There is a clinical need for large numbers of phenotypes which are suitable for tissue engineering and cell therapy applications. Pluripotent stem cells (PSCs) are readily expanded in vitro and can differentiate into any somatic phenotype, making them a potential cell source. However, generating clinicallyrelevant numbers of phenotypes requires culture in stir-based bioreactor systems which expose cells to shear stress.

Here we use a parallel plate bioreactor as a surrogate model system to better understand the effects of shear stress on pluripotent embryonic stem cells (ESCs). Initial studies examined the impact of cell deformation by shear stress during early ESC differentiation. Shear-treatment regulated specification into the three germ lineages and promoted mesodermal differentiation. Next we examined the response to shear stress during later specification events. The application of shear stress was found to promote mesodermal differentiation towards both definitive hematopoietic and mature endothelial phenotypes, although delayed applications were less effective at promoting hematopoietic specification. The next studies used low oxygen treatment to study the impact of another differentiation cue in the presence and absence of shear stress. Hypoxia promoted mesodermal phenotypes but the response was highly dependent on the physical microenvironment such as the culture method and the presence of shear stress. The next group of studies examined the impact of shear stress on ESC expansion. ESCs expanded under flow conditions maintained pluripotency but mesodermal specification was regulated in a manner that was dependent on the presence or absence of a ROCK inhibitor. The final studies used small molecule inhibitors to determine the role of specific signaling molecules during the shear-mediated differentiation of ESCs. Although inhibition of ROCK had little effect, inhibition c-SRC, JNK, or ERK modulated the shear-response.

These studies highlight the important effects of shear stress during PSC culture and increase the basic science understanding of stem cell regulation by the physical microenvironment. The systematic approach to analyze multiple parameters allows for an improved translation of techniques from the bench-top into large-scale bioprocessing systems. Altogether these studies can inform large-scale differentiation techniques and bioreactor design in order to help establish the cell banks needed for clinical applications.