In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.

环境因素对诱导多能干细胞 (iPSC) 的体外操作对于三维 (3D) 组织/器官诱导具有重要意义。机械力的影响取决于许多因素，包括力和细胞类型。然而，缺乏有关 iPSC 中此类影响的信息。本研究的目的是通过分析全局基因表达谱来确定 iPSC 响应间歇压缩力 (ICF) 的分子机制。以 3D 形式附着在组织培养板上的小鼠 iPSC 的胚胎体在无血清培养基中进行 24 小时的 ICF。RNA测序数据的基因本体分析表明，受ICF差异调节的基因主要与代谢过程、膜和蛋白质结合有关。基于拓扑的分析表明，ICF 诱导细胞周期类别中的基因和与代谢过程相关的下调基因。京都基因百科全书和基因组数据库揭示了与 p53 信号通路和细胞周期相关的差异调节基因。qPCR 分析显示显着上调Ccnd1、Cdk6和Ccng1。流式细胞术显示ICF诱导细胞周期和增殖，同时减少凋亡细胞的数量。ICF 还在 mRNA 和蛋白质水平上调转化生长因子 β1 (Tgfb1)，并且在 ICF 之前用 TGF-β 抑制剂 (SB431542) 预处理消除了 ICF 诱导的Ccnd1和Cdk6表达。总之，这些发现表明，iPSCs 中的 TGF-β 信号传导可增强增殖并减少响应 ICF 的细胞凋亡，这可能会产生一种有效的方案来操纵 iPSCs 进行类器官制造。