Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

We describe derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum-and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension culture-reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor-expressing cells based on their differential survival and proliferation in suspension culture. Seamless integration of SiPSC reprogramming and directed differentiation enabled scalable production of beating cardiac cells in a continuous single cell-and small aggregate-based process. This method is an important step toward the development of robust PSC generation, expansion and differentiation technology.