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.