithelial (stromal and mesenchymal) organoids. Given that the organoids can reconstitute intestinal epithelial tissues when placed back into animals, organoid technologies are expected to accelerate regenerative medicine for human intestinal diseases (17). In addition, transplantation experiments have clearly shown that the cells cultured in organoids retain many physiological features including their tissue regeneration capabilities, which further provides a rationale to use organoids as a model system to study the intestinal epithelium in vitro. In this article, I first summarize advances in the development of intestinal organoid technology. Then, I describe Correspondence to: T. Nakamura; E-mail: REVIEW Department of Advanced Therapeutics for GI Diseases, Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan 14 Organoids to model intestinal barrier functions recent work that utilizes organoids to study intestinal barrier functions, describing the protective functions of particular epithelial cell types and also the interactions of IECs with microorganisms or immune cells. Intestinal organoids Tissue regeneration capabilities of ISCs in intestinal organoids Intestinal organoids have immense potential for therapeutic use in human intestinal diseases. Our group first described that mouse colonic epithelial organoids can regenerate colonic epithelia when transplanted back into the host animal (11). In this study, acute colitis was induced in recipient mice to generate ulcerations in the colon, and the cultured colon epithelial organoids, labeled by enhanced green fluorescent protein (EGFP), were infused in recipient mice through an enema. The transplanted cells adhered to the injured recipient tissue and, by 4 weeks, they reconstituted a healthy colonic epithelium that contained proliferating cells and all terminally differentiated cell types. Notably, the engrafted crypts were entirely EGFP+ at this point, suggesting that each crypt formed a clonal population derived from EGFP+ stem cells. When tetramethylrhodamine isothiocyanate (TRITC)conjugated dextran (TRITC-dextran) was administered orally, blood TRITC concentrations in transplanted mice were comparable to those in control mice, indicating the maintenance of epithelial barrier function in graft tissues (11). Furthermore, colonic epithelial organoids, whose culture was initiated from a single Lgr5+ colonic stem cell, engrafted in multiple recipients as normal colonic epithelia (11). This study thus provides proof of principle that damaged intestinal epithelia can be repaired by transplanting IECs that are isolated and then expanded as organoids in vitro. Studies have now shown that various types of intestinal epithelial organoids, such as those derived from fetal mouse small intestines (12), adult mouse small intestines (30, 31), adult human colons (32) or even those generated from mouse fibroblasts by direct lineage reprogramming (15, 33), can all regenerate the intestinal epithelium in vivo. Intriguingly, Fukuda et al. demonstrated that small intestinal epithe (...truncated)