Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells

Abstract

The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Canonical Wnt activation by GSK3β inhibitors and mesoderm induction.
Figure 2: VEGF-A and PDGF-BB-mediated differentiation of hPSCs into vascular endothelial or vascular smooth muscle cells.
Figure 3: Global transcriptome and metabolomic analyses confirm vascular cell identity of differentiated hPSCs.
Figure 4: In vitro characterization of hPSC ECs and hPSC VSMCs.
Figure 5: Co-culture experiments and in vivo characterization of hPSC ECs.

References

  1. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  PubMed  Google Scholar 

  3. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Grskovic, M., Javaherian, A., Strulovici, B. & Daley, G. Q. Induced pluripotent stem cells–opportunities for disease modelling and drug discovery. Nat. Rev. Drug Discov. 10, 915–929 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Tiscornia, G., Vivas, E. L. & Belmonte, J. C. Diseases in a dish: modeling human genetic disorders using induced pluripotent cells. Nat. Med. 17, 1570–1576 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Zhu, H., Lensch, M. W., Cahan, P. & Daley, G. Q. Investigating monogenic and complex diseases with pluripotent stem cells. Nat. Rev. Genet. 12, 266–275 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. James, D. et al. Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1 dependent. Nat. Biotechnol. 28, 161–166 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Levenberg, S., Golub, J. S., Amit, M., Itskovitz-Eldor, J. & Langer, R. Endothelial cells derived from human embryonic stem cells. Proc. Natl Acad. Sci. USA 99, 4391–4396 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kane, N. M. et al. Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscler. Thromb. Vasc. Biol. 30, 1389–1397 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Vodyanik, M. A., Bork, J. A., Thomson, J. A. & Slukvin, II Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 105, 617–626 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Li, Z. et al. Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects. Stem Cells 26, 864–873 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang, H. et al. Gene expression profile signatures indicate a role for Wnt signaling in endothelial commitment from embryonic stem cells. Circ. Res. 98, 1331–1339 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Levenberg, S., Zoldan, J., Basevitch, Y. & Langer, R. Endothelial potential of human embryonic stem cells. Blood 110, 806–814 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kelly, M. A. & Hirschi, K. K. Signaling hierarchy regulating human endothelial cell development. Arterioscler. Thromb. Vasc. Biol. 29, 718–724 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang, Z. Z. et al. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nat. Biotechnol. 25, 317–318 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Sumi, T., Tsuneyoshi, N., Nakatsuji, N. & Suemori, H. Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling. Development 135, 2969–2979 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Inman, G. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Orlova, V. V. et al. Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts. Arterioscler. Thromb. Vasc. Biol. 34, 177–186 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Yamashita, J. et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92–96 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Tam, P. P. & Loebel, D. A. Gene function in mouse embryogenesis: get set for gastrulation. Nat. Rev. Genet. 8, 368–381 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Huelsken, J. et al. Requirement for beta-catenin in anterior-posterior axis formation in mice. J. Cell Biol. 148, 567–578 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu, Y., Festing, M. H., Hester, M., Thompson, J. C. & Weinstein, M. Generation of novel conditional and hypomorphic alleles of the Smad2 gene. Genesis 40, 118–123 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Woll, P. S. et al. Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells. Blood 111, 122–131 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Brade, T., Manner, J. & Kuhl, M. The role of Wnt signalling in cardiac development and tissue remodelling in the mature heart. Cardiovasc. Res. 72, 198–209 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Park, C. et al. A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells. Development 131, 2749–2762 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Tada, S. et al. Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132, 4363–4374 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Nostro, M. C., Cheng, X., Keller, G. M. & Gadue, P. Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood. Cell Stem Cell 2, 60–71 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bone, H. K., Nelson, A. S., Goldring, C. E., Tosh, D. & Welham, M. J. A novel chemically directed route for the generation of definitive endoderm from human embryonic stem cells based on inhibition of GSK-3. J. Cell Sci. 124, 1992–2000 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. & Brivanlou, A. H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55–63 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Fabian, M. A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329–336 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Soker, S., Takashima, S., Miao, H. Q., Neufeld, G. & Klagsbrun, M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92, 735–745 (1998).

