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Proc Natl Acad Sci U S A. 2018 Jun 19;115(25):6369-6374. doi: 10.1073/pnas.1714099115. Epub 2018 Jun 4.

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

Author information

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada.
Department of Molecular Genetics, Weizmann Institute for Science, Rehovot 76100, Israel.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada;
CCRM, Toronto, ON M5G 1M1, Canada.
The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada.
Medicine by Design: A Canada First Research Excellence Fund Program, University of Toronto, Toronto, ON M5G 1M1, Canada.
School of Biomedical Engineering, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.


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.


cell state; human pluripotent stem cells; manufacturing; suspension culture

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