Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium

Therapeutic application of stem cell derivatives requires large quantities of cells produced in defined media that cannot be produced via conventional adherent culture. We have applied human induced pluripotent stem (hiPS) cells expressing eGFP under control of the OCT4 promoter to establish the expansion of undifferentiated human embryonic stem (hES) and hiPS cells in suspension culture. A defined culture medium has been identified that results in up to six-fold increase in cell numbers within four days. Our culture system is based on initial single cell dissociation which is critical for standardized process inoculation. HES / hiPS cells were expanded for up to 17 passages. The cells maintained a stable karyotype, their expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers. The ability to expand HES / hiPS cells in a scalable suspension culture represents a critical step towards standardized production in stirred bioreactors.