Transcriptional profile of human iPSC and HIOs with APC mutations. Homo sapiens

Background and Aims: mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. Methods: we have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects we also established an isogenic platform using TALEN-mediated gene editing. Results: comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. Conclusions: we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis. Overall design: Four independent biological replicates of each (APC+/+ vs APC+/-) were analyzed in triplicates, before and after differentiation into intestinal organoids, for a total of 48 samples

研究背景与目的：腺瘤性结肠息肉病（Adenomatous Polyposis Coli, APC）基因突变见于绝大多数散发性结直肠癌病例，亦是导致遗传性家族性腺瘤性息肉病（Familial Adenomatous Polyposis, FAP）最常见的突变类型。目前APC突变促使结直肠癌发生的具体分子机制尚未完全阐明，部分原因在于缺乏能够重现APC介导的肠道癌变早期事件的合适模型。
实验方法：本研究构建了一套全新的研究平台，分别从健康个体及FAP特异性APC突变携带者处获取人类诱导多能干细胞（human induced Pluripotent Stem cells, iPSC），并在细胞分化为肠道类器官前后，评估该突变对细胞的影响。为尽可能消除遗传背景的干扰，本研究同时构建了基于转录激活因子样效应物核酸酶（TALEN）介导基因编辑的同基因平台。
实验结果：对正常及APC突变型iPSC的比较分析显示，由于APC呈杂合性突变，同时伴随包括异常分裂后期及中心体数目异常在内的染色体畸变，细胞的身份识别与极性出现显著缺陷。值得注意的是，在分化为肠道祖细胞谱系后，APC杂合性突变导致细胞的转录特征发生重大改变，关键信号通路出现失调，包括代谢重编程、异常脂质代谢以及肠道特异性钙粘蛋白表达异常。
研究结论：本研究开发了一套全新的APC诱变iPSC/肠道模型，为杂合性APC突变会引发明确的表型与分子缺陷提供了有力证据，该缺陷会影响细胞的基本功能与完整性，为APC介导的肿瘤发生早期事件提供了全新的研究视角。
实验整体设计：本研究对每组（APC野生型与APC杂合突变型）的4个独立生物学重复样本分别进行三次平行检测，涵盖细胞分化为肠道类器官前后的样本，总计48个检测样本。