Derivation and Culture of Naïve Pluripotent Stem Cells in Stirred Suspension Bioreactors

人类多能干细胞（hPSC）为人类疾病建模和再生医学提供了巨大的潜力。为了解决预期的对hPSC的高需求，我们已经开发了在搅拌悬浮生物反应器中将它们作为聚集体扩增的方法。使用小鼠模型，我们已经确定，在生物反应器中搅拌细胞聚集体引起的流体剪切促进多能性，允许去除多能性维持因子LIF。然而，对于hPSC来说，情况并非如此，hPSC需要多能性维持因子bFGF和ROCK抑制剂Y-27632，以便在生物反应器中保持活力。我们将培养要求的这些差异归因于最近发现小鼠和人PSC不是相同类型的细胞。人类PSC（称为启动多能性）比小鼠PSC（称为幼稚多能性）发育更成熟。最近的实验已经揭示了这一点，其中使用化学抑制剂或缺氧将hPSC转化为幼稚状态。在本申请中，我们将确定通过将引发的hPSC转化回幼稚状态，hPSC是否可以在不使用多能性维持因子或抑制剂的情况下维持在生物反应器中。我们将研究流体剪切如何通过引发膜相关β-连环蛋白的核转位来促进生物反应器中的幼稚多能状态。我们的实验将为hPSC的未来临床应用提供支持。我们的研究不仅将降低hPSC扩增的成本，还将消除使用化学抑制剂的要求，否则会损害hPSC质量。

Human pluripotent stem cells (hPSCs) offer huge potential for modeling human diseases and in regenerative medicine. Toaddress the anticipated high demand for hPSCs, we have developed methods to expand them as aggregates in stirredsuspension bioreactors. Using the mouse model, we have determined that fluid shear caused by stirring cellular aggregatesin the bioreactor promotes pluripotency, allowing for the removal of the pluripotency maintenance factor, LIF. However thesame 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 recentobservation that mouse and human PSCs are not the same type of cell. Human PSCs (known as primed pluripotency) aredevelopmentally more mature than mouse PSCs (known as naïve plurpotency). This has been revealed by recentexperiments where hPSCs have been converted to the naïve state using either chemical inhibitors or hypoxia. In thisapplication we will determine if by converting primed hPSCs back to the naïve state, hPSCs can be maintained in thebioreactor without the use of pluripotency maintenance factors or inhibitors. We will investigate how fluid shear promotes thenaïve pluripotent state in bioreactors by eliciting the nuclear translocation of membrane associated beta-catenin. Ourexperiments will support the future clinical applications of hPSCs. Not only will our research drive down the cost of hPSCexpansion, it will also remove a requirement for using chemical inhibitors, which otherwise compromise hPSC quality.