Lengthened G1 Phase Indicates Differentiation Status in Human Embryonic Stem Cells
Lengthened G1 Phase Indicates Differentiation Status in Human Embryonic Stem Cells
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DOI：
10.1089/scd.2012.0168
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发表时间：
2013-01-01
影响因子：
4
通讯作者：
Draper, Jonathan S.
中科院分区：
文献类型：
--
作者：
Calder, Ashley;Roth-Albin, Ivana;Draper, Jonathan S.
多能干细胞中的细胞周期以G1期的短暂而著称，允许快速增殖并减少与G1期相关的分化信号敏感性的持续时间。G1期长度的变化被认为是伴随着人类胚胎干细胞（hESC）的分化，但这种变化的时间和程度是不明确的。了解hESC分化的早期步骤将有助于更好地控制再生医学和药物发现应用的分化。在这里，我们报告的第一次使用实时细胞周期报告人胚胎干细胞。我们共表达染色质装饰H2 B-GFP融合蛋白和荧光泛素化细胞周期指示剂（FUCCI）-G1融合蛋白，G1期特异性报告，在hESC中测量活细胞的细胞周期状态。我们发现FUCCI-G1的表达在未分化的hESC中检测到微弱，但在分化后迅速增加。处于G1期的hESC显示出未分化集落起始细胞功能的降低，强调了G1期驻留和分化之间的关系。重要的是，我们证明了在响应诱导分化的化学物质，这意味着广泛的细胞-细胞的变化，改变G1期长度所需的阈值之间的变化。最后，分化标志物的获得似乎与G1期延长一致，不同的G1期特征与早期hESC分化的不同标志物相关。我们的数据表明，细胞周期的变化与人胚胎干细胞分化的紧密耦合，并强调了我们已经实施的细胞周期报告系统和测定作为一种新的途径，用于研究多能性和分化。
The cell cycle in pluripotent stem cells is notable for the brevity of the G1 phase, permitting rapid proliferation and reducing the duration of differentiation signal sensitivity associated with the G1 phase. Changes in the length of G1 phase are understood to accompany the differentiation of human embryonic stem cells (hESCs), but the timing and extent of such changes are poorly defined. Understanding the early steps governing the differentiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discovery applications. Here we report the first use of real-time cell cycle reporters in hESCs. We coexpressed the chromatin-decorating H2B-GFP fusion protein and the fluorescence ubiquitination cell cycle indicator (FUCCI)-G1 fusion protein, a G1 phase-specific reporter, in hESCs to measure the cell cycle status in live cells. We found that FUCCI-G1 expression is weakly detected in undifferentiated hESCs, but rapidly increases upon differentiation. hESCs in the G1 phase display a reduction in undifferentiated colony-initiating cell function, underscoring the relationship between G1 phase residence and differentiation. Importantly, we demonstrate inter-and intracolony variation in response to chemicals that induce differentiation, implying extensive cell-cell variation in the threshold necessary to alter the G1 phase length. Finally, gain of differentiation markers appears to be coincident with G1 phase lengthening, with distinct G1 phase profiles associated with different markers of early hESC differentiation. Our data demonstrate the tight coupling of cell cycle changes to hESC differentiation, and highlight the cell cycle reporter system and assays we have implemented as a novel avenue for investigating pluripotency and differentiation.