A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

Jason Lee,Mitchell Wong,Pablo Maceda,Miguel Armenta Ochoa,Lara Samarneh,Eun Yoon,Aaron B Baker

doi:10.1038/s41598-020-72158-5

Jason Lee, Mitchell Wong + Show 5 more

Open Access

https://doi.org/10.1038/s41598-020-72158-5

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Journal: Scientific Reports	Publication Date: Sep 22, 2020
Citations: 5	License type: open-access

Affiliation: The University of Texas at Austin

Abstract

Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. The application of mechanical forces to cultured cells is often performed using specialized systems that lack the flexibility and throughput of other biological techniques. In this study, we developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously. Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the increase in reprograming-related gene expression in mouse fibroblasts when combined with mechanical loading. Taken together, our findings demonstrate a new powerful tool for investigating the mechanobiological mechanisms of disease and performing drug screening in the presence of applied mechanical load.

Highlights

Mechanical forces are important in the regulation of physiological homeostasis and the development of disease
Generation of induced pluripotent stem cells (iPSCs) is typically achieved through the expression of octamer-binding transcription factor-4 (Oct-4), Kruppel-like factor 4 (Klf4), Sex Determining Region Y Box 2 (Sox2), and v-Myc Myelocytomatosis Viral Oncogene Homolog (c-Myc)[1]
We found that high level of mechanical strain and physiological loading induced the expression of reprograming factors in mouse embryonic fibroblasts (MEFs)

Summary

Introduction

Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Pneumatic systems are commercially available and have limitations in the dynamics of the pneumatic system and ability to apply only a single strain at one time[36] The throughput for these systems is often a major limitation, which prevents the performance of screening studies for pathway and drug discovery. The system allows the application of arbitrary waveforms with highly controlled dynamics, allowing the simulation of complex physiological forces

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