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. 2015 May 7;521(7550):43-7.
doi: 10.1038/nature14415. Epub 2015 Apr 29.

Sequential cancer mutations in cultured human intestinal stem cells

Jarno Drost  1 , Richard H van Jaarsveld  2 , Bas Ponsioen  2 , Cheryl Zimberlin  3 , Ruben van Boxtel  1 , Arjan Buijs  4 , Norman Sachs  1 , René M Overmeer  2 , G Johan Offerhaus  5 , Harry Begthel  1 , Jeroen Korving  1 , Marc van de Wetering  6 , Gerald Schwank  1 , Meike Logtenberg  1 , Edwin Cuppen  1 , Hugo J Snippert  2 , Jan Paul Medema  3 , Geert J P L Kops  2 , Hans Clevers  1
Affiliations

Sequential cancer mutations in cultured human intestinal stem cells

Jarno Drost et al. Nature. .

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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