In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells

Abstract

Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Recently, we derived stem cells with blastomere-like features from mouse cleavage-stage embryos, which we named expanded-potential stem cells (EPSCs). Here, we provide detailed protocols that describe how to establish EPSCs from single eight-cell-stage blastomeres or whole eight-cell pre-implantation mouse embryos, or by conversion of mouse ES cells or induced pluripotent stem (iPS) cells reprogrammed from fibroblasts. It takes 2–3 weeks to derive EPSCs from each cell source. The EPSCs derived from these protocols can differentiate into all embryonic and extra-embryonic lineages when implanted into chimeras. Furthermore, bona fide TS and XEN cell lines can be derived from EPSCs in vitro.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Dynamic of OCT4 and CDX2 expression in embryos cultured in EPSCM.
Fig. 2: Proliferation and apoptosis in embryos cultured in EPSCM.
Fig. 3: Colony formation assay of single cells from filled blastocyst.
Fig. 4: Embryo collection and blastomere preparation.
Fig. 5: Derivation of EPSCs from eight-cell embryos on SNL76/7 feeders.
Fig. 6: Derivation of feeder-free EPSCs from eight-cell embryos.
Fig. 7: Derivation of EPSCs from single blastomeres.
Fig. 8: Conversion of ES cells to EPSCs.
Fig. 9: MEF reprogramming and conversion of iPS cells to EPSCs.
Fig. 10: RT–qPCR analysis of Axin2 and H19 expression.
Fig. 11: Examples of unsuccessful EPSC chimera formation.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data for Fig. 10 are available online.

References

  1. 1.

    Hamatani, T., Carter, M. G., Sharov, A. A. & Ko, M. S. Dynamics of global gene expression changes during mouse preimplantation development. Dev. Cell 6, 117–131 (2004).

    CAS  Article  Google Scholar 

  2. 2.

    Lyon, M. F. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190, 372–373 (1961).

    CAS  Article  Google Scholar 

  3. 3.

    Gu, T. P. et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606–610 (2011).

    CAS  Article  Google Scholar 

  4. 4.

    Guo, F. et al. Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell 15, 447–459 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Rougier, N. et al. Chromosome methylation patterns during mammalian preimplantation development. Genes Dev. 12, 2108–2113 (1998).

    CAS  Article  Google Scholar 

  6. 6.

    Bedzhov, I., Graham, S. J., Leung, C. Y. & Zernicka-Goetz, M. Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369, 20130538 (2014).

  7. 7.

    Saiz, N. & Plusa, B. Early cell fate decisions in the mouse embryo. Reproduction 145, R65–R80 (2013).

    CAS  Article  Google Scholar 

  8. 8.

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

    CAS  Article  Google Scholar 

  9. 9.

    Kunath, T. et al. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132, 1649–1661 (2005).

    CAS  Article  Google Scholar 

  10. 10.

    Martin, G. R. 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).

    CAS  Article  Google Scholar 

  11. 11.

    Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A. & Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072–2075 (1998).

    CAS  Article  Google Scholar 

  12. 12.

    Macfarlan, T. S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57–63 (2012).

    CAS  Article  Google Scholar 

  13. 13.

    Choi, Y. J. et al. Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells. Science 355, eaag1927 (2017).

    Article  Google Scholar 

  14. 14.

    Morgani, S. M. et al. Totipotent embryonic stem cells arise in ground-state culture conditions. Cell Rep. 3, 1945–1957 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Yang, J. et al. Establishment of mouse expanded potential stem cells. Nature 550, 393–397 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    Tarkowski, A. K. & Wroblewska, J. Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage. J. Embryol. Exp. Morphol. 18, 155–180 (1967).

    CAS  PubMed  Google Scholar 

  17. 17.

    Yamanaka, Y., Ralston, A., Stephenson, R. O. & Rossant, J. Cell and molecular regulation of the mouse blastocyst. Dev. Dyn. 235, 2301–2314 (2006).

    CAS  Article  Google Scholar 

  18. 18.