    Article  CAS  PubMed  Google Scholar 

  32. DeAlmeida, V. I. et al. The soluble wnt receptor Frizzled8CRD-hFc inhibits the growth of teratocarcinomas in vivo. Cancer Res. 67, 5371–5379 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Winnier, G., Blessing, M., Labosky, P. A. & Hogan, B. L. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105–2116 (1995).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  35. Yamamizu, K., Kawasaki, K., Katayama, S., Watabe, T. & Yamashita, J. K. Enhancement of vascular progenitor potential by protein kinase A through dual induction of Flk-1 and Neuropilin-1. Blood 114, 3707–3716 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Englund, M. C. et al. The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking. In Vitro Cell. Dev. Biol. Anim. 46, 217–230 (2010).

    Article  PubMed  Google Scholar 

  37. Burridge, P. W. et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS ONE 6, e18293 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Chan, M. C. et al. Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression. EMBO J. 29, 559–573 (2010).

    Article  CAS  PubMed  Google Scholar 

  39. Owens, G. K., Kumar, M. S. & Wamhoff, B. R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol. Rev. 84, 767–801 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Cheung, C., Bernardo, A. S., Trotter, M. W., Pedersen, R. A. & Sinha, S. Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility. Nat. Biotechnol. 30, 165–173 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Engert, S., Liao, W. P., Burtscher, I. & Lickert, H. Sox17-2A-iCre: a knock-in mouse line expressing Cre recombinase in endoderm and vascular endothelial cells. Genesis 47, 603–610 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Atienza, J. M. et al. Dynamic and label-free cell-based assays using the real-time cell electronic sensing system. Assay Drug Dev. Technol. 4, 597–607 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Solly, K., Wang, X., Xu, X., Strulovici, B. & Zheng, W. Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays. Assay Drug Dev. Technol. 2, 363–372 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Rabiet, M. J. et al. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler. Thromb. Vasc. Biol. 16, 488–496 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. Galkina, E. & Ley, K. Vascular adhesion molecules in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 27, 2292–2301 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Lusis, A. J. Atherosclerosis. Nature 407, 233–241 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Rasmussen, L. M., Wolf, Y. G. & Ruoslahti, E. Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor-beta activity. Am. J. Pathol. 147, 1041–1048 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Grainger, S. J., Carrion, B., Ceccarelli, J. & Putnam, A. J. Stromal cell identity influences the in vivo functionality of engineered capillary networks formed by co-delivery of endothelial cells and stromal cells. Tissue Eng. Part A 19, 1209–1222 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Cowan, C. A. et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Challet Meylan, L., Patsch, C. & Thoma, E. C. Endothelial cells differentiation from hPSCs. Nat. Protoc. Exch. (2015) http://dx.doi.org/10.1038/protex.2015.055

  52. Thoma, E. C., Challet Meylan, L. & Patsch, C. Vascular smooth muscle cells differentiation from hPSCs. Nat. Protoc. Exch. (2015) http://dx.doi.org/10.1038/protex.2015.056

  53. Gotlieb, A. I. & Spector, W. Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation. Am. J. Pathol. 103, 271–282 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Collins, S. J., Gallo, R. C. & Gallagher, R. E. Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270, 347–349 (1977).

