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

Jeeranan Manokawinchoke,Thanaphum Osathanon,Hiroshi Egusa,Hiroko Okawa,Prasit Pavasant,Phoonsuk Limraksasin

doi:10.1038/s41368-021-00151-3

Jeeranan Manokawinchoke, Thanaphum Osathanon + Show 4 more

Open Access

https://doi.org/10.1038/s41368-021-00151-3

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Abstract

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.

Highlights

Induced pluripotent stem cells, which can be generated by reprogramming of somatic cells including oral tissue cells,[1] possess unlimited self-renewal property and can differentiate into any type of cell and tissue
Differential gene expression profile in intermittent compressive force (ICF)-treated Induced pluripotent stem cells (iPSCs) implied a reduction of cell apoptosis
To examine the pathways regulated by ICF in iPSCs in a 3D form, apoptosis, the cells were fluorescently stained with annexin V and embryoid bodies formed from mouse iPSCs were plated on a tissue propidium iodide (PI) to identify cells in early and late apoptosis

Summary

Introduction

Induced pluripotent stem cells (iPSCs), which can be generated by reprogramming of somatic cells including oral tissue cells,[1] possess unlimited self-renewal property and can differentiate into any type of cell and tissue. IPSCs are considered to be a promising tool for tissue regeneration and for disease modeling by in vitro fabrication of three dimensional (3D) tissues/organs (organoids).[2] Recently, technologies for engineered cell manipulation have been actively investigated to control 3D cell-cell interactions of stem cells to generate organoids. Mechanical stress is a promising manipulation technique for organoid formation of iPSCs.[3]. Mechanical force regulates numerous biological responses in stem cells.[4] Mechanical stimulation induces differentiation and maturation of iPSCs toward bone and cartilage cell lineages.[5,6]

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