Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Gene Expression Omnibus

Data deposits

Microarray, RNA-seq, ChIP-seq and MethylC-seq data have been deposited in the Gene Expression Omnibus under accession number GSE60605.


  1. 1.

    & Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981)

  2. 2.

    Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981)

  3. 3.

    & The nature of embryonic stem cells. Annu. Rev. Cell Dev. Biol. 30, 647–675 (2014)

  4. 4.

    et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007)

  5. 5.

    et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007)

  6. 6.

    et al. The transcriptional and functional properties of mouse epiblast stem cells resemble the anterior primitive streak. Cell Stem Cell 14, 107–120 (2014)

  7. 7.

    & Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009)

  8. 8.

    et al. HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition. EMBO J. 31, 2103–2116 (2012)

  9. 9.

    & Gene function in mouse embryogenesis: get set for gastrulation. Nature Rev. Genet. 8, 368–381 (2007)

  10. 10.

    & The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo. Dev. Biol. 178, 124–132 (1996)

  11. 11.

    , & Epiblast stem cells contribute new insight into pluripotency and gastrulation. Dev. Growth Differ. 52, 293–301 (2010)

  12. 12.

    et al. Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell 6, 215–226 (2010)

  13. 13.

    , , , & Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011)

  14. 14.

    et al. Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors. Development 141, 1209–1221 (2014)

  15. 15.

    , , & Epiblast ground state is controlled by canonical Wnt/β-catenin signaling in the postimplantation mouse embryo and epiblast stem cells. PLoS ONE 8, e63378 (2013)

  16. 16.

    & et al. Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells. Nature Cell Biol. 13, 1–8 (2011)

  17. 17.

    et al. Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat. Commun. 4, 2403 (2013)

  18. 18.

    et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008)

  19. 19.

    et al. Isolation of epiblast stem cells from preimplantation mouse embryos. Cell Stem Cell 8, 318–325 (2011)

  20. 20.

    et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnol. 25, 681–686 (2007)

  21. 21.

    , , & In vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation. Cell Rep. 2, 1571–1578 (2012)

  22. 22.

    et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009)

  23. 23.

    , , & XCMS Online: a web-based platform to process untargeted metabolomic data. Anal. Chem. 84, 5035–5039 (2012)

  24. 24.

    et al. An accelerated workflow for untargeted metabolomics using the METLIN database. Nature Biotechnol. 30, 826–828 (2012)

  25. 25.

    , , & Cadherin-mediated cell interaction regulates germ cell determination in mice. Development 130, 6423–6430 (2003)

  26. 26.

    , & Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm. Mol. Reprod. Dev. 70, 20–29 (2005)

  27. 27.

    & Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. Development 118, 1255–1266 (1993)

  28. 28.

    et al. Transcriptomic landscape of the primitive streak. Development 137, 2863–2874 (2010)

  29. 29.

    , & A transcriptome landscape of mouse embryogenesis. Dev. Cell 13, 761–762 (2007)

  30. 30.

    , , , & Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. J. Clin. Invest. 119, 1438–1449 (2009)

  31. 31.

    et al. Isolation and characterization of novel rhesus monkey embryonic stem cell Lines. Stem Cells 24, 2177–2186 (2006)

  32. 32.

    et al. Differential L1 regulation in pluripotent stem cells of humans and apes. Nature 503, 525–529 (2013)

  33. 33.

    In search of naivety. Cell Stem Cell 15, 543–545 (2014)

  34. 34.

    & Axis development and early asymmetry in mammals. Cell 96, 195–209 (1999)

  35. 35.

    & Stem cells: a designer’s guide to pluripotency. Nature 516, 172–173 (2014)

  36. 36.

    et al. Divergent reprogramming routes lead to alternative stem-cell states. Nature 516, 192–197 (2014)

  37. 37.

    No reference here

  38. 38.

    et al. Germline‐specific expression of the Oct‐4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675–684 (1999)

  39. 39.

