In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multi-cellular 3D structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumor-derived extracellular matrix (ECM), which severely limits use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs such that 3D structures are never embedded in tumor-derived ECM. We also demonstrate that the hydrogel serves as an injectable HIO vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.

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The intestinal epithelium forms a highly dynamic and selective barrier that controls absorption of fluid and solutes while restricting pathogen access to underlying tissues. Barrier properties are achieved by intercellular junctions that include an apical tight junction (TJ) and subjacent adherens junction and desmosomes. The TJ tetraspan claudin proteins form pores between epithelial cells to control paracellular fluid and ion movement. In addition to regulation of barrier function, claudin family members control epithelial homeostasis and are expressed in a spatiotemporal manner in the intestinal and crypt-luminal axis. This delicate balance of physiologic differential claudin protein expression is altered during mucosal inflammation. Inflammatory mediators influence transcriptional regulation, as well as endocytic trafficking, targeting, and retention of claudins in the TJ. Increased expression of intestinal epithelial claudin-1, -2, and -18, with downregulation of claudin-3, -4, -5, -7, -8, and -12, has been observed in intestinal inflammatory disorders. Such changes in claudin proteins modify the epithelial barrier function in addition to influencing epithelial and mucosal homeostasis. An improved understanding of the regulatory mechanisms that control epithelial claudin proteins will provide strategies to strengthen the epithelial barrier function and restore mucosal homeostasis in inflammatory disorders.

The mammalian intestine houses a complex microbial community, which influences normal epithelial growth and development, and is integral to the repair of damaged intestinal mucosa1–3. Restitution of injured mucosa involves the recruitment of immune cells, epithelial migration and proliferation4,5. Although microenvironmental alterations have been described in wound healing6, a role for extrinsic influences, such as members of the microbiota, has not been reported. Here, we show that a distinct subpopulation of the normal mucosal-associated gut microbiota expands and preferentially colonizes sites of damaged murine mucosa in response to local environmental cues. Our results demonstrate that formyl peptide receptor 1 (FPR1) and neutrophilic NADPH oxidase (NOX2) are required for the rapid depletion of microenvironmental oxygen and compensatory responses, resulting in a dramatic enrichment of an anaerobic bacterial consortium. Furthermore, the dominant member of this wound-mucosa-associated microbiota, Akkermansia muciniphila (an anaerobic, mucinophilic gut symbiont7,8), stimulated proliferation and migration of enterocytes adjacent to the colonic wounds in a process involving FPR1 and intestinal epithelial-cell-specific NOX1-dependent redox signalling. These findings thus demonstrate how wound microenvironments induce the rapid emergence of ‘probiont’ species that contribute to enhanced repair of mucosal wounds. Such microorganisms could be exploited as potential therapeutics.

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2 9 8 4 jci.org Volume 129 Number 8 August 2019 degrade ECM components. Humans express 24 MMPs that regulate diverse activities important for ECM remodeling and forward movement of the epithelium (reviewed in ref. 19). MMP endoproteinase activity facilitates removal of disorganized structural proteins from healing wounds to make room for newly synthesized collagen. Furthermore, MMP-mediated conversion of type III collagen to more stable type I collagen increases wound tensile strength. Fibroblast-and keratinocyte-derived MMP-1 promotes breakdown of excess collagen in murine and rabbit models of skin repair (20-22). Though not expressed in skin, epithelial cellderived matrilysin (MMP-7) is reportedly the key MMP involved in repairing injured intestinal mucosa in humans (23, 24). Signals that trigger epithelial migration and proliferation from injured sites are incompletely understood. Loss or modification in cell-cell contact and release of intracellular molecules initiates repair (25). These events set the stage for recruiting leukocytes and mesenchymal cells that orchestrate wound repair. Formylated peptides and ATP released by damaged cells, also referred to as damage-associated molecular patterns (DAMPs), orchestrate repair by promoting epithelial cell migration and proliferation. Epithelial wounds are also a source of intracellular Ca ++ waves that are rapidly transmitted into surrounding tissues to influence repair. Furthermore, ROS signaling and wound-associated physical cues influence epithelial repair. Small GTPases in the Rho family regulate remodeling of F-actin, intercellular junctions, and cell-matrix adhesions (26) and are crucial for epithelial cell migration and wound sealing. Similarly, the Rho GTPase Rac1 promotes intestinal epithelial proliferation by targeting β 1-integrin in cellular protrusions and modulating actin dynamics (26). Reparative signaling events are also regulated by extracellular mediators in the epithelial milieu, including annexin A1, annexin A2, and serum amyloid A1, which have been shown to influence integrin localization, focal adhesion kinase activation, and cell matrix remodeling in mouse and human intestinal mucosa (27-30). After injury, chemokines/cytokines and growth factors play crucial roles in epithelial c ell adhesion, migration, proliferation, and differentiation. TGF-β-dependent signaling pathways mediate the regulatory effects of many repair mediators, including PDGF, EGF, VEGF, IL-1, IL-2, IL-6, and IFN-γ (6). Canonical and noncanonical Wnt proteins also modulate epithelial wound repair. A recent in vivo study revealed a role of Wnt5a in orchestrating colonic crypt

In vitro differentiation of human pluripotent stem cell (hPSC)-derived organoids (HOs) facilitates the production of multicellular three-dimensional structures analogous to native human tissues. Most current methods for the generation of HOs rely on Matrigel, a poorly defined basement membrane derivative secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, limiting the potential use of HOs for regenerative medicine applications. Here, we describe a protocol for the synthesis of a fully defined, synthetic hydrogel that supports the generation and culture of HOs. Modular, cell-encapsulating hydrogels are formed from a four-armed poly(ethylene glycol) macromer that has maleimide groups at each terminus (PEG-4MAL) and is conjugated to cysteine-containing adhesive peptides and cross-linked via protease-degradable peptides. The protocol also includes guidelines for the localized in vivo delivery of PEG-4MAL hydrogel-encapsulated HOs to injured mouse colon. The PEG-4MAL hydrogel supports the engraftment of the HOs and accelerates colonic wound repair. This culture and delivery strategy can thus be used to develop HO-based therapies to treat injury and disease. Hydrogel and tissue preparation and subsequent encapsulation can be performed within 2.5-3.5 h. Once HOs have been cultured in synthetic hydrogels for at least 14 d, they can be prepared and delivered to the mouse colon in under 5 h.

Epithelial cells form regulated and selective barriers between distinct tissue compartments. The Apical Junctional Complex (AJC) consisting of the tight junction (TJ) and adherens junction (AJ) control epithelial homeostasis, paracellular permeability and barrier properties. The AJC is composed of mutliprotein complexes consisting of transmembrane proteins that affiliate with an underlying perijunctional F-actin myosin ring through cytoplasmic scaffold proteins. AJC protein associations with the apical actin-myosin cytoskeleton are tightly controlled by a number of signaling proteins including the Rho family of GTPases that orchestrate junctional biology, epithelial homeostasis and barrier function. This review highlights the vital relationship of Rho GTPases and AJCs in controlling the epithelial barrier. The pathophysiologic relationship of Rho GTPases, AJC, apical actomyosin cytoskeleton and epithelial barrier function is discussed.