Complete Suspension Culture of Human Induced Pluripotent Stem Cells Supplemented with Suppressors of Spontaneous Differentiation

AbstractHuman induced pluripotent stem cells (hiPSCs) are promising resources for producing various type of tissues in regenerative medicine; however, a scalable culture system that can precisely control the cell status for hiPSCs is not developed yet. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without any microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed culture processes of hiPSCs including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of hiPSC status in suspension culture conditions paves the way for their stable and automated clinical application.