Tissue geometry drives deterministic organoid patterning

Abstract

Epithelial organoids are stem cell–derived tissues that approximate aspects of real organs, and thus they have potential as powerful tools in basic and translational research. By definition, they self-organize, but the structures formed are often heterogeneous and irreproducible, which limits their use in the lab and clinic. We describe methodologies for spatially and temporally controlling organoid formation, thereby rendering a stochastic process more deterministic. Bioengineered stem cell microenvironments are used to specify the initial geometry of intestinal organoids, which in turn controls their patterning and crypt formation. We leveraged the reproducibility and predictability of the culture to identify the underlying mechanisms of epithelial patterning, which may contribute to reinforcing intestinal regionalization in vivo. By controlling organoid culture, we demonstrate how these structures can be used to answer questions not readily addressable with the standard, more variable, organoid models.

Figure 1:. Spatiotemporal control over organoid crypt formation through photopatterning.

(A) Composite image showing Lgr5-GFP expression in a symmetric colony and photopattern visible with transmitted 405 nm light immediately after spatially restricted light exposure. (B) Mechanical characterization of gel regions with atomic force microscopy corresponds to conversion of photocleavable moieties within the gel. (C-E) Spatially defined crypt formation within photopatterned gels 24 h (C), 48 h (D) and 72 h (E) after light-induced softening. (F) Quantification of fraction of photo-softened gel regions containing a crypt. Individual data points and mean are shown. Lgr5-eGFP expression (G, H) and proliferation (I) are localized within the buds extending into the softened regions. Enterocytes are found in the central regions (J) of the organoids Paneth cells are restricted to the buds (K) of the organoids. Enteroendocrine cells (L) are also present. Scale bars, 30 µm.

Figure 2:. Geometrically controlled symmetry-breaking and epithelial patterning within intestinal organoids.

(A) Schematic depicting the generation of microfabricated tissues of controlled size and shape. (B) An array of intestinal organoids formed from engineered intestinal tissues of rod-like geometry and magnification. (C) Frequency map, showing average Lgr5 expression over ~80 tissues. (D) An array of intestinal organoids at day 5 and (E), quantification of the average number of buds per location within tubular intestinal tissues. (F) Paneth cells staining by lysozyme in the array of intestinal organoids and (G) average Paneth cell distribution. (H) AldoB-expressing enterocytes within rod-shaped organoids and (I) average enterocyte distribution. (B, D, F, H) Scale bars, 100 μm. (C, G, I) Scale bars, 25 μm.

Figure 3:. Tissue geometry controls organoid patterning through cell shape-mediated regulation of YAP and Notch signaling.

(A) Brightfield and Lgr5-eGFP time-lapse imaging of the representative organoid development and (B) frequency maps, showing average Lgr5 expression over ~80 tissues. (C) Relative changes in the Lgr5-eGFP expression in curved ends and flat sides of the organoids over time and (D) Lgr5-eGFP localization along length of the averaged tissue over time. (E) Immunofluorescence images showing the difference in internuclear distance, cell shape and the subcellular distribution of YAP between cells of the end and the side regions, 24 hours after cell loading. (F) Quantification of internuclear distance within the end and side regions of the tissues. Individual points, which represent the distance between neighboring nuclei, and means are shown. **** P < 0.0001 (G) Quantification of the nuclear localization of YAP within cells of the different organoid regions. Individual points and means are shown. **** P < 0.0001. (H) Immunofluorescence images showing the difference in the subcellular distribution of YAP cells between cells of the end and the side regions and appearance of the first DLL+, 36 hours after cell loading. (I) Quantification of DLL+ cells localization. **** P < 0.0001 (J, K) Immunofluorescence images showing YAP expression and localization of the enterocytes (AldoB), Paneth cells (Lys) and DLL+ cells in the representative organoids. (L) Schematic illustration summarizing the proposed mechanism of the geometry driven organoids patterning. Scale bars, 25 μm.

Figure 4:. Bioengineered organoids with an in vivo-like tissue architecture.

(A) Scheme of the designed topography resembling the native tissue with characteristic intestinal crypt-villus architecture. (B, C) SEM images of the poly(dimethylsiloxane) (PDMS) template used for fabricating bioengineered hydrogel substrates featuring a crypt-villus architecture, top and side view. Scale bars, 200 μm. (D) Top view of the hydrogel substrate shaped according to the topology of the native intestinal mucosa. (E) Brightfield time-course images of the intestinal epithelium development. (D,E) Extended depth of field for a z-stack ~600 μm. (F) Localization of the Lgr5+ stem cells in the engineered crypts. (D-F) Scale bars, 100 μm. (G) 3D reconstruction of the immunofluorescence images showing confluent monolayer of E-Cadherin expressing epithelial cells covering hydrogel substrates, harboring villi composed of enterocytes (AldoB) and other differentiated cell types.