Sequential cancer mutations in cultured human intestinal stem cells
Sequential cancer mutations in cultured human intestinal stem cells
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DOI：
10.1038/nature14415
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发表时间：
2015-05-07
期刊：
影响因子：
64.8
通讯作者：
Clevers, Hans
中科院分区：
文献类型：
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作者：
Drost, Jarno;van Jaarsveld, Richard H.;Clevers, Hans
隐窝干细胞代表肠肿瘤的起源细胞。小鼠和人类肠道干细胞都可以在含有干细胞小生境因子WNT、R-spondin、表皮生长因子（EGF）和头蛋白的培养基中长时间培养，作为保持遗传和表型稳定的上皮类器官。在这里，我们利用CRISPR/Cas9技术对培养的人类肠道干细胞中四种最常见的突变结直肠癌基因（APC，P53（也称为TP 53），KRAS和SMAD 4）进行靶向基因修饰。可以通过从培养基中去除单独的生长因子来选择突变的类器官。四重突变体生长独立于所有干细胞生态位因子，并耐受P53稳定剂nutlin-3的存在。在异种移植到小鼠中时，四重突变体生长为具有侵袭性癌特征的肿瘤。最后，APC和P53的联合丢失足以出现广泛的非整倍体，这是肿瘤进展的标志。
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.