Tissue and functional complexity of the intestine

The small and large intestines have a staggering number of physiological functions, including but not limited to digestion, absorption, endocrine hormone-mediated release of digestive enzymes, peristalsis, satiety and insulin secretion, antigen presentation, control of microbial growth and excretion. To support these physiological functions, the small and large intestines have a diverse set of cell types and a unique tissue architecture (Figure 1a). The intestine is comprised of an inner lumen that is entirely enclosed by a complex epithelium folded into villi aimed at increasing the absorptive surface area. Next to the epithelium is a muscularis mucosa and submucosa containing a layer of enteric nerves termed the submucosal plexus, which runs the entire length of the small and large intestines. The outer layer of enteric nerves, the myenteric plexus, lies between a layer of circular and longitudinal smooth muscle. In addition, blood vessels are found throughout the layers of the intestine and each villus contains a capillary bed that is involved in uptake of absorbed nutrients and drugs.

There is significant cellular diversity in each layer of the gut. The epithelium is composed of four differentiated cell types (Figure 1b). Most epithelial cells are absorptive enterocytes that transport macromolecules, ions and water. The three remaining cell types are secretory in nature: goblet cells secrete mucins into the lumen that form a protective barrier; Paneth cells secrete anti-microbial peptides such as lysozyme; and enteroendocrine cells secrete hormones that regulate insulin secretion, satiety, motility, and release of digestive enzymes from the gall bladder and pancreas, among other things. The epithelium is highly regenerative and turns over approximately every 7 days. The renewal of the epithelium is driven by intestinal stem cells that reside in the crypts of Lieberkühn at the base of the villi. The submucosal layers contain smooth muscle myocytes, fibroblasts, subepithelial myofibroblasts, as well as enteric ganglia. There is a rudimentary understanding of how the complex architecture and cellular diversity of the intestine arise during embryonic development, which has helped efforts towards directing differentiation of human induced and embryonic pluripotent stem cells, collectively called pluripotent stem cells (PSCs), into the intestine. However, there is still much to be learned.

Intestinal development

Development of the intestine can be broadly subdivided into several steps including endoderm formation, midgut and hindgut specification, gut tube morphogenesis, assembly of mesenchyme, colonization by neural crest cells, crypt-villus morphogenesis and cytodifferentiation [1] (Figure 2a). Several important signaling pathways have been identified as being required for directing these early stages of intestinal development in a broad range of vertebrate species, including birds, frogs and mice [2–4]. For example, Wnt and fibroblast growth factor signaling pathways direct endoderm into a midgut and hindgut fate, and this process is required for subsequent intestinal development. Moreover, the synergistic activity of both pathways is involved in the initiation of gut tube morphogenesis and formation of the gut mesenchyme [5]. Inhibition of either of these pathways results in abnormal intestinal morphogenesis and the loss of expression of posterior/hindgut markers, including Cdx genes. Once formed, the simple cuboidal epithelium of the hindgut and the surrounding mesenchyme undergo a series of reciprocal signaling events resulting in formation of a polarized columnar epithelium containing villi, a proliferative progenitor zone, and distinct intestinal lineages [5, 6]. Along with development of the epithelium, there is a coordinated development of gut mesenchyme into the submucosal and smooth muscle layers. As discussed below, vagal neural crest cells that give rise to the enteric nervous system (ENS) migrate ventrally, undergo extensive proliferation, and incorporate into the developing gut shortly after gut tube formation [7, 8] (Figure 2a).

Building a better system: engineering additional complexity into human intestinal organoids

Low-throughput, limited genetic diversity, and species differences are recognized limitations for the use of animal models to study gastrointestinal (GI) disease. Recent advances in the understanding of human intestinal stem cells now allows for the derivation of human intestinal epithelial enteroids from patient biopsies [9]. Human enteroids have been cultured from both the small and large intestines and contain all major epithelial cell types. However, enteroids do not contain any additional cell and tissue types such as smooth muscle, supporting fibroblasts, endothelial cells, or enteric nerves. While there are more complex organoid systems derived from adult mouse intestines [10], it would be advantageous to have a more functional human intestinal organoid system as a screening platform to monitor drug absorption and the impact on intestinal motility.

Generation of more biologically complex human intestinal tissue has been accomplished through the directed differentiation of human PSCs (Figure 2b) [5, 11]. The process is initiated using the Nodal-related protein activin, which directs differentiation of human PSCs into definitive endoderm [12, 13]. Synergistic activity of the fibroblast growth factor and Wnt signaling pathways was then used to promote a posterior gut tube fate [2–4], to induce gut tube morphogenesis, and to promote growth of the intestinal mesenchyme. The resulting gut tube spheroids were strikingly similar to the gut tube of an embryonic day 9 mouse embryo, consisting of a CDX2-expressing cuboidal epithelium surrounded by a CDX2-expressing mesenchyme. Growth of gut tube spheroids in three-dimensional conditions that favor intestinal growth [14] resulted in the efficient production of human intestinal organoids (HIOs) that have both secretory and absorptive function. Moreover, the cellular diversity and architecture was strikingly similar to that of the developing gut. The epithelium contained crypt and villus-like structures as well as all of the cell types normally found in the gut. The mesenchyme underwent differentiation into stratified layers. Some layers expressed smooth muscle markers whereas others expressed markers of fibroblasts and subepithelial myofibroblasts, both found in the submucosal layer. Furthermore, the HIOs generated via these methods were capable of basic intestinal function, including absorption of amino acids and secretion of mucus, and were able to be passaged in vitro for over 1 year, resulting in 50,000-fold expansion.

Despite the significant level of complexity, PSC-derived HIOs lack both an enteric nervous system and a vascular system. This is probably due to the absence of vascular and ENS precursors, specifically neural crest stem cells. PSCs can be directed to differentiate into vascular and neural crest stem cells [15–19], raising the intriguing possibility that additional tissue complexity can be engineered into HIOs by addition of neural and/or vascular precursors in a contrived manner to recapitulate the normal development of the intestinal vasculature and ENS.