Generating human intestinal tissues from pluripotent stem cells to study development and disease - PubMed (original) (raw)

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Generating human intestinal tissues from pluripotent stem cells to study development and disease

Katie L Sinagoga et al. EMBO J. 2015.

Abstract

As one of the largest and most functionally complex organs of the human body, the intestines are primarily responsible for the breakdown and uptake of macromolecules from the lumen and the subsequent excretion of waste from the body. However, the intestine is also an endocrine organ, regulating digestion, metabolism, and feeding behavior. Intricate neuronal, lymphatic, immune, and vascular systems are integrated into the intestine and are required for its digestive and endocrine functions. In addition, the gut houses an extensive population of microbes that play roles in digestion, global metabolism, barrier function, and host-parasite interactions. With such an extensive array of cell types working and performing in one essential organ, derivation of functional intestinal tissues from human pluripotent stem cells (PSCs) represents a significant challenge. Here we will discuss the intricate developmental processes and cell types that are required for assembly of this highly complex organ and how embryonic processes, particularly morphogenesis, have been harnessed to direct differentiation of PSCs into 3-dimensional human intestinal organoids (HIOs) in vitro. We will further describe current uses of HIOs in development and disease research and how additional tissue complexity might be engineered into HIOs for better functionality and disease modeling.

Keywords: endoderm; intestinal development; organoids; stem cells; tissue engineering.

© 2015 The Authors.

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Figures

Figure 1

Figure 1. Generation of HIOs from PSCs

Early stages of embryonic development guide the generation of HIOs from pluripotent stem cells into intestine. Definitive endoderm is established by the end of gastrulation (e7.5 in the mouse) and requires TGFβ molecule Nodal signaling. Differentiation of PSCs into definitive endoderm is achieved using the Nodal mimetic Activin A, which promotes differentiation into definitive endoderm as marked by Foxa2 (green) and Sox17 (red). The 2-dimensional sheet of DE folds to form a 3-dimensional primitive gut tube that is functionally subdivided along the anterior–posterior axis into a foregut, mid-gut, and hindgut (e8.5 in the mouse). Morphogenesis and posteriorization are induced in DE cultures via activation of FGF/WNT signaling. This causes the 2-D endoderm monolayers to undergo morphogenesis to form 3-D spheroids that additionally express the posterior marker CDX2. Spheroids that are grown in 3-D culture conditions for 4 weeks form HIOs that express CDX2 and have all major secretory and absorptive lineages of the fetal intestine. HIOs can be routinely split and re-plated in fresh 3-D matrigel every 2 weeks and have been passaged in this manner for up to a year.

Figure 2

Figure 2. Unique advantages and limitations of current intestinal organoid systems

HIOs grown in vitro are similar to fetal intestine and thus are uniquely suited to study human intestinal development using both genetic and pharmacologic manipulation of genes and signaling pathways. HIOs also contain mesenchyme allowing for studies of epithelial–mesenchymal interactions. HIOs grown in vivo contain intestinal stem cells, crypts, villi, and differentiated smooth muscle layers and can be used for functional studies of human intestine. Primary cultures of human intestinal crypts (called enteroids (ENOs) or adult intestinal organoids) have more mature characteristics and can be used to study intestinal stem cell biology. Lastly, direct analysis of patient tissues provides an important snap shot of normal and pathological states of patient tissue; there is no opportunity for experimental manipulation. Both in vitro systems share many advantages in studying intestinal biology and disease modeling.

Figure 3

Figure 3. Complexity of the intestine

The intestine is an assembly of multiple cell types from all 3 germ layers. The endoderm gives rise to the epithelium of the intestine (insert). This region is known as the mucosa. This includes cell subtypes such as enterocytes (yellow), enteroendocrine cells (blue), Paneth cells (pink), and goblet cells (green). Capillaries and blood vasculature as well as myofibroblasts and Peyer's patches/M cells control transport of nutrients, epithelial integrity, and immune responses, respectively. The epithelium is circled by three smooth muscle layers that are arranged in alternating longitudinal–circular–longitudinal orientations. Embedded within the muscle is the enteric neural system (ENS), the submucosal plexus and the myenteric plexus. While HIOs contain most of these epithelial and mesodermal cell types, other cell types such as neurons will need to be incorporated in order to more accurately mimic in vivo development and adult function. In addition, microbial components can be injected into HIOs to study immunity, gut barrier functions, and metabolism by the microbiome.

Figure 4

Figure 4. Using HIOs to study intestinal development and model diseases

The in vitro culture of human intestinal tissues provides new opportunities to study cancer, infection, and genetic diseases. This is essential since certain human diseases, such as colon cancer, and enteric pathogens, such as C. cayetanensis, are not effectively studied in other model systems. This makes studying human intestinal disease very difficult. In addition, HIOs could be used as a rapid primary screen for drug absorption and GI toxicity, the most common off-target effect of new drugs.

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