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Synthetic hydrogels for human intestinal organoid generation and colonic wound repair

Abstract

In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the 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 three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection 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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Figure 1: PEG-4MAL polymer density and adhesive ligand type control HIO viability.
Figure 2: Engineered PEG-4MAL supports HIO development.
Figure 3: PEG-4MAL polymer density regulates HIO generation from intestinal spheroids in the absence of Matrigel embedding.
Figure 4: PEG-4MAL-generated HIOs develop a mature intestinal tissue structure in vivo.
Figure 5: PEG-4MAL-generated HIOs differentiate into mature intestinal tissue in vivo.
Figure 6: PEG-4MAL serves as an injectable delivery vehicle to promote HIO engraftment and wound closure.

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Acknowledgements

This research was supported by the National Institute of Health (A.J.G. was supported by R01 AR062368 and R01 AR062920; A.N. was supported by DK055679, DK059888 and DK089763) and a seed grant from the Regenerative Engineering and Medicine Research Center between Emory University, Georgia Tech and the University of Georgia. J.R.S. was supported by the Intestinal Stem Cell Consortium (U01DK103141), a collaborative research project funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the National Institute of Allergy and Infectious Diseases (NIAID), by the NIAID Novel, Alternative Model Systems for Enteric Diseases (NAMSED) consortium (U19AI116482) and PHS Grant UL1TR000454 from the Clinical and Translational Science Award Program. R.C.-A. is supported by the National Science Foundation Graduate Research Fellowship (DGE-1650044) and the Alfred P. Sloan Foundation’s Minority Ph.D. (MPHD) Program (G-2016-20166039). M.Q. is supported by a fellowship from the Crohn’s and Colitis Foundation of America (CCFA 326912). A.E.F. is supported by János Bolyai Research Fellowship (BO/00023/17/8). We would like to thank Y.-H. Tsai (University of Michigan, USA) for providing tissue sections for immunohistochemical analysis.

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Authors

Contributions

R.C.-A. and M.Q. conducted all experiments, collected data and performed data analyses. A.E.F. assisted with in vitro and in vivo intestinal crypt experiments. P.H.D. performed the in vivo HIO implantation under the kidney capsule experiment. S.H. performed the culture and differentiation of PSCs into intestinal spheroids. D.S. assisted with in vivo HIO delivery to mouse colonic wounds. V.G.-H. and A.J.M. performed the in situ hybridization experiments. A.N., A.J.G. and J.R.S. conceptualized and designed the project and experiments. R.C.-A., A.J.G., A.N., J.R.S. and M.Q. wrote the manuscript.

Corresponding authors

Correspondence to Jason R. Spence or Asma Nusrat or Andrés J. García.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PEG-4MAL hydrogel preparation and mechanical properties.

(a) PEG-4MAL macromers are conjugated with thiol-containing adhesive peptide to produce a functionalized PEG-4MAL macromer, which is then crosslinked in the presence of HIOs/spheroids using protease-cleavable peptides containing terminal cysteines to form (b) hydrogel network. (c,d) Relationship between polymer density (wt%) and (c) storage modulus or (d) loss modulus (mean ± SEM; n = 10 independently prepared hydrogels per condition). (e) Schematic of spheroid development into HIOs within MatrigelTM and further growth within PEG-4MAL hydrogel. (f) Schematic of spheroid development into HIOs within hydrogel. Source data are available in Supplementary Table 1.

Supplementary Figure 2 PEG-4MAL hydrogel supports hiPSC-derived intestinal spheroid development into HIOs comparable to hESC-derived spheroids.

Transmitted light and fluorescence microscopy images of hiPSC-derived HIO generation within (a) 4.0% PEG-4MAL hydrogels, (b) MatrigelTM, or (c) 8.0% PEG-4MAL hydrogels. hiPSC-derived spheroid and HIO viability was assessed at different time-points after encapsulation. (d) Transmitted light microscopy images of hESC-derived HIO generation within 4.0% PEG-4MAL hydrogels. (a, c, d) These organoids were never encapsulated within MatrigelTM. Black arrows show epithelial budding. Bar, 500 μm. Three independent experiments were performed and data is presented for one of the experiments. Every experiment was performed with 12 gel samples per experimental group (PEG-4MAL, MatrigelTM).

