How to make an intestine

Abstract

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.

Keywords: Directed differentiation; Embryonic stem cells; Gastrointestinal disease; Gut tube; Intestinal morphogenesis; Pluripotent stem cells.

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 three-dimensional (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⁺ gut/intestinal tissue by inducing high levels of FGF and WNT signaling (for example, by using recombinant FGF4+WNT3A). During this differentiation process, endoderm and mesoderm undergo morphogenesis to form CDX2⁺ tube-like structures (middle panel) and 3D spheres that delaminate from the tissue culture dish (fourth image from the left shows a delaminated spheroid). Three-dimensional spheroids contain CDX2⁺ endoderm and mesoderm, and are grown in a 3D matrix in the presence of EGF, noggin (NOG) and R-spondin (RSPO1). Over the course of 1 month, spheroids expand and differentiate into HIOs, which contain multiple differentiated cell types. HIOs can be repeatedly passaged every 10-14 days for more than 1 year.

Fig. 2.

Gut tube formation in the developing mouse embryo. Three primary germ layers, mesoderm (Mes), ectoderm (Ec) and endoderm (En), are specified during gastrulation. The gut tube is specified from the endoderm, which has a flat and squamous-like morphology (left, E7.5). The developing gut tube epithelium forms along the anterior-posterior axis (middle, E8.5), stained here by whole-mount in situ hybridization for NM_029639 (Moore-Scott et al., 2007). At this point in time, the anterior endoderm has formed the foregut tube, the posterior endoderm has formed hindgut tube and the middle region is forming the midgut. Stained sections of the mid/hindgut region show that the epithelium has acquired a cuboidal-like morphology. Gut tube formation is completed by E9.5 in the mouse embryo (right), with the pseudo-stratified epithelium fully encased in mesoderm-derived mesenchyme. Staining for desmoplakin highlights the pseudo-stratified morphology of the gut tube. The boxed area and lines indicate the regions or sections enlarged in the panels underneath.

Fig. 3.

Milestones in murine and HIO intestinal development. The intestinal epithelium is present as a pseudostratified epithelium at ∼E12.5 through E13.5 in the mouse, and within 14 days of HIO specification. After this point, the appearance of mesenchymal clusters (orange), which respond to hedgehog (Hh) and platelet-derived growth factor A (Pdgfa) secreted from the epithelium, is coincident with the onset of villus morphogenesis. In the mouse, villus morphogenesis proceeds between E14.5 and E16.5, whereas in HIOs this stage takes considerably longer. As the epithelium remodels, the columnar epithelium forms with stereotypical villus (green) and proliferative intervillus (yellow) domains. Mesenchymal clusters remain associated with the villus tip and act as a postmitotic signaling center, signaling to the epithelium via secreted growth factors (including Bmp ligands, among others), which prevent epithelial proliferation. The fetal intervillus domain gives rise to the adult crypt by postnatal day 14 in the mouse, and after approximately 8 weeks in HIOs. The mature adult crypt houses the proliferative intestinal stem cells (yellow) and secretory Paneth cells (red), as well as several additional cell types, the most prevalent of which are enterocytes (green), goblet cells (blue) and enteroendocrine cells (purple).