小鼠和人类多能干细胞(PSC)的谱系规范伴随着染色体结构域的空间整合和其复制时机的时间整合。复制时间和染色质组织均在G1期的时间决定点(TDP)建立。这里,我们开发了活细胞成像工具来跟踪分化过程中的时空复制域整合。首先,我们证明荧光泛素化细胞周期指示(Fucci)系统无法区分G1/S或G2/M细胞周期转换。相反,我们采用荧光PCNA的组合来监测S期进展,胞质分裂来区分有丝分裂,和荧光核苷酸标记早期和晚期复制灶,并在所有细胞周期过渡中跟踪其3D组织进入亚核染色质区室。我们发现,随着人类PSC的分化,用于空间聚集复制焦点复制的S期长度增加,与域的全局划分为时间上聚集的染色质块一致。重要的是,域的重新定位和锚定在S期开始之前完成,即使在简短的PSCG1期的背景下。这种方法也可用于研究单个PSC中的细胞命运转变,可以看出这与G1期优先区分。一起,我们的结果建立了实时,用于追踪人类PSC分化过程中细胞周期转变的活细胞成像方法,可用于研究染色体结构域巩固和谱系规范的其他方面。
Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.