Grant: Collaborative Health Research Projects (NSERC partnered)

Human pluripotent stem cells (hPSCs) offer huge potential for modeling human diseases and in regenerative medicine. To address the anticipated high demand for hPSCs, we have developed methods to expand them as aggregates in stirred suspension bioreactors. Using the mouse model, we have determined that fluid shear caused by stirring cellular aggregates in the bioreactor promotes pluripotency, allowing for the removal of the pluripotency maintenance factor, LIF. However the same is not true for hPSCs, which require both the pluripotency maintenance factor, bFGF, and the ROCK inhibitor, Y-27632, in order to remain viable in the bioreactor. We attribute these differences in culturing requirements to the recent observation that mouse and human PSCs are not the same type of cell. Human PSCs (known as primed pluripotency) are developmentally more mature than mouse PSCs (known as naïve plurpotency). This has been revealed by recent experiments where hPSCs have been converted to the naïve state using either chemical inhibitors or hypoxia. In this application we will determine if by converting primed hPSCs back to the naïve state, hPSCs can be maintained in the bioreactor without the use of pluripotency maintenance factors or inhibitors. We will investigate how fluid shear promotes the naïve pluripotent state in bioreactors by eliciting the nuclear translocation of membrane associated beta-catenin. Our experiments will support the future clinical applications of hPSCs. Not only will our research drive down the cost of hPSC expansion, it will also remove a requirement for using chemical inhibitors, which otherwise compromise hPSC quality.