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Designer matrices for intestinal stem cell and organoid culture

Abstract

Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease1,2,3. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown4. Here we used modular synthetic hydrogel networks5,6 to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.

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Figure 1: Building a synthetic ISC niche.
Figure 2: Matrix mechanical properties control ISC proliferation.
Figure 3: ISC proteolytic activity influences proliferation, morphology and fate.
Figure 4: Intestinal organoid formation within well-defined matrices.

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Acknowledgements

We thank M. Knobloch (University of Zurich) for helpful discussions, A. Negro (EPFL) for help in the development of PEG–alginate hybrid hydrogels, D. Ossipov (Uppsala University) for providing hyaluronic acid, the Lausanne Genomic Technologies Facility (K. Harshman) for RNA-seq and D. Pioletti (EPFL) for rheometer use. N.G. was supported by an EMBO Long-Term Postdoctoral Fellowship. This work was also supported by funding from the Ecole Polytechnique Fédérale de Lausanne (EPFL). Work performed in the laboratory of H.C. was supported by the NWO Translational Adult Stem Cell Research Grant 40-41400-98-1108 and by a NWO VENI Grant 916.15.182.

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Authors

Contributions

N.G. and M.P.L. conceived the study, designed experiments, analysed data and wrote the manuscript. N.G. was involved in performing and analysing all experiments in the manuscript except for those involving human organoids. P.O.M. helped design experiments and analyse RNA-seq data. A.M. performed qPCR gene expression experiments and analysed data and produced lentiviruses. S.G. performed flow cytometry analysis of integrin expression of ISCs culture in Matrigel and PEG RGD. M.E.B. designed and characterized PEG–alg hydrogel system and helped N.G. perform experiments with ISCs in these matrices. N.S. and H.C. designed experiments and analysed data with human cells. N.S. performed experiments with human cells.

Corresponding author

Correspondence to Matthias P. Lutolf.

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Competing interests

Ecole Polytechnique Fédérale de Lausanne (with M.P.L. and N.G.) has filed patent applications pertaining to synthetic gels for epithelial stem cell and organoid cultures. H.C. is an inventor of several patents on organoid technology.

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Reviewer Information

Nature thanks L. Li, J. Mills and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Primary crypt culture in synthetic matrices, characterization of ISCs grown in PEG RGD culture with or without CHIR99021 and valproic acid.

a, ISCs cultured in Matrigel and unmodified PEG gels for 24 h. b, ISC colonies formed from freshly isolated mouse intestinal crypts embedded in PEG RGD. c, Relative mRNA levels of intestinal genes, quantified by qPCR. d, ISC colonies cultured under self-renewal conditions in Matrigel, but not PEG gels, contain lysozyme-expressing Paneth cells. e, Stiffness-dependent colony formation and quantification of ISCs cultured with EGF, noggin, R-spondin and Wnt3a. Graphs show individual data points derived from n = 3 independent experiments and means. Scale bars, 50 μm.

Source data

Extended Data Figure 2 Effect of controlled matrix softening on ISC colony morphology, YAP activity and growth.

a, Mechanical properties of control and softened PEG alginate (PEG–alg) hybrid gels obtained by selective degradation of the alginate network. Graph shows individual data points derived from 3 independently prepared gels. b, Morphology of day 1 colonies in control and softened PEG–alg gels. c, d, Distribution of YAP in day 1 colonies in control and softened PEG–alg gels (c) and quantification (d). n = 28 colonies (control) and n = 31 (softened). Data are represented as mean ± s.e.m. e, Alginate lyase treatment does not affect colony growth in PEG RGD gels. Softening of PEG–alg gels by alginate-lyase-mediated digestion blocks colony growth. f, Quantification of shRNA-mediated knockdown of YAP. Graph shows individual data points derived from 2 independent experiments and means. g, Effect of verteporfin on ISC colony formation in PEG RGD. Graph shows individual data points derived from 3 independent experiments and means.*P < 0.05; **P < 0.01; ***P < 0.01. Scale bars, 50 μm.

