This project examined the response of embryonic stem cells (ESCs) to various hydrodynamic conditions generated via rotary orbital suspension culture, in particular investigating the formation and maintenance of cell aggregates, the differentiation of cells within these aggregates, and the modulation of the β-catenin signaling pathway implicated in regulating differentiation.
Embryoid bodies can be formed at many rotary orbital speeds with a lower limit of 20 rpm and an upper limit of 60 rpm. EB size was inversely regulated by rotary speed, with slower rotary speeds generating larger EBs than faster rotary speeds, while EB yield was directly regulated by rotary speed. In addition to controlling the overall size of EBs, rotary orbital speed also altered the kinetics of EB formation, with slower rotary speeds generating distinct, primitive EBs within 6 hours of inoculation and faster speeds requiring as much as 24 hours to attain primitive EB formation. Shear stresses within rotary orbital culture were relatively uniform and mild (≤ 2.5 dyn/cm2 ) as determined by computational fluid dynamics; however the three speeds examined (25, 40, and 55 rpm) did result in unique shear stress profiles under which the EBs were formed and cultured. The differences observed in EB formation kinetics and size were accompanied by alterations in the global gene expression patterns between static and rotary culture conditions as well as between rotary orbital speeds.
Rotary orbital culture was compared static suspension and hanging drop culture to further assess the effects of hydrodynamic conditions on EB differentiation. EBs formed under rotary orbital culture at 40 rpm were found to enhance mesoderm differentiation, particularly cardiomycoyte differentiation, compared to static culture, exhibiting increased levels cardiogenic-related genes (Nkx2.5, MLC-2v, and α-MHC) and protein (α-sarcomeric actin) expression as well as increased contractile frequency. The differences of rotary orbital culture compared to static and hanging drop culture demonstrated the ability of hydrodynamic conditions to affect cardiomyocyte differentiation and led to the investigation of how rotary speed may also alter cardiomyocyte differentiation.
Rotary orbital speed modulated the temporal pattern of mesoderm and endoderm related genes (Nkx2.5, Gata-4, and AFP) with slower speeds exhibiting earlier increases in expression levels. Similarly, cardiogenic-related genes (MLC-2v and α-MHC ) were increased earlier within slower rotary speed culture conditions, and increased contractile frequency was also observed within EBs from slower rotary speeds. These differences in gene expression, specifically related to cardiogenic differentiation, due to rotary orbital culture conditions suggested the regulation of EB formation and size by rotary orbital speeds may modulate signaling pathways that govern mesoderm and cardiogenic differentiation.
Beta-catenin signaling is an important signaling pathway active in early embryonic development, aiding in the regulation of embryonic patterning, axis formation, primitive streak formation, and mesoderm development. In differentiating ESCs, β-catenin signaling is required for initial mesoderm differentiation, and temporal regulation of β-catenin signaling is necessary for cardiomyocyte development from ESCs (early activation leads to cardiomyocyte progenitor development, followed by later inhibition, resulting in mature cardiomyocyte differentiation).
The effects of hydrodynamic conditions from rotary orbital culture on β-catenin expression and cardiogenic gene transcription were examined. Rotary orbital speed modulated both spatiotemporal location of β-catenin and the phosphorylation state of β-catenin with slower rotary speeds resulting in earlier nuclear dephosphorylated β-catenin expression. Hydrodynamic culture in general appeared to increase β-catenin-regulated transcription (Mesp-1, Mef-2c) and cardiogenic gene expression (Nkx2.5, MLC-2v) compared to static culture conditions. These results led to a proposed model of hydrodynamic regulation of cardiomyocyte differentiation, suggesting that enhanced EB formation resulted in increased β-catenin availability from E-cadherin for transcriptional regulation upon activation. (Abstract shortened by UMI.)