复制应激的实质背景水平是胚胎和诱导多能干细胞(iPSCs)的特征,这可能会导致染色体的数值和结构不稳定,包括12号染色体的反复畸变。在分化的细胞中,复制应激敏感基因组区域,包括常见的脆弱地点,通过温和的蚜虫霉素处理通过有丝分裂染色体断裂诱导广泛定位,复制聚合酶的抑制剂。IPSC表现出较低的凋亡阈值和较高的修复能力,阻碍了脆弱的位点定位。咖啡因可增强基因毒性作用,并消除化学和物理诱变剂诱导的G2/M检查点延迟。使用5-乙炔基-2'-脱氧尿苷(EdU)进行复制标记,我们表征了暴露于蚜虫菌素和/或咖啡因的异步iPSCs的有丝分裂进入动力学。在复制应激暴露的调整时间下,说明细胞周期延迟,与原代淋巴细胞相比,iPSC的中期染色体断裂率更高。使用差异染色体染色和随后的基因座特异性荧光原位杂交,我们将跨越大神经元ANKS1B基因的FRA12L脆性位点定位在12q23.1,这可能导致iPSCs中12号染色体反复发生的错误分离和重排.综述了有关ANKS1B遗传改变及其可能的功能影响的公开数据。我们的研究提供了iPSC中常见脆性位点诱导的第一个证据,并揭示了在早期人类发育和体外细胞扩增过程中临床相关基因的潜在体细胞不稳定性。
Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2\'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal
ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the
ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.