Source data

Extended Data Figure 3 Stiff degradable matrices induce an inflammation-like state in ISCs.

a, Gene set enrichment analysis (GSEA) comparing RNA-seq gene expression data of ISCs cultured in degradable compared to non-degradable matrices to published gene signatures (see Methods for details). b, Functional annotation of signalling pathway significantly upregulated in degradable matrices (details and statistics shown in Extended Data Tables 2, 3).

Extended Data Figure 4 Comparison of organoid formation and YAP activity in Matrigel and PEG RGD.

a, Organoid formation does not occur within synthetic PEG RGD matrices. ISC colonies cultured in non-degradable (PEG N-DG) and degradable (PEG DG) PEG RGD matrices, with or without GM6001 for 4 d, and subsequently cultured under organoid formation conditions for 2 d. b, Time-course analysis of morphology and Lgr5–eGFP expression during organoid formation. ISC colonies formed in Matrigel and PEG hydrogels were monitored for 48 h, following a switch to differentiation and organoid formation conditions. c, Localization of YAP at day 1 and day 4 in ISC colonies formed in 300 Pa and 1.3 kPa PEG RGD gels. d, Quantification of nuclear translocation of YAP as a function of time in 300 Pa, 1.3 kPa PEG RGD and Matrigel. Data are shown as means ± s.e.m. To assess the extent of YAP nuclear translocation, n = 21 (day 1, 1.3 kPa); 27 (day 2, 1.3 kPa); 22 (day 3, 1.3 kPa); 22 (day 4, 1.3 kPa); 27 (day 1, 300 Pa); 6 (day 2, 300 Pa); 23 (day 3, 300 Pa); 22 (day 4, 300 Pa); 30 (day 1, Matrigel); 28 (day 2, Matrigel); 30 (day 3, Matrigel); 30 (day 4, Matrigel). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars, 50 μm.

Source data

Extended Data Figure 5 Effect of laminin-derived peptides on organoid formation, and human organoid expansion in PEG RGD.

a, Effect of different laminin-111-derived sequences on intestinal organoid viability. b, Morphology of intestinal organoids grown in Matrigel, plain PEG or PEG-AG73 gels. c, AG73-conjugated PEG matrices significantly enhance the growth of intestinal organoids. d, Effect of AG73 on intestinal organoid viability and growth is concentration-dependent. e, Quantification of intestinal organoid viability in Matrigel, TG PEG-AG73 and MT PEG-AG73. f, Morphology and Lgr5–eGFP expression in intestinal organoids grown in Matrigel and MT PEG-AG73 gels. g, Quantification of Lgr5–eGFP expression in intestinal organoids expanded in Matrigel, TG PEG-AG73 and MT PEG-AG73. h, Establishment of apicobasal polarity and presence of Paneth (lysozyme) cells within organoids grown in MT PEG-AG73. i, AG73 peptides are not capable of supporting differentiation and organoid formation from ISC colonies, whereas full-length laminin-111 is. j, Quantification of nuclear translocation of YAP as a function of time in cells cultiured within mechanically stable and softening gels. Data are shown as means ± s.e.m. To assess the extent of YAP nuclear translocation, n = 21 (day 1, 100% sPEG), 27 (day 2, 100% sPEG), 22 (day 3, 100% sPEG), 22 (day 4, 100% sPEG), 30 (day 1, 75% dPEG), 30 (day 2, 75% dPEG), 30 (day 3, 75% dPEG), and 30 (day 4, 75% dPEG) colonies were analysed. k, l, Phase contrast images (k) and Ki67 expression (l) of human ISC colonies and human patient-derived colorectal cancer organoids grown in PEG RGD. Graphs show individual data points derived from n = 2 (a), n = 3 (ce, g) independent experiments and means. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars, 50 μm.

Source data

Extended Data Table 1 Primers used for qPCR analysis of intestinal gene expression
Extended Data Table 2 Functional annotation of genes significantly upregulated in degradable PEG RGD matrices versus non-degradable PEG RGD matrices, in terms of cellular pathways
Extended Data Table 3 Functional annotation of genes significantly upregulated in degradable PEG RGD matrices versus non-degradable PEG RGD matrices, in terms of cellular processes
Extended Data Table 4 Laminin-derived peptides

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Gjorevski, N., Sachs, N., Manfrin, A. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016). https://doi.org/10.1038/nature20168

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