Discussion

Generation of clinically relevant quantities of human pluripotent stem (hPS) cells is essential for clinical use. However, the lack of robust methods for clinical expansion of hPS cells continues to limit clinical use. Here we report the development of a culture system for the expansion of hPS cells that is free of human- or animal-derived components and is chemically defined; it contains only raw materials
of clinical quality with complete traceability during manufacturing processes. By using this complete, clinical-grade culture system, hPS cells can easily be expanded as a 2D monolayer and maintain high
expression of pluripotent stem cell markers and lack expression of differentiation markers for over 10 passages. These cells display stem cell characteristics such as the long-term capacity for self-renewal and the capability to differentiate into all three germ layers. Moreover, the
culture system utilizes a single-cell enzymatic passaging protocol that enables expansion from single hPS cells, making it amenable to genome engineering and selection applications. We also demonstrate that the culture system supports large-scale, non-adherent expansion of hPS cells in suspension culture in a perfusion bioreactor. In summary, our
culture system supports the expansion of hPS cells in 2D monolayer and 3D non-adherent suspension culture formats, under GMP conditions.