Use of organoid cultures to assess genome editing technology and model human physiology

使用类器官培养物评估基因组编辑技术并模拟人类生理学

• 批准号: 1943428
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1943428
• 关键词: Use; organoid; cultures; assess; genome

Assessment of cell transformation (i.e. the acquisition of malignant characteristics in morphology, growth control orfunction) has proven problematic to model in vitro. The soft agar cell transformation assay has been validated, but only in alimited number of immortalised or mixed population embryonic cells and the specificity of the assay has been questioned.Furthermore, the genetic basis of cell transformation in this model has not been fully delineated. The use of genetically andphenotypically stable organoids derived from single human stem cells should provide a better model for cell proliferation andtransformation.Organoids have the advantage of 3D morphology, being able to be derived from several major organs and can be engineeredto demonstrate proliferative effects by introducing a cell type specific ki67 fluorescent protein to tag. Organoid stability isalso dependent on several growth factors and inhibitors e.g. EGF, Wnt, BMP inhibitor, TGFb inhibitor. Growth followingwithdrawal of these factors, which are known pathways implicated in cancer genesis, will be indicative of cell phenotypicand/or transformative changes. It is also proposed that this model could be used to investigate mechanisms of prooncogenicityand also the potential off-target effect of genome editing by CRISPR/Cas9, for which there is currently an unmetneed for in vitro assays to assess concerns around inappropriate editing in oncogenes.The project will develop organoids from a variety of tissue sources, including organoids with the ki67 tag, and assessment ofproliferative and morphological changes following withdrawal of growth factors after treatment with reference carcinogensand following transfection with specific and promiscuous guide RNA and associated Cas9 protein. Comparative data will begenerated from alternative in vitro methodologies to analyse cell transformation e.g. the soft agar assay. Referencecarcinogens will be analysed in this assay along with the potential of CRISPR/Cas9 editing to induce cell transformation(which has not been previously assessed). The student will also use next generation sequencing technologies to identifyspecific genes involved in morphological changes in both cell and organoid cultures. Success will provide new and morerelevant model for understanding and assessment of morphological changes and carcinogenesis.

细胞转化的评估（即获得形态学、生长控制或功能方面的恶性特征）已证明在体外建模方面存在问题。软琼脂细胞转化试验已得到验证，但仅限于有限数量的永生化或混合群体胚胎细胞，且该试验的特异性受到质疑，此外，该模型中细胞转化的遗传基础尚未完全阐明。利用遗传和表型稳定的单个人类干细胞衍生的类器官应该为细胞增殖和转化提供一个更好的模型。类器官具有三维形态的优势，能够衍生自几个主要器官，并且可以通过引入细胞类型特异性ki 67荧光蛋白来标记来展示增殖效应。类器官的稳定性还依赖于几种生长因子和抑制剂，例如EGF、Wnt、BMP抑制剂、TGF β抑制剂。这些因子是已知的与癌症发生有关的途径，在这些因子消失后生长将指示细胞表型和/或转化性变化。还提出该模型可用于研究原癌发生的机制以及CRISPR/Cas9基因组编辑的潜在脱靶效应，目前还需要体外测定来评估致癌基因中不适当编辑的担忧。该项目将从各种组织来源开发类器官，包括带有ki 67标签的类器官，以及评估在用参考致癌物处理后以及在用特异性和混杂的引导RNA和相关的Cas9蛋白转染后，在撤回生长因子后的增殖和形态学变化。比较数据将从分析细胞转化的替代体外方法中产生，例如软琼脂试验。将在该试验中分析参考致癌物沿着CRISPR/Cas9编辑诱导细胞转化的潜力（先前尚未评估）。学生还将使用下一代测序技术来鉴定细胞和类器官培养中形态变化所涉及的特定基因。这一成功将为理解和评价形态学变化和癌变提供新的和更相关的模型。

项目成果

Three-Dimensional Human Alveolar Stem Cell Culture Models Reveal Infection Response to SARS-CoV-2.

• DOI: 10.1016/j.stem.2020.10.004
• 发表时间: 2020-12-03
• 期刊: Cell stem cell
• 影响因子: 23.9
• 作者: Youk J;Kim T;Evans KV;Jeong YI;Hur Y;Hong SP;Kim JH;Yi K;Kim SY;Na KJ;Bleazard T;Kim HM;Fellows M;Mahbubani KT;Saeb-Parsy K;Kim SY;Kim YT;Koh GY;Choi BS;Ju YS;Lee JH
• 通讯作者: Lee JH

Emergence of a High-Plasticity Cell State during Lung Cancer Evolution.

肺癌演化过程中高塑性细胞状态的出现。

• DOI: 10.1016/j.ccell.2020.06.012
• 发表时间: 2020-08-10
• 期刊: Cancer cell
• 影响因子: 50.3
• 作者: Marjanovic ND;Hofree M;Chan JE;Canner D;Wu K;Trakala M;Hartmann GG;Smith OC;Kim JY;Evans KV;Hudson A;Ashenberg O;Porter CBM;Bejnood A;Subramanian A;Pitter K;Yan Y;Delorey T;Phillips DR;Shah N;Chaudhary O;Tsankov A;Hollmann T;Rekhtman N;Massion PP;Poirier JT;Mazutis L;Li R;Lee JH;Amon A;Rudin CM;Jacks T;Regev A;Tammela T
• 通讯作者: Tammela T

Development of an in vitro human lung organoid system to study alveolar type 2 stem cell maintenance and dysfunction during disease

开发体外人肺类器官系统来研究疾病期间肺泡 2 型干细胞的维持和功能障碍

• DOI: 10.17863/cam.85289
• 发表时间: 2021
• 作者: Evans K