How to make an intestine

James M. Wells () Jason R. Spence With the high prevalence of gastrointestinal disorders, there is great interest in establishing in vitro models of human intestinal disease and in developing drug-screening platforms that more accurately represent the complex physiology of the intestine. We will review how recent advances in developmental and stem cell biology have made it possible to generate complex, three-dimensional, human intestinal tissues in vitro through directed differentiation of human pluripotent stem cells. These are currently being used to study human development, genetic forms of disease, intestinal pathogens, metabolic disease and cancer. - Introduction The intestinal tract is one of the most architecturally and functionally complex organs of the body. The human intestine is 8 m in length (Hounnou et al., 2002) and is subdivided into five functional domains along the proximal-to-distal axis: the duodenum, jejunum and ileum are segments of the small intestine; and the cecum and colon make up the large intestine. The intestine comprises specialized cell and tissue types from all three germ layers. Intestinal tissues include endoderm-derived epithelium, which houses specialized intestinal stem cells (ISCs) (Sato and Clevers, 2013), mesoderm-derived smooth muscle, vasculature, lymphatics and immune cells, and the ectoderm-derived enteric nervous system (ENS). The contributions from all of the germ layers are required to help coordinate a myriad of complex intestinal functions. The intestine is best known for its digestive function to orchestrate the breakdown of macronutrients, regulate absorption and secrete waste. Less appreciated is the central role of the intestinal endocrine system in coordinating systemic nutrient levels and feeding behavior with other organ systems via endocrine hormones. In addition, the epithelium of the intestine acts as a selective barrier by restricting microbes to the gut lumen (Turner, 2009). These epithelial functions are largely carried out by four cell types: absorptive enterocytes and the three secretory lineages. Secretory cells involved in barrier function include goblet and Paneth cells, which secrete mucin and antimicrobial factors. The enteroendocrine cells are a rare but diverse population of cell types that secrete hormones that regulate satiety, motility, absorption, -cell proliferation, secretion of other hormones and digestive enzymes, among other things (Engelstoft et al., 2013). The epithelium of the intestine turns over every 5 days in mice, and this regenerative capacity is driven by a resident population of ISCs (Creamer et al., 1961; Sato et al., 2009). The mechanical functions of the intestine are controlled by complex interactions between the epithelium, smooth muscle and ENS, which regulate peristalsis to ensure unidirectional movement of luminal contents. Given the cellular and functional complexity of the intestine, it is no wonder that there are a staggering number of people impacted by intestinal dysfunction. Common intestinal disorders include infections, irritable bowel syndrome (IBS), malabsorption and fecal incontinence. Other debilitating diseases include inflammatory bowel disease (IBD), colon cancer and diseases that, in some cases, have a genetic basis, such as Hirschsprungs Disease. Additionally, since most oral drugs are absorbed in the intestine, the most common drug side effects are intestinal. Given the plethora of functions carried out by the intestine, there is significant interest in preventing or reversing intestinal disease by manipulation of intestinal cell biology; for example, by stimulating ISC growth as a means to protect or re-establish epithelial integrity and barrier function following injury (Zhou et al., 2013). However, gastrointestinal (GI) disease research has largely relied on in vivo animal studies, which are intrinsically low throughput and sometimes do not adequately mimic human physiology. Therefore, in vitro-derived human intestinal tissues represent a powerful tool for functional modeling of congenital defects in human intestinal development, preclinical screening for drug efficacy and toxicity testing, and for establishing models to study the mechanistic basis of diseases including IBD and cancer. In this Primer, we discuss the current understanding of intestine development and how this information has been used to direct the differentiation of human pluripotent stem cells (PSCs) into intestinal tissue in vitro. This approach requires the temporal manipulation of signaling pathways in a step-wise manner that recapitulates early intestinal development (Fig. 1). These main developmental steps include the formation of definitive endoderm, posterior endoderm patterning, gut tube formation, and intestinal growth and morphogenesis (Fig. 2). Success in this area is largely due to a shift from two- to three-dimensional growth conditions and the presence of mesenchymal cell types that result in a level of tissue complexity that more closely resembles the developing intestine in vivo. We will also evaluate how these tissues can be used to model human intestinal development and disease. The first step: endoderm formation Generating intestinal cells from PSCs requires a step to mimic the process of gastrulation and formation of definitive endoderm (DE) (Fig. 1). Studies of gastrulation using frog, fish, chick and mouse embryos have been essential in identifying a conserved molecular pathway that directs gastrulating cells into the endoderm and mesoderm lineages (reviewed in Zorn and Wells, 2009; Zorn and Wells, 2007). Central to these processes is the Nodal family of EGF NOG 1 month Fig. 1. Intestinal differentiation and morphogenesis in a dish. (A) Directed differentiation of human PSCs into human intestinal organoids (HIOs). Pluripotent stem cells (PSCs) are first differentiated into definitive endoderm (DE) (yellow) and, during differentiation, a small population of cells differentiate into mesoderm (red). Upon the activation of WNT and FGF signaling, endoderm begins to express gut-specific transcription factors (CDX2, red nucleus), which persists in the epithelium throughout intestinal development. In addition, the mesenchymal cells proliferate and coalesce with the endoderm to form threedimensional (3D) spheroids, consisting of a mesenchymal layer and a polarized epithelial layer with a lumen. Spheroids are then grown under 3D conditions in vitro and form HIOs. HIOs contain most epithelial cell types of the developing intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. Other cell types have not been explicitly identified. The mesenchyme (light red) also differentiates into smooth muscle and fibroblastic cell types. (B) Directed differentiation of human PSCs (far left image shows one colony) is induced by the nodal mimetic activin A, resulting in the formation of SOX17+/FOXA2+ DE. Human DE is then differentiated into CDX2+ gu (...truncated)