Abstract
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.
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Acknowledgements
We thank members of the Toronto Centre for Phenogenomics for chimera generation and K. Woltjen (Kyoto University) for providing 6C secondary fibroblasts as well as providing PB-TET-cMyc and PB-TET-MKOS plasmids. PMF351 was provided by M. Fussenegger (Swiss Federal Institute of Technology). This work was funded by Natural Sciences and Engineering Research Council of Canada, the Canadian Institute of Health Research (MOP-57885) and an Ontario Research Fund Global Leadership Round in Genomics and Life Sciences award to A.N.; D.A.F. was supported by a McLean award to P.W.Z.; P.W.Z. was supported as the Canada Research Chair in Stem Cell Bioengineering.
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D.A.F. designed and performed most experiments. P.D.T. performed chimera experiments, part of quantitative PCR experiments and contributed to drafting the manuscript. H.S. performed cTnT differentiation experiments and did confocal imaging. R.P.B. performed western blot analysis and did part of the secondary MEF, spleen and adult fibroblast reprogramming experiments. N.S. did part of the secondary MEF and spleen reprogramming experiments. S.S. performed secondary DN1 blood reprogramming experiments. G.C. performed statistical analysis. A.N. helped to design the project. D.A.F. and P.W.Z. designed the project and wrote the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–8, Supplementary Tables 1–3 (PDF 7538 kb)
Supplementary Video 1
Spontaneously beating aggregates generated from primary fibroblast cells reprogrammed and differentiated towards the cardiac lineage in an integrated suspension-culture process. (MOV 391 kb)
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Fluri, D., Tonge, P., Song, H. et al. Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures. Nat Methods 9, 509–516 (2012). https://doi.org/10.1038/nmeth.1939
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DOI: https://doi.org/10.1038/nmeth.1939
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