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A comparison of non-integrating reprogramming methods

Nature Biotechnology volume 33, pages 58–63 (2015)Cite this article

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Abstract

Human induced pluripotent stem cells (hiPSCs1,2,3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.

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Figure 1: Performance comparison of non-integrating reprogramming methods.
Figure 2: Comparison of the genetic integrity of hiPSCs derived by different reprogramming methods.

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Acknowledgements

We would like to thank B. Hamilton (Stemgent) for miRNA reprogramming agents and protocols, G. Mostoslavsky (Boston University) for human STEMCCA lentiviral plasmid constructs, M. Armant (Boston Children's Hospital) for MRC5 and hCD34+ cells, G. MacLean (Boston Children's Hospital) for advice on episomal blood reprogramming and S. D'Souza (Icahn School of Medicine at Mount Sinai) for a list of contacts for human cell reprogramming laboratories. This work was supported in part by grants R01HL75737, U01HL107440, UO1-HL100001, U01HL87402 and U01HL100408 (National Heart, Lung, and Blood Institute (NHLBI) Progenitor Cell Biology Consortium), R24DK092760 (the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)), PO1NS066888 (the National Institute of Neurological Disorders and Stroke (NINDS)), by the SMA Foundation, the Jerome Le Jeune Foundation, an EMBO postdoctoral fellowship (E.B.) and the Harvard Stem Cell Institute.

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Authors and Affiliations

  1. Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Thorsten M Schlaeger, Karrie Chan, Amelia Cianci, Alexander DeVine, Andrew Ettenger, Kelly Fitzgerald, Michelle Godfrey, Dipti Gupta, Jade McPherson, Prerana Malwadkar, Manav Gupta, Blair Bell, Emily Brookes, Anne B C Cherry, Leonard I Zon & George Q Daley

  2. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Thorsten M Schlaeger, Laurence Daheron, Thomas R Brickler, Samuel Entwisle, Maureen S Lynes, Alexander M Tsankov, Leonard I Zon, Lee L Rubin, Alexander Meissner, Chad A Cowan & George Q Daley

  3. Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Akiko Doi, Namyoung Jung, Xin Li & Andrew P Feinberg

  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Akiko Doi

  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Anne B C Cherry & George Q Daley

  6. Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, Massachusetts, USA

    Didem Demirbas

  7. Broad Institute, Cambridge, Massachusetts, USA

    Alexander M Tsankov, Alexander Meissner & Chad A Cowan

  8. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Lee L Rubin & Chad A Cowan

  9. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Chad A Cowan

  10. Howard Hughes Medical Institute, Children's Hospital Boston and Dana Farber Cancer Institute, Boston, Massachusetts, USA

    George Q Daley

Authors
  1. Thorsten M Schlaeger
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  2. Laurence Daheron
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  18. Xin Li
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  19. Maureen S Lynes
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  20. Emily Brookes
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  22. Didem Demirbas
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  24. Leonard I Zon
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  25. Lee L Rubin
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  26. Andrew P Feinberg
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  27. Alexander Meissner
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  28. Chad A Cowan
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  29. George Q Daley
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Contributions

T.M.S., L.D., C.A.C., A.M., L.L.R., A.P.F., L.I.Z. and G.Q.D. contributed to the conception and design of the study; T.M.S., L.D. and G.Q.D. wrote the manuscript; T.R.B., S.E., K.C., A.C., A.D., A.E., K.F., M.G., D.G., J.M., P.M., M.G. and B.B. performed reprogramming and cell culture experiments; A.D., N.J., X.L. and A.P.F. performed epigenetics analyses, T.M.S., K.F., D.G., P.M., L.D., T.R.B. and S.E. performed PCR assays; M.S.L., E.B., A.B.C.C. and D.D. provided cell samples and reprogramming data; A.M.T. and A.M. performed the scorecard analysis; T.M.S. performed statistical analyses; T.M.S. and L.D. performed the survey.

Corresponding authors

Correspondence to Thorsten M Schlaeger or George Q Daley.

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The authors declare no competing financial interests.

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods (PDF 3485 kb)

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Schlaeger, T., Daheron, L., Brickler, T. et al. A comparison of non-integrating reprogramming methods. Nat Biotechnol 33, 58–63 (2015). https://doi.org/10.1038/nbt.3070

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