    Johnson, M. H. & Ziomek, C. A. The foundation of two distinct cell lineages within the mouse morula. Cell 24, 71–80 (1981).

    CAS  Article  Google Scholar 

  19. 19.

    Yagi, R. et al. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development 134, 3827–3836 (2007).

    CAS  Article  Google Scholar 

  20. 20.

    Nishioka, N. et al. The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev. Cell 16, 398–410 (2009).

    CAS  Article  Google Scholar 

  21. 21.

    Piccolo, S., Dupont, S. & Cordenonsi, M. The biology of YAP/TAZ: hippo signaling and beyond. Physiol. Rev. 94, 1287–1312 (2014).

    CAS  Article  Google Scholar 

  22. 22.

    Azzolin, L. et al. YAP/TAZ incorporation in the beta-catenin destruction complex orchestrates the Wnt response. Cell 158, 157–170 (2014).

    CAS  Article  Google Scholar 

  23. 23.

    Sonderegger, S., Pollheimer, J. & Knofler, M. Wnt signalling in implantation, decidualisation and placental differentiation--review. Placenta 31, 839–847 (2010).

    CAS  Article  Google Scholar 

  24. 24.

    Chan, S. W. et al. Hippo pathway-independent restriction of TAZ and YAP by angiomotin. J. Biol. Chem. 286, 7018–7026 (2011).

    CAS  Article  Google Scholar 

  25. 25.

    Wang, W., Huang, J. & Chen, J. Angiomotin-like proteins associate with and negatively regulate YAP1. J. Biol. Chem. 286, 4364–4370 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Zhao, B. et al. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev. 25, 51–63 (2011).

    Article  Google Scholar 

  27. 27.

    Wang, W. et al. Tankyrase inhibitors target YAP by stabilizing angiomotin family proteins. Cell Rep. 13, 524–532 (2015).

    Article  Google Scholar 

  28. 28.

    Lu, C. W. et al. Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos. Nat. Genet. 40, 921–926 (2008).

    CAS  Article  Google Scholar 

  29. 29.

    Maekawa, M. et al. Requirement of the MAP kinase signaling pathways for mouse preimplantation development. Development 132, 1773–1783 (2005).

    CAS  Article  Google Scholar 

  30. 30.

    Li, X. et al. Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos. Cell Stem Cell 8, 46–58 (2011).

    CAS  Article  Google Scholar 

  31. 31.

    Kapoor, A. et al. Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer. Cell 158, 185–197 (2014).

    CAS  Article  Google Scholar 

  32. 32.

    Shao, D. D. et al. KRAS and YAP1 converge to regulate EMT and tumor survival. Cell 158, 171–184 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J. & Smithgall, T. E. Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis. Oncogene 21, 8075–8088 (2002).

    CAS  Article  Google Scholar 

  34. 34.

    Martello, G. et al. Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal. Cell Stem Cell 11, 491–504 (2012).

    CAS  Article  Google Scholar 

  35. 35.

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

    CAS  Article  Google Scholar 

  36. 36.

    Zimmerlin, L. et al. Tankyrase inhibition promotes a stable human naive pluripotent state with improved functionality. Development 143, 4368–4380 (2016).

    CAS  Article  Google Scholar 

  37. 37.

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

    Article  Google Scholar 

  38. 38.

    Ryan, D. J., Yang, J., Lan, G. & Liu, P. Derivation and maintenance of mouse expanded potential stem cells. Protoc. Exch. https://doi.org/10.1038/protex.2017.102 (2017).

  39. 39.

    Baker, C. L. & Pera, M. F. Capturing totipotent stem cells. Cell Stem Cell 22, 25–34 (2018).

    CAS  Article  Google Scholar 

  40. 40.

    Niakan, K. K., Schrode, N., Cho, L. T. & Hadjantonakis, A. K. Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells. Nat. Protoc. 8, 1028–1041 (2013).

    CAS  Article  Google Scholar 

  41. 41.

