Mechanopluripotency – Adherens Junction Mechanotransduction Supports mESC Pluripotency Gene Expression via β-catenin Nuclear Translocation in Response to Fluid Shear Stress
Date
2023-11-07
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Abstract
Pluripotent stem cells are increasingly being investigated as a base platform from which effector cells can be differentiated and utilized in various contexts as whole-cell therapeutics. Current culture methodologies require 2D plate culture and this represents a bottleneck in the form of increased costs associated with consumable usage and difficulties with standardization in manipulation. These difficulties cascade into ballooning therapy costs and troubles with cGMP compliance required for all biopharmaceutical manufacturing. To this end, there has been interest in culturing pluripotent stem cells in bioreactors, shown in previous therapeutic biopharmaceutical platforms to achieve cost-effective scale and compliance. In our bioreactor work, we have observed an increase in core pluripotency gene expression in mouse embryonic stem cells (mESCs) cultured as aggregates at specific impeller speeds in stirred tank bioreactors. We have termed this phenomenon “mechanopluripotency”. This project aimed to investigate the mechanism(s) responsible for our observation of induced pluripotency in the bioreactor. Using mutant cell lines containing a β-catenin reporter system, we show in both the bioreactor and a defined model system that in response to shear stress of ~6 dyne/cm2 β-catenin translocates to the nucleus where it is responsible for co-activation of the Wnt signaling pathway, known to affect pluripotency gene expression. Additionally, we show evidence that this response is mediated via altered molecular dynamics at the adherens junction, a key hub for cell-cell connections. By knocking out the cytoskeleton protein vinculin we show that β-catenin signaling is impaired in both the bioreactor and defined model system and a decrease in core pluripotency gene expression in the absence of exogenous maintenance factors. These results indicate that mechanopluripotency is mediated primarily through β-catenin and by impairing the adherens junction’s ability to adapt to mechanical force the response is attenuated.
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Keywords
Stem Cells, Biomedical Engineering, Bioreactor, Biochemistry, Flow Chamber
Citation
Harper, L. W. (2023). Mechanopluripotency – adherens junction mechanotransduction supports mESC pluripotency gene expression via β-catenin nuclear translocation in response to fluid shear stress (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.