Abstract

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.

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Acknowledgements

We thank the high-quality cell sorting service and technical input provided by Monash Flowcore Facility and J. Hatwell-Humble for conducting the teratomas experiments. We also thank the Cytogenetics department of Monash Pathology for help with G-banding karyotype analysis. Furthermore, the authors thank the ACRF Centre for Cancer Genomic Medicine at the MHTP Medical Genomics Facility for assistance with next-generation library preparation and Illumina sequencing. This work was supported by National Health and Medical Research Council (NHMRC) project grants APP1104560 to J.M.P. and A.L.L., APP 1069830 to R.L. and a Monash University strategic grant awarded to C.M.N. X.L. was supported by a Monash International Postgraduate Research Scholarship and a Monash Graduate Scholarship. A.S.K. was supported by an NHMRC Early Career Fellowship APP1092280. J.M.P. and R.L. were supported by Silvia and Charles Viertel Senior Medical Research Fellowships.

Author information

Author notes

    • Xiaodong Liu
    •  & Christian M Nefzger

    These authors contributed equally to this work.

Affiliations

  1. Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, Victoria, Australia.

    • Xiaodong Liu
    • , Christian M Nefzger
    • , Fernando J Rossello
    • , Joseph Chen
    • , Anja S Knaupp
    • , Jaber Firas
    • , Jacob M Paynter
    • , Ketan Mishra
    • , Margeaux Hodgson-Garms
    • , Sarah M Williams
    •  & Jose M Polo
  2. Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, Victoria, Australia.

    • Xiaodong Liu
    • , Christian M Nefzger
    • , Fernando J Rossello
    • , Joseph Chen
    • , Anja S Knaupp
    • , Jaber Firas
    • , Jacob M Paynter
    • , Ketan Mishra
    • , Margeaux Hodgson-Garms
    • , Sarah M Williams
    •  & Jose M Polo
  3. Australian Regenerative Medicine Institute, Monash University, Wellington Road, Clayton, Victoria, Australia.

    • Xiaodong Liu
    • , Christian M Nefzger
    • , Fernando J Rossello
    • , Joseph Chen
    • , Anja S Knaupp
    • , Jaber Firas
    • , Jacob M Paynter
    • , Hun S Chy
    • , Carmel M O'Brien
    • , Ketan Mishra
    • , Margeaux Hodgson-Garms
    • , Sarah M Williams
    • , Susan K Nilsson
    • , Andrew L Laslett
    •  & Jose M Polo
  4. ARC Center of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Western Australia, Australia.

    • Ethan Ford
    • , Jahnvi Pflueger
    •  & Ryan Lister
  5. The Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia.

    • Ethan Ford
    • , Jahnvi Pflueger
    •  & Ryan Lister
  6. Manufacturing, CSIRO, Clayton, Victoria, Australia.

    • Hun S Chy
    • , Carmel M O'Brien
    • , Susan K Nilsson
    •  & Andrew L Laslett
  7. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

    • Cheng Huang
    •  & Ralf B Schittenhelm
  8. The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

    • Natasha Jansz
    •  & Marnie E Blewitt
  9. University of Melbourne, Melbourne, Victoria, Australia.

    • Natasha Jansz
    •  & Marnie E Blewitt
  10. Monash Bioinformatics Platform, Monash University, Wellington Road, Clayton, Victoria, Australia.

    • Sarah M Williams

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Contributions

X.L., C.M.N. and J.M.P. conceived the study and designed experiments. X.L. performed somatic cell reprogramming in different culture conditions and tissue culture experiments with support from C.M.N., J.C., J.F., K.M., J.M.P. and M.H.-G. S.K.N. supported the teratomas experiments. X.L. and C.M.N. performed FACS experiments, SPADE/viSNE and the molecular experiments of the cells with support from A.S.K. and J.C.; A.S.K., C.H. and R.B.S. performed cell preparation for mass spectrometry experiments and analysis. F.J.R., S.M.W. and C.M.N. analyzed Fluidigm and RNA sequencing data under the guidance of J.M.P. J.P., F.J.R., E.F. and R.L. performed targeted methylation experiments and analyzed the results. N.J. and M.E.B. performed molecular characterization. H.S.C. and C.M.O′B. provided reagents and technical assistance. A.L.L. provided reagents and assisted with writing. X.L., C.M.N. and J.M.P. wrote the manuscript. All authors approved of and contributed to the final version of the manuscript.

Competing interests

J.M.P. is a director in Cell Mogrify; however, the work presented in this manuscript is not related to Cell Mogrify.

Corresponding author

Correspondence to Jose M Polo.

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    Supplementary Figures 1–7 and Supplementary Tables 1, 2, 4–7.

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    Supplementary Protocol

    Establishment and maintenance of human naive pluripotent stem cells by primed to naive conversion and reprogramming of fibroblasts.

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    Supplementary Table 3

    Proteins detected in primed and naive rt2iLGöY iPSCs by mass spectometry.

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DOI

https://doi.org/10.1038/nmeth.4436