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

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

Abstract

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.

Keywords: cell state; human pluripotent stem cells; manufacturing; suspension culture.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bioreactors
  • Cell Culture Techniques / methods
  • Cell Differentiation / drug effects
  • Cell Line
  • Cytokines / pharmacology
  • Germ Layers / cytology
  • Germ Layers / drug effects
  • Humans
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / drug effects
  • Small Molecule Libraries / pharmacology

Substances

  • Cytokines
  • Small Molecule Libraries

Grant support