Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells

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 i PSCs is lacking. The aim of this study was to identify a molecular mechanism in i PSCs responding to intermittent compressive force(ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse i PSCs, 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. q PCR 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 m RNA 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 i PSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate i PSCs for organoid fabrication.

Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.