    , & Myeloid cells limit production of antibody-secreting cells after immunization in the lymph node. J. Immunol. 192, 1004–1012 (2014)

  40. 40.

    & FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev. Cell 1, 37–49 (2001)

  41. 41.

    , , , & Generating gene knockout rats by homologous recombination in embryonic stem cells. Nature Protocols 6, 827–844 (2011)

  42. 42.

    , & Successful whole embryo culture with commercially available reagents. Int. J. Dev. Biol. 57, 61–67 (2013)

  43. 43.

    et al. Feeder-independent culture of human embryonic stem cells. Nature Methods 3, 637–646 (2006)

  44. 44.

    & Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nature Protocols 8, 1513–1524 (2013)

  45. 45.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013)

  46. 46.

    et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nature Biotechnol. 29, 149–153 (2011)

  47. 47.

    et al. A more efficient method to generate integration-free human iPS cells. Nature Methods 8, 409–412 (2011)

  48. 48.

    et al. Efficient correction of hemoglobinopathy-causing mutations by homologous recombination in integration-free patient iPSCs. Cell Res. 21, 1740–1744 (2011)

  49. 49.

    et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010)

  50. 50.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)

  51. 51.

    et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007)

  52. 52.

    et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68–73 (2011)

  53. 53.

    et al. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511, 177–183 (2014)

  54. 54.

    et al. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013)

  55. 55.

    , , & Identification of active regulatory regions from DNA methylation data. Nucleic Acids Res. 41, e155 (2013)

  56. 56.

    et al. GREAT improves functional interpretation of cis-regulatory regions. Nature Biotechnol. 28, 495–501 (2010)

  57. 57.

    et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell. 5, 97–110 (2009)

  58. 58.

    Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nature Protocol. 4, 495–505 (2009)

Download references


We would like to thank S. Mitalipov and J. Thomson for providing rhesus ESCs and iPSCs, respectively, F. Gage for providing chimpanzee iPSCs, K. Zhang for assistance with cell line derivation, M. Ku of the H. A. and Mary K. Chapman Charitable Foundations Genomic Sequencing Core for performing RNA-seq and mouse ChIP-seq experiments, M. Chang of the Integrative Genomic and Bioinformatics Core for bioinformatics analysis, W. T. Berggren and the staff of the Salk STEM Core for preparation of custom-mTeSR1 base medium and supply of validated stem culture materials, G. Pao and K. Hasegawa for discussions, Y. Dayn from transgenic core facility and J. Luo for blastocyst injections, Y. Tsunekawa for providing the mutant eGFP human ESCs reporter line, E. O’Connor and K. Marquez of Human Embryonic Stem Cell Core Facility of Sanford Consortium for Regenerative Medicine for FACS sorting, R. H. Benitez, A. Goebl, R. D. Soligalia for assistance with genome editing, M. F. Pera for critical reading of the manuscript, and M. Schwarz, and P. Schwarz for administrative help. M.L. and K.S. are supported by a California Institute for Regenerative Medicine Training Grant. We thank J. L. Mendoza for his support on this project. This work was funded in part by UCAM (mouse studies). J.R.E. is an Investigator of the Howard Hughes Medical Institute. P.G. was supported by Fundacion Pedro Guillen. Work in the laboratory of J.C.I.B. was supported by G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust and The Moxie Foundation.

Author information

Author notes

    • Daiji Okamura

    Present address: Department of Advanced Bioscience, Graduate School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.

    • Jun Wu
    •  & Daiji Okamura

    These authors contributed equally to this work.