    Matsunari, H. et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc. Natl. Acad. Sci. USA 110, 4557–4562 (2013).

    CAS  Article  Google Scholar 

  42. 42.

    Yang, Y. et al. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 169, 243–257.e25 (2017).

    CAS  Article  Google Scholar 

  43. 43.

    Hemberger, M. et al. Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives. Dev. Biol. 257, 371–381 (2003).

    CAS  Article  Google Scholar 

  44. 44.

    Yang, J. et al. Signalling through retinoic acid receptors is required for reprogramming of both mouse embryonic fibroblast cells and epiblast stem cells to induced pluripotent stem cells. Stem Cells 33, 1390–1404 (2015).

    CAS  Article  Google Scholar 

  45. 45.

    Hung, S. S. et al. Repression of global protein synthesis by Eif1a-like genes that are expressed specifically in the two-cell embryos and the transient Zscan4-positive state of embryonic stem cells. DNA Res. 20, 391–402 (2013).

    CAS  Article  Google Scholar 

  46. 46.

    Sasaki, H., Ferguson-Smith, A. C., Shum, A. S., Barton, S. C. & Surani, M. A. Temporal and spatial regulation of H19 imprinting in normal and uniparental mouse embryos. Development 121, 4195–4202 (1995).

    CAS  PubMed  Google Scholar 

  47. 47.

    Thomas, K. R. & Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512 (1987).

    CAS  Article  Google Scholar 

  48. 48.

    McMahon, A. P. & Bradley, A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62, 1073–1085 (1990).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank our colleagues from the research support facility (M. Woods, C. Sinclair, E. Brown, B. Doe, S. Newman, E. Grau and others) at the Sanger Institute and the animal facility at CRUK-CI. The P.L. lab was supported by the Wellcome Trust (grant numbers: 098051 and 206194) and internal funding from the University of Hong Kong.

Author information

Affiliations

Authors

Contributions

J.Y. and D.J.R. drafted the protocol. J.Y., D.J.R. and P.L. wrote the protocol with inputs from G.L. and X.Z. The initial observations of pre-implantation embryos in EPSCM were made by D.J.R. Most experiments presented in this protocol were performed by J.Y. All chimeric blastocysts were generated by G.L. and X.Z.

Corresponding authors

Correspondence to Jian Yang or Pentao Liu.

Ethics declarations

Competing interests

Genome Research Limited has filed a provisional patent application that covers the derivation and maintenance of EPSCs (WO2016079146). P.L., D.J.R. and J.Y. are listed as inventors. The other authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related link

Key reference using this protocol

Yang, J. et al. Nature 550, 393–397 (2017) https://doi.org/10.1038/nature24052

Integrated supplementary information

Supplementary Figure 1 Diagrams of constructs.

These constructs include episomal vectors used in MEF reprogramming experiment and PB vectors used in EPSCs transfection. CAG: CMV early enhancer/chick β-actin; SV40/pA: SV40 polyA; Orip: origin of plasmid replication; EBNA-1: EBV nuclear antigen 1; T2A, E2A: 2A peptides; bpA: bovine growth hormone polyA.

Supplementary Figure 2 Gating strategy.

Non-transfected EPSCs (DR25) were used as negative control. In all sorting experiments, cell population was initially identified on a FSC/SSC plot, using a polygon gate to separate distinct cell population from debris and feeder (over 85%). The boundary between positive and negative was determined by negative control. And negative population in control was over 99.5%. Using these gates, the sample, DR25-EPSCs transfected with an mCherry fluorescence reporter construct was sorted (76.5% mCherry positive) and plated. a. Negative control, untransfected DR25-EPSCs. b. Sample, DR25-EPSCs transfected with PBCAGmCherry.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, and Supplementary Table 1

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yang, J., Ryan, D.J., Lan, G. et al. In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells. Nat Protoc 14, 350–378 (2019). https://doi.org/10.1038/s41596-018-0096-4

Download citation

Further reading

Comments

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.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing