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Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny

Nature Methods volume 11, pages 106–112 (2014)Cite this article

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Abstract

Although Lgr5+ intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5+ intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ∼100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5+ cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.

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Figure 1: The combination of CHIR and VPA promotes self-renewal of Lgr5+ stem cells.
Figure 2: The combination of CHIR and VPA maintains the stem cell state of Lgr5+ stem cells.
Figure 3: Differentiation of ISCs cultured under the CV condition.
Figure 4: Exploring the mechanism of action for CHIR and VPA.
Figure 5: Model for ISC self-renewal and differentiation.

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Acknowledgements

This research was supported by US National Institutes of Health (NIH) grant DE013023 to R.L. and a Harvard Institute of Translational Immunology/Helmsley Trust Pilot Grant in Crohn's Disease to J.M.K. H.F.F. was supported by an EMBO long-term fellowship. We also thank D. Breault, R. Montgomery and R. Shivdasani for critically reviewing the manuscript; W. Cho, M. Haraguchi and Q. Wang for helpful discussions; W. Salmon and N. Watson of the W.M. Keck Biological Imaging Facility at the Whitehead Institute for assistance with imaging; G. Paradis, X. Song and M. Jennings of the MIT Flow Cytometry Core Facility for assistance with flow cytometry and CCSG NIH grant CA014051 for support; A. Bhan, V. Yajnik, M. Miri and A. Brunelle at Massachusetts General Hospital for providing human tissue; and all of the staff of the MIT animal care facility.

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Authors and Affiliations

  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA

    Xiaolei Yin & Robert Langer

  2. Department of Medicine, Center for Biomedical Engineering, Brigham and Women's Hospital, Cambridge, Massachusetts, USA

    Xiaolei Yin & Jeffrey M Karp

  3. Harvard Medical School, Boston, Massachusetts, USA

    Xiaolei Yin & Jeffrey M Karp

  4. Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA

    Xiaolei Yin, Robert Langer & Jeffrey M Karp

  5. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Xiaolei Yin & Jeffrey M Karp

  6. Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht, Utrecht, The Netherlands

    Henner F Farin, Johan H van Es & Hans Clevers

  7. Department of Chemical Engineering, MIT, Cambridge, Massachusetts, USA

    Robert Langer

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Contributions

X.Y., H.F.F., H.C., R.L. and J.M.K. conceived of and designed the experiments. X.Y. and H.F.F. performed the experiments. J.H.v.E. provided Dll1EGFP-IRES-CreERT2, Atoh1 knockout organoids and Rbpj/Apc knockout-mice intestine. X.Y., H.F.F., J.H.v.E., H.C., R.L. and J.M.K. analyzed the data. X.Y., H.F.F., R.L. and J.M.K. wrote the manuscript.

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Results (PDF 4841 kb)

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Yin, X., Farin, H., van Es, J. et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat Methods 11, 106–112 (2014). https://doi.org/10.1038/nmeth.2737

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