Subjects

Abstract

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

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Primary accessions

European Nucleotide Archive

Data deposits

Sequencing data have been deposited in the EMBL European Nucleotide Archive under accession number ERP009240.

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Acknowledgements

We would like to thank H. M. Rodermond for help with in vivo transplantation assays and members of the contributing laboratories for support. We thank A. Pronk, W. van Houdt and J. van Gorp for facilitating human colon tissue. We are grateful for support from the following: The Netherlands Organisation for Scientific Research (NWO-ZonMw) VENI grant to J.D. (91614138); University of Amsterdam (2012-5735) and The Dutch Digestive Diseases Foundation (MLDS) (FP13-07) to C.Z. and J.P.M.; Netherlands Institute of Regenerative Medicine (N.S. and G.S.); Dutch Cancer Society (KWF) (KWF/PF-HUBR 2007-3956 for H.B.; KWF Fellowship UU2013-6070 for H.J.S.); Stand Up to Cancer/Stichting Vrienden van het Hubrecht (M.v.d.W.); NWO-ZonMw (116.005.002 for R.v.B.); and the CancerGenomics.nl (NWO Gravitation) program.

Author information

Author notes

    • Richard H. van Jaarsveld
    • , Bas Ponsioen
    •  & Cheryl Zimberlin

    These authors contributed equally to this work.

Affiliations

  1. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands

    • Jarno Drost
    • , Ruben van Boxtel
    • , Norman Sachs
    • , Harry Begthel
    • , Jeroen Korving
    • , Marc van de Wetering
    • , Gerald Schwank
    • , Meike Logtenberg
    • , Edwin Cuppen
    •  & Hans Clevers
  2. Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands

    • Jarno Drost
    • , Richard H. van Jaarsveld
    • , Bas Ponsioen
    • , Cheryl Zimberlin
    • , Ruben van Boxtel
    • , Norman Sachs
    • , René M. Overmeer
    • , Harry Begthel
    • , Jeroen Korving
    • , Marc van de Wetering
    • , Gerald Schwank
    • , Meike Logtenberg
    • , Edwin Cuppen
    • , Hugo J. Snippert
    • , Jan Paul Medema
    • , Geert J. P. L. Kops
    •  & Hans Clevers
  3. Molecular Cancer Research, Centre for Molecular Medicine, UMC Utrecht, 3584CG, Utrecht, The Netherlands

    • Richard H. van Jaarsveld
    • , Bas Ponsioen
    • , René M. Overmeer
    • , Hugo J. Snippert
    •  & Geert J. P. L. Kops
  4. Laboratory of Experimental Oncology and Radiobiology, Centre for Experimental Molecular Medicine, AMC, 1105AZ Amsterdam, The Netherlands

    • Cheryl Zimberlin
    •  & Jan Paul Medema
  5. Department of Medical Genetics, UMC Utrecht, 3508AB Utrecht, The Netherlands

    • Arjan Buijs
  6. Department of Pathology, UMC Utrecht, 3584CX Utrecht, The Netherlands

    • G. Johan Offerhaus
  7. Foundation Hubrecht Organoid Technology (HUB), 3584CT Utrecht, The Netherlands

    • Marc van de Wetering

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Contributions

J.D. and H.C. conceived the project and wrote the manuscript. J.D. engineered and characterized all mutant organoid lines. R.H.v.J., B.P., H.J.S., R.M.O. and G.J.P.L.K. designed and performed live-cell imaging experiments. C.Z. and J.P.M. performed in vivo transplantation assays. R.v.B. and E.C. performed off-target analyses. J.D. performed karyotyping. A.B. made karyograms. G.J.O. staged subcutaneous tumours. H.B. and J.K. performed immunohistochemistry. N.S. optimized matrix for organoid growth. G.S. designed APC sgRNAs. M.v.d.W. established normal human colon organoid line. M.L. helped genotype the mutant small intestinal organoids.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hans Clevers.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure

    This file contains Supplementary Figure 1, uncropped images of western blots.

Excel files

  1. 1.

    Supplementary Table 1

    This table shows off-target analysis of KRAS, APC, P53 and SMAD4 sgRNAs. Off-targets were assessed by amplicon-based NGS sequencing of candidate off-target sites in the indicated organoid cultures. The columns in the table indicate the position of the candidate off-target sites (chromosome, start and end position), the alignment of the off-target sequence with the sequence of the sgRNA (indicated are bases that differ), the off-target score (a score of 1 indicates complete sequence similarity, the lower the score the more sequence differences), the mean base coverage per sample over the indicated off-target sequence, coordinates of identified small insertions and deletions (indels) in the indicated candidate off-target site and the variant allele frequencies (VAF) for the identified indels in the different samples, respectively. Only indels at the target sites for which the sgRNAs were designed (KRAS, APC, P53 and SMAD4; marked in yellow) were identified. One indel was observed at a candidate off-target site of the APC sgRNA in all samples (non-coding region). However, as the KRASG12D organoid line was not transfected with this sgRNA, this indel is not considered as an off-target effect. No additional indels were observed.

Videos

  1. 1.

    Live cell imaging of chromosome segregations in human intestinal stem cell organoids

    Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and RASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video.

  2. 2.

    Live cell imaging of chromosome segregations in human intestinal stem cell organoids

    Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video.

  3. 3.

    Live cell imaging of chromosome segregations in human intestinal stem cell organoids

    Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video.

  4. 4.

    Live cell imaging of chromosome segregations in human intestinal stem cell organoids

    Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video.

  5. 5.

    Live cell imaging of chromosome segregations in human intestinal stem cell organoids

    Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video.

  6. 6.

    Live cell imaging of chromosome segregations in human intestinal stem cell organoids

    Representative live cell imaging experiments of wild-type (1), APCKO (2), APCKO/P53KO (3), KRASG12D/APCKO/P53KO (4) and KRASG12D/APCKO/P53KO/SMAD4KO (5) organoids and P53KO in the second human intestinal organoid line (6). Organoids were subjected to confocal imaging for 16 – 20 hours. Shown are representative parts of this time window and organoids presented are depicted as green-lined dots in quantification plot Figure 4a. Upper right quadrant shows H2B-mNeon fluorescence after maximum-projection of 3D z-stacks; upper left represents same data, color-coded for depth (z), facilitating tracking of individual events; lower quadrants include transmitted light images with and without merged H2B-mNeon (green). Scale bars as indicated in video.

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DOI

https://doi.org/10.1038/nature14415

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