a Laboratorios de Investigación Aplicada en Neurociencias (LIAN-CONICET) , Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia (FLENI) , Belén de Escobar , Provincia de Buenos Aires , Argentina.
Cell Cycle. 2018;17(14):1721-1744. doi: 10.1080/15384101.2018.1496740. Epub 2018 Aug 10.
Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3β activity.
hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III β-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.
人类多能干细胞（hPSCs），包括胚胎和诱导多能干细胞（hESCs 和 hiPSCs），表现出独特的细胞周期特征，例如由于 G1 期缩短而导致的倍增时间短。核心细胞周期机制是否直接调节 hPSCs 的干性和/或分化潜能仍有待确定。迄今为止，已经提出了几种描述 hPSCs 非典型细胞周期的情况，因此，关于细胞周期蛋白如何表达和调节仍存在争议。此外，尚未研究 hESCs 衍生的神经祖细胞（NP）中细胞周期谱和主要细胞周期蛋白的表达模式。因此，本文我们描述了 hPSCs 和 NP 中主要细胞周期蛋白的表达模式。我们确定所有研究的细胞周期蛋白 mRNA 表达水平沿细胞周期波动。特别地，在对同步化细胞群体进行彻底分析后，我们观察到 hPSCs 和 NP 中 cyclin E1 mRNA 水平在 G1/S 急剧增加，同时 cyclin E1 蛋白积累。此外，我们证明 cyclin E1 mRNA 表达水平涉及 hPSCs 中 MEK/ERK 途径和转录因子 c-Myc 和 E2Fs 的激活。最后，我们的结果表明，蛋白酶体通过需要功能性 CDK2 但不需要 GSK3β 活性的机制介导 G2/M 中观察到的 cyclin E1 蛋白的显著下调（降解）。
hPSCs：人类多能干细胞；hESCs：人类胚胎干细胞；hiPSCs：人类诱导多能干细胞；NP：神经祖细胞；HF：人包皮成纤维细胞；MEFs：小鼠胚胎成纤维细胞；iMEFs：辐照的小鼠胚胎成纤维细胞；CDKs：细胞周期蛋白依赖性激酶；CKIs：CDK 抑制剂；CNS：中枢神经系统；Oct-4：八聚体-4；EB：类胚体；AFP：甲胎蛋白；cTnT：心肌肌钙蛋白 T；MAP-2：微管相关蛋白；TUJ-1：神经元特异性 III 类 β-微管蛋白；bFGF：碱性成纤维细胞生长因子；PI3K：磷脂酰肌醇 3-激酶；KSR：敲除血清替代物；CM：iMEF 条件培养基；E8：必需 E8 培养基。
