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

Nature Methods volume 9, pages 509–516 (2012)Cite this article

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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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Figure 1: Secondary fibroblasts inducibly expressing reprogramming factors survived and proliferated in suspension culture.
Figure 2: Suspension-culture reprogramming of multiple cell types to pluripotency.
Figure 3: Whole-genome analysis revealed high similarity between reprogramming under adherent and suspension-culture conditions.
Figure 4: Primary MEFs reprogrammed under suspension culture conditions.
Figure 5: Integrated derivation, expansion and differentiation of SiPSCs toward cardiac progenitors.

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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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Authors and Affiliations

  1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    David A Fluri, Hannah Song, Ricardo P Baptista, Nika Shakiba, Shreya Shukla, Geoffrey Clarke & Peter W Zandstra

  2. Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    David A Fluri, Hannah Song, Ricardo P Baptista, Nika Shakiba, Shreya Shukla, Geoffrey Clarke & Peter W Zandstra

  3. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Peter D Tonge & Andras Nagy

  4. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Andras Nagy

  5. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Peter W Zandstra

  6. McEwen Centre for Regenerative Medicine, University Health Network and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario, Canada

    Peter W Zandstra

Authors
  1. David A Fluri
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  7. Geoffrey Clarke
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  9. Peter W Zandstra
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Contributions

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.

Corresponding author

Correspondence to Peter W Zandstra.

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The authors declare no competing financial interests.

Supplementary information

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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