  1. The Salk Institute for Biological Studies, Gene Expression Laboratory, La Jolla, California 92037, USA

    • Jun Wu
    • , Daiji Okamura
    • , Mo Li
    • , Keiichiro Suzuki
    • , Li Ma
    • , Zhongwei Li
    • , Isao Tamura
    • , Marie N. Krause
    • , Tomoaki Hishida
    • , Yuta Takahashi
    • , Emi Aizawa
    • , Na Young Kim
    • , Concepcion Rodriguez Esteban
    •  & Juan Carlos Izpisua Belmonte
  2. Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California 92037, USA

    • Chongyuan Luo
    •  & Joseph R. Ecker
  3. The Salk Institute for Biological Studies, Genomic Analysis Laboratory, La Jolla, California 92037, USA

    • Chongyuan Luo
    • , Yupeng He
    • , Joseph R. Nery
    • , Zhuzhu Zhang
    •  & Joseph R. Ecker
  4. The Salk Institute for Biological Studies, Integrated Genomics, La Jolla, California 92037, USA

    • Chris Benner
  5. Ludwig Institute for Cancer Research, University of California, San Diego School of Medicine, Department of Cellular and Molecular Medicine, 9500 Gilman Drive, La Jolla, California 92093-0653, USA

    • Tingting Du
    •  & Bing Ren
  6. Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8577, Japan

    • Yuta Takahashi
  7. Grado en Medicina, Universidad Católica, San Antonio de Murcia, Campus de los Jerónimos, 135, Guadalupe 30107, Spain

    • Jeronimo Lajara
    •  & Pedro Guillen
  8. Fundacion Pedro Guillen, Clínica Cemtro, Avenida Ventisquero de la Condesa, 42, 28035 Madrid, Spain

    • Pedro Guillen
  9. Hospital Clinic of Barcelona, Carrer Villarroel, 170, 08036 Barcelona, Spain

    • Josep M. Campistol
  10. University of California, Davis, Davis, California 95616, USA

    • Pablo J. Ross
  11. The Salk Institute for Biological Studies, Peptide Biology Laboratory, La Jolla, California 92037, USA

    • Alan Saghatelian


  1. Search for Jun Wu in:

  2. Search for Daiji Okamura in:

  3. Search for Mo Li in:

  4. Search for Keiichiro Suzuki in:

  5. Search for Chongyuan Luo in:

  6. Search for Li Ma in:

  7. Search for Yupeng He in:

  8. Search for Zhongwei Li in:

  9. Search for Chris Benner in:

  10. Search for Isao Tamura in:

  11. Search for Marie N. Krause in:

  12. Search for Joseph R. Nery in:

  13. Search for Tingting Du in:

  14. Search for Zhuzhu Zhang in:

  15. Search for Tomoaki Hishida in:

  16. Search for Yuta Takahashi in:

  17. Search for Emi Aizawa in:

  18. Search for Na Young Kim in:

  19. Search for Jeronimo Lajara in:

  20. Search for Pedro Guillen in:

  21. Search for Josep M. Campistol in:

  22. Search for Concepcion Rodriguez Esteban in:

  23. Search for Pablo J. Ross in:

  24. Search for Alan Saghatelian in:

  25. Search for Bing Ren in:

  26. Search for Joseph R. Ecker in:

  27. Search for Juan Carlos Izpisua Belmonte in:


J.W., D.O. and J.C.I.B. conceived the study. J.W. and D.O. derived mESC, EpiSC and rsEpiSC lines. J.W., D.O. and C.R.E. designed and performed in vivo embryo grafting experiments. J.W., D.O., M.L., K.S., L.M., Z.L., T.H. and P.R. designed and performed all in vitro experiments; J.M.C., J.L. and P.G. helped project design and discussions and performed microarray experiments. I.T., Y.T. performed bisulfite sequencing experiments; M.K. performed teratoma studies; C.L., Y.H., Z.Z., J.R.N. and J.E. performed whole-genome bisulfite sequencing experiments and analysed data. T.D. and B.R. performed ChIP-seq experiments. C.B. and M.L. performed bioinformatics analysis. A.S. analysed global metabolic profiling data; E.A. and N.K. provided technical support. J.W., D.O., M.L. and J.C.I.B. prepared the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1 and Western blots for Figure 4b and Extended Data Figure 4c.

  2. 2.

    Supplementary Tables

    This file contains Supplementary Tables 1-8.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.