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PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery

A Publisher Correction to this article was published on 24 October 2018

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Abstract

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.

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Fig. 1: Preparation of the solutions for the precursors of the PEG-4MAL hydrogel.
Fig. 2: Synthesis of the PEG-4MAL hydrogel and organoid generation.
Fig. 3: Typical results of rheometric characterization of PEG-4MAL hydrogels.
Fig. 4: Preparation of PEG-4MAL hydrogel-generated organoids and setup for mucosal injection.
Fig. 5: PEG-4MAL hydrogel serves as an injectable delivery vehicle in a colonic mucosal wound model.
Fig. 6: PEG-4MAL hydrogel promotes HIO engraftment into mucosal wounds.
Fig. 7: PEG-4MAL hydrogel supports hPSC-derived intestinal spheroid development into HIOs.

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Zixuan Zhao, Xinyi Chen, … Hanry Yu

Change history

  • 24 October 2018

    In the version of this protocol originally published, the caption for Fig. 3 was erroneously placed with Fig. 4, and that for Fig. 4 was placed with Fig. 3. This error has been corrected in the HTML and PDF versions of the paper.

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Acknowledgements

This research was supported by the National Institutes of Health (A.J.G. was supported by R01 AR062368 and R01 AR062920; A.N. was supported by R01 DK055679, R01 DK059888, DK055679, 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), 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. was supported by a National Science Foundation Graduate Research Fellowship (DGE-1650044) and the Alfred P. Sloan Foundation’s Minority PhD (MPHD) Program (G-2016-20166039). M.Q. was supported by a fellowship from the Crohn’s and Colitis Foundation of America (CCFA 326912). We thank A. Heinen for technical support.

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Authors

Contributions

R.C.-A. and M.Q. conducted all experiments, collected data, and performed data analyses. S.H. performed the culture and differentiation of hPSCs into intestinal spheroids. D.S. assisted with in vivo HIO delivery to mouse colonic wounds. 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, Asma Nusrat or Andrés J. García.

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Competing interests

R.C.-A., M.Q., J.R.S., A.N., and A.J.G. are inventors on a patent application related to this work and co-owned by the Georgia Tech Research Corp. and the University of Michigan. The remaining authors declare no competing interests.

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Cruz-Acuña, R. et al. Nat. Cell Biol., 1326–1335 (2017) https://doi.org/10.1038/ncb3632.

Supplementary information

41596_2018_36_MOESM1_ESM.mov

Supplementary Video 1 Injection of HO-containing PEG-4MAL hydrogel into colonic mucosal wound via colonoscope. The wound is observed through the camera, the needle is inserted at the distal end of the wound by carefully penetrating the mucosa, and the contents of the custom-made device are injected into the site. A protrusion at the injection site is observed through the camera after a successful injection of HIO-containing hydrogel precursor solutions. All experiments with mice were performed while following national and institutional guidelines.

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Cruz-Acuña, R., Quirós, M., Huang, S. et al. PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery. Nat Protoc 13, 2102–2119 (2018). https://doi.org/10.1038/s41596-018-0036-3

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