Supplementary Figure 3 PEG-4MAL hydrogels with different macromer sizes and mediators of mechanotransduction are essential for hESC-derived spheroid survival.

(a) Transmitted light and fluorescence microscopy images of HIO generation within 20 kDa (4.0%) or 40 kDa (8.0%) PEG-4MAL hydrogels. HIO viability was assessed at 5 d after encapsulation. (b) Percentage of total organoid area stained for live or dead (mean ± SEM) after 5 d of encapsulation (n = 6 organoids analyzed per condition). (c) HIO projected area and Feret diameter normalized to Day 0 values (mean ± SEM) after 5 d of encapsulation (n = 5 organoids analyzed per condition). (b,c) Unpaired two-tailed t-test with Welch’s correction showed no significant differences between HIO viability or HIO size parameters within 20 kDa (4.0%) and 40 kDa (8.0%) PEG-4MAL (P > 0.05). (d) Transmitted light and fluorescence microscopy images of spheroids cultured within 4.0% PEG-4MAL hydrogels supplemented with (d) 10 μM or (e) 30 μM of verteporfin, Y-27632 or blebbistatin, or (f) DMSO (vehicle control). Spheroids death was assessed by annexin-V (apoptosis) and propidium iodide (dead) labeling at 1 d after encapsulation. Bar, 100 μm. One experiment was performed with 12 PEG-4MAL hydrogel samples per experimental group. Source data are available in Supplementary Table 1.

Supplementary Figure 4 Gene expression levels of PEG-4MAL-encapsulated spheroids are comparable to those embedded in MatrigelTM.

RNA levels of pluripotency (OCT4), endoderm (FOXA2), and epithelial junction (ZO1, ECAD and CLDN2) genes, as quantified by RT-qPCR (mean ± SEM; n = 6 samples per group). Unpaired two-tailed t-test was used to identify statistical differences between matrix types (P < 0.01; ns, not significant). One experiment was performed. Source data are available in Supplementary Table 1. Primer sequences are provided in Supplementary Table 2.

Supplementary Figure 5 PEG-4MAL hydrogel supports HLO development comparable to MatrigelTM.

(a) Transmitted light and fluorescence microscopy images of HLOs cultured in 4.0% PEG-4MAL hydrogels or MatrigelTM. HLO viability was assessed at 7 d after encapsulation. Bar, 500 μm. (b) Fluorescence microscopy images of HLO at 7 d after encapsulation in 4.0% PEG-4MAL hydrogel or MatrigelTM and labeled for e-cadherin (ECAD), lung epithelia (NKX2.1), and basal cells (P63). DAPI, counterstain. “L” indicates HLO lumen. Bars, 25 μm. (a,b) One experiment was performed with 6 gel samples per experimental group (PEG-4MAL, MatrigelTM).

Supplementary Figure 6 PEG-4MAL hydrogel serves as an injectable delivery vehicle in colonic mucosal wound model and promotes HIO engraftment.

(a) Mechanically-induced submucosal wounds were performed in the distal colon of mice using a mechanical probe through a mouse colonoscope. One day post-wounding HIOs generated in engineered 4% PEG-4MAL hydrogels or MatrigelTM were recovered from the matrix, mixed with the engineered hydrogel precursor solutions, and injected underneath the submucosal wounds. A group with no injections, HIOs injected in saline, or injection of HIO-free hydrogel precursor solutions were used as control groups. Distal colon tissue harvest, immunostaining and imaging was performed 4 weeks post-wounding. (b) Fluorescence microscopy images labeled for human mitochondria (HUMIT) of murine colonic tissue at the wound site at 4 weeks post-injection or control tissue. DAPI, counterstain. “L” indicates HIO lumen. Bar, 100 μm. (c) In situ hybridization images of (c) control adult human colon or sections taken at the mouse colonic wound site stained for human OLFM4 + cells. (d) In situ hybridization images of tissue sections from mice colon that did not undergo colonic injuries or received HIO injections (control) and sections taken at the mouse colonic wound site stained for mouse Lgr5 + intestinal stem cells. Bar, 50 μm. Two independent experiments were performed and data is presented for one of the experiments. Experiments performed with 4 mice per experimental group (five colonic wounds/injections per mouse; bd).

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Cruz-Acuña, R., Quirós, M., Farkas, A. et al. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair. Nat Cell Biol 19, 1326–1335 (2017). https://doi.org/10.1038/ncb3632

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