Biologically inspired approaches to enhance human organoid complexity

Abstract

Organoids are complex three-dimensional in vitro organ-like model systems. Human organoids, which are derived from human pluripotent stem cells or primary human donor tissue, have been used to address fundamental questions about human development, stem cell biology and organ regeneration. Focus has now shifted towards implementation of organoids for biological discovery and advancing existing systems to more faithfully recapitulate the native organ. This work has highlighted significant unknowns in human biology and has invigorated new exploration into the cellular makeup of human organs during development and in the adult - work that is crucial for providing appropriate benchmarks for organoid systems. In this Review, we discuss efforts to characterize human organ cellular complexity and attempts to make organoid models more realistic through co-culture, transplantation and bioengineering approaches.

Keywords: Human development; Organoids; Stem cells.

Fig. 1.

Increasing organoid complexity through co-culture. (A) Approaches to incorporate enteric nerves into HIOs through co-culture. Top: HIOs were seeded with hPSC-derived ENCC spheres onto PGA/PLLA scaffolds and immediately transplanted into the intestinal mesentery. Bottom: HIOs were co-cultured with hPSC-derived vagal NCCs in vitro and were transplanted beneath the kidney capsule of an immunocompromised mouse for further maturation. (B) Microinjection is a common approach to introduce various microbes into the lumen of 3D gastrointestinal organoids. For example, a non-pathogenic strain of E. coli is depicted being injected into the lumen of hPSC-derived HIOs. (C) 2D monolayer cultures have been established from 3D biopsy-derived human intestinal and colon epithelial organoids. These 2D systems allow for easy access to both apical and basal regions of organoid epithelium. Recently, microbial pathogen-immune crosstalk was assessed across this polarized primary organoid-derived epithelium. (D) Liver bud formation can be achieved through mesenchyme-endothelial-hPSC-derived hepatic endoderm co-culture, which undergoes 3D self-organization after only a few days in culture. Transplantation and transwell culture systems have been used to further study hepatocyte maturation within the liver buds.

Fig. 2.

Orthotopic and ectopic transplantation of human organoids. Both hPSC and primary tissue has been transplanted into immunocompromised mice. The colors represent different types of organoids, both primary and/or hPSC-derived, that have been transplanted into the indicated locations. Orthotopic transplantation refers to transplantation into the analogous in vivo location, whereas ectopic transplantation refers to transplantation into a region outside of the native in vivo environment (i.e. beneath kidney capsules, within fat pads).

Fig. 3.

Bioengineered 3D environments support human intestinal organoid cultures. (A) Mechanically dynamic PEG hydrogels support intestinal epithelial organoid formation. LGR5+ intestinal stem cells were expanded in PEG hydrogels with an initial stiffness that softened over time via hydrolytic degradation to accommodate differentiation. (B) Generation of polarized crypt/villus structures through a combination of micropatterning and signaling gradients. Isolated human primary intestinal crypts were cultured on a microengineered collagen scaffold to produce crypt/villus architecture that became patterned into proliferative/differentiated domains through exposure to differential signaling gradients across a transwell system. DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (γ-secretase inhibitor); W, Wnt; N, noggin; R, R-spondin.