Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Miyagi, 980-8575, Japan.
Dental Stem Cell Biology Research Unit and Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, 10330, Thailand.
Int J Oral Sci. 2022 Jan 4;14(1):1. doi: 10.1038/s41368-021-00151-3.
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.
体外环境因素对诱导多能干细胞（iPSCs）的操纵对于三维（3D）组织/器官的诱导非常重要。力的作用取决于许多因素，包括力和细胞类型。然而，关于 iPSCs 中这种效应的信息还很缺乏。本研究的目的是通过分析全基因表达谱来确定 iPSCs 对间歇压缩力（ICF）响应的分子机制。将附着在 3D 组织培养板上的小鼠 iPSCs 胚状体置于无血清培养基中进行 24 小时 ICF。RNA 测序数据的基因本体分析表明，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 诱导细胞周期分类中的基因，并下调与代谢过程相关的基因。京都基因与基因组百科全书数据库揭示了差异调节的基因与 p53 信号通路和细胞周期有关。qPCR 分析表明，Ccnd1、Cdk6 和 Ccng1 显著上调。流式细胞术显示 ICF 诱导细胞周期和增殖，同时减少凋亡细胞数量。ICF 还在 mRNA 和蛋白水平上上调转化生长因子 β1（Tgfb1），并且在 ICF 之前用 TGF-β 抑制剂（SB431542）预处理可消除 ICF 诱导的 Ccnd1 和 Cdk6 表达。总之，这些发现表明 TGF-β 信号在 iPSCs 中增强了增殖并减少了对 ICF 的凋亡反应，这可能为操纵 iPSCs 进行类器官制造提供了一种有效的方案。
