Abstract
Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. 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 the 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 the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.
Competing Interest Statement
YHA received collaborative research grant from KANEKA corporation during this study. SK, KT, MI, YK, TN, TK, RM, MS, and NN were employed with KANEKA corporation during this study. MMT, SK, KT, MI, YK, and YHA. are inventors of patents arising from this work (WO/2021/162090 and PCT/JP2020/005255). The other authors declare no competing interests.
Footnotes
We have completed our revision to meet all the helpful comments by the reviewers. Major points are below. 1. We tested several cell seeding densities and several stirring speeds with or without WNT/PKCβ inhibitors (Figure 6-figure supplement 1). 2. We have performed additional experiments using conventional media, mTeSR1 (Stem Cell Technologies, Vancouver, Canada), comparing with the adherent feeder-free culture system in four different hiPSC lines simultaneously (Figure 4 - figure supplement 2). 3. We have performed additional experiments on hiPSC maintenance across 5 hiPSC lines in suspension culture using StemFit AK02N medium simultaneously (Figure 3C - E). 4. We found that the expression of naive pluripotency markers, KLF2, KLF4, KLF5, and DPPA3, were up-regulated in the suspension conditions treated with LY333531 and IWR-1-endo while the expression of OCT4 and NANOG was at the same levels (Figure 5-figure supplement 2). 5. We added the data on the cloning efficiency, which are compared with adherent cultures (Figure 7B). 6. We have added more detailed explanation in the Discussion section
