Modulating cell state to enhance suspension expansion of human pluripotent stem cells

Significance Efficient manufacturing is critical for the translation of cell-based therapies to clinical applications. To date, high-yield expansion of human pluripotent stem cells (hPSC) in suspension bioreactors has not been reported. Here, we present a strategy to shift suspension-grown hPSC to a high-yield state without compromising their ability to differentiate to all three germ layers. In this new state, hPSC expand to densities 5.7 ± 0.2-fold higher than conventional hPSC each passage in suspension bioreactors. High-density suspension cultures enable process intensification, cost reduction, and more efficient manufacturing. This work advances cell-state engineering as a valuable tool to overcome current challenges in therapeutic cell production and processing. The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.