Effect of Cell Division on Cell Differentiation (RNA-Seq H9 cells UD E24 E24RO E24woTET E24TET)

We addressed the role of cell cycle regulator CDK1 during differentiation by either inhibiting its activity with the specific inhibitor RO3306 (10µM) or by knocking its expression down using specific shRNAs, during mesendoderm differentiation. Human pluripotent stem cells (H9 cells) were synchronized in their cell cycle and then induced to differentiate into mesendoderm for 24h. shRNAs against CDK1 were induced 4 days before starting the differentiation. The CDK1 inhibitor was added to the medium 8h after the induction of differentiation. At the end of the experiment, the cells were harvested and processed for RNA extraction. Overall design: Four different conditions were analyzed: Undifferentiated (UD), Mesendoderm (E24), Mesendoderm + CDK1 inhibityor (E24RO), Mesendoderm without induction of shRNA_CDK1 with tetracycline (E24woTET), and Mesendoderm plus induction of shRNA_CDK1 with tetracycline (E2TET). Two biological replicates were generated for each condition.

本研究探究了细胞周期调控因子CDK1在细胞分化过程中的功能：在中内胚层分化阶段，分别通过特异性抑制剂RO3306（浓度10μM）抑制其活性，或利用特异性短发夹RNA（short hairpin RNA，shRNA）敲低其表达。本实验采用人类多能干细胞（human pluripotent stem cells，H9细胞），先将其进行细胞周期同步化处理，随后诱导分化为中内胚层，诱导时长为24小时。靶向CDK1的短发夹RNA将在分化启动前4天完成诱导表达，而CDK1抑制剂则于分化诱导完成后的8小时添加至细胞培养基中。实验结束后，收集全部细胞并进行RNA提取操作。实验整体设计如下：本次实验共分析四组不同处理条件，分别为未分化组（Undifferentiated，UD）、中内胚层诱导24小时组（Mesendoderm，E24）、中内胚层诱导+CDK1抑制剂处理组（Mesendoderm + CDK1 inhibitor，E24RO）、未通过四环素诱导shRNA_CDK1表达的中内胚层组（Mesendoderm without tetracycline-induced shRNA_CDK1 expression，E24woTET）以及经四环素诱导shRNA_CDK1表达的中内胚层组（Mesendoderm with tetracycline-induced shRNA_CDK1 expression，E2TET）。每组处理均设置两个生物学重复。