Fluid Flow Modulation of Murine Embryonic Stem Cell Pluripotency Gene Expression in the Absence of LIF

Abstract

Fluid forces are strong modulators of cell fate and fundamental components of spinner flask bioreactors used for stem cell expansion and differentiation. Here, we investigated the effects of fluid forces on murine embryonic stem cells (mESCs) in the absence of Leukemia Inhibitory Factor (LIF) using parallel-plate flow chambers. Cells were seeded onto gelatin-coated glass slides and grown for 2.5 days before exposure to fluid forces. Pluripotency marker gene expression was quantified by qPCR. An average shear stress of 0.6 Pa applied for 24 h in the absence of LIF and presence of high molecular weight dextran increased Oct4 and Sox2, decreased Nanog, and did not change in Rex1 mRNA levels in comparison to statically cultured cells in the presence of LIF. At 0.3 Pa shear stress, Oct4 and Sox2 expression increased, with a reduction in Nanog and Rex1 levels. The presence of pulsation significantly increased expression of Rex1 and Nanog, but not expression of Oct4 or Sox2, compared to cells exposed to steady flow for 24 h. This study suggests incorporation of high cell–cell contact, viscosity elevation with dextran, moderate shear stress (0.6 Pa), and the presence of pulsatility in bioreactor expansion protocols for mESCs to support maintenance of pluripotency.