Fluid Shear Stress Promotes Embryonic Stem Cell Pluripotency via Interplay between β-catenin and Vinculin in Bioreactor Culture.

The expansion of pluripotent stem cells (PSCs) as aggregates in stirred suspension bioreactors is garnering attention as an alternative to adherent culture. However, the hydrodynamic environment in the bioreactor can modulate PSC behavior, pluripotency and differentiation potential in ways that need to be well understood. In this study, we investigated how murine embryonic stem cells (mESCs) sense fluid shear stress and modulate a non-canonical Wnt signaling response to promote pluripotency. mESCs showed higher expression of pluripotency marker genes, Oct4, Sox2 and Nanog in the absence of LIF in stirred suspension bioreactors compared to adherent culture, a phenomenon we have termed mechanopluripotency. In bioreactor culture, fluid shear promoted the nuclear translocation of the less well-known pluripotency regulator β-catenin and concomitant increase of c-Myc expression, an upstream regulator of Oct4, Sox2 and Nanog. We also observed similar β-catenin nuclear translocation in LIF-free mESCs cultured on E-cadherin substrate under defined fluid shear stress conditions in flow chamber plates. mESCs showed lower shear-induced expression of pluripotency marker genes when β-catenin was inhibited, suggesting that β-catenin signaling is crucial to mESC mechanopluripotency. Key to this process is vinculin, which is known to rearrange and associate more strongly with adherens junctions in response to fluid shear. When the vinculin gene is disrupted, we observe that nuclear β-catenin translocation and mechanopluripotency are abrogated. Our results indicate that mechanotransduction through the adherens junction complex is important for mESC pluripotency maintenance. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Fluid shear stress generated in bioreactors can modulate cell behavior, pluripotency, and differentiation capability. In this study, we have revealed that fluid shear stress upregulates mESC pluripotency, which obviates the necessity of adding LIF in bioreactor culture. We have termed this phenomenon as mechanopluripotency. Translocation of β-catenin to the nucleus is the key to mechanopluripotency in bioreactor culture. In addition, adherens junction-associated vinculin is another important factor as vinculin gene disruption results in abrogation of mechanopluripotency in bioreactor culture.