    Article  CAS  PubMed  Google Scholar 

  55. Wang, T. J. et al. Metabolite profiles and the risk of developing diabetes. Nat. Med. 17, 448–453 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A. Einhaus, S. Zimmermann, C. Stücki and N. Dahm for excellent technical support and all members of the Roche Stem Cell Platform for helpful discussions. We thank C. MacGillivray at the HSCRB Histology Core facility. We also thank S. Aigner and C. Warren for critically reviewing the manuscript and A. Gündner, C. Hudak and K. Song for help with the illustrations. We acknowledge the continued support of H.-L. Roche and thank M. Steger from Roche Pharma Partnering for his input and support of the project. C.P. and E.C.T. were supported by Roche Postdoctoral Fellowships (RPF). A part of the research received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement number 115439, resources of which are composed of financial contribution from the European Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA companies’ in kind contribution. L.C.-M. was supported by a fellowship from the Swiss National Science Foundation. F.G.K. was supported by a fellowship from the German Cancer Aid. This work was supported by HHMI and NIH grants R01 HL04880, 5P30 DK49216, 5R01 DK53298, 5U01 HL10001-05, and R24 DK092760 (to L.I.Z.); HL106018, HL083867, HL60963 (E.L.C.); 2R01DK081572 and an AHA Established Investigator Award (R.E.G.); R01DK097768, U01HL100408, the Harvard Stem Cell Institute and Harvard University (C.A.C.).

Author information

Authors and Affiliations

Authors

Contributions

C.P., L.C.-M., E.C.T. and E.U. designed and performed experiments, analysed and interpreted data and wrote the manuscript. S.J.G., F.G.K., L.S., K.C., Y.X., M.H.C.F., W.H., W.P., C.R.W., R.J.-R. and I.A. designed and performed experiments and analysed data. M.P. performed high-content imaging analysis. T.H. designed and performed gene expression experiments, performed and interpreted bioinformatic analyses and wrote the manuscript. J.F.O’S. designed and performed metabolomic experiments, interpreted and analysed the data and wrote the manuscript. U.C. and R.J. provided scientific input. P.-O.F., D.K., P.H., L.I.Z., E.L.C. and R.E.G. analysed and interpreted the data and supervised experiments. M.G. and R.I. analysed and interpreted the data and supervised the project. C.A.C. interpreted the data, supervised the project and wrote the manuscript. C.P., L.C.-M., E.C.T., E.U., J.F.O’S., M.P., D.K., T.H. and W.H. contributed to description of online methods.

Corresponding authors

Correspondence to Christoph Patsch or Chad A. Cowan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Canonical Wnt activation by GSK3β inhibitors and mesoderm induction.

(A) Molecular structure of different GSK3β inhibitors. (B) Schematic illustration of the luciferase reporter assay used in this study to detect the expression of β-catenin. (C) Cell viability assay on treatment with different concentration of GSK3β inhibitors. A 6-point 3-fold serial dilution of each compound was performed (10, 3, 1, 0.3, 0.1, 0.03 μm, last 2 concentration data not shown). Columns show mean +/− s.d. of 5 independent experiments. (D) Binding constant (Kd) determination for the GSK3β inhibitors tested in this study. An 11-point 3-fold serial dilution of each test compound was prepared in 100% DMSO at 100× final test concentration and subsequently diluted to 1x in the assay (final DMSO concentration = 2.5%). Representative curve of four independent experiments is shown. (E) Bright field pictures of hESCs treated with increasing concentration of the GSK3β inhibitor BIO illustrating its cell toxicity. Representative images of one experiment are shown.

Supplementary Figure 2 VEGF and PDGF-BB-mediated differentiation of hPSCs into vascular endothelial or smooth muscle cells.

(A) BMP4-dependent expression of mesoderm markers T, MIXL, and EOMES at day 4 of differentiation. Columns show mean of 3 technical replicates of a single well of a single experiment. (B) Comparison of the ability of different GSK3β inhibitors to induce endothelial cell differentiation as shown by immunostaining for the endothelial marker VE-Cadherin. Representative images of 3 independent experiments. (C) Effect of BMP4 on hPSC-ECs differentiation. 3 wells per conditions of a single experiment. (D) Potency of different GSK3β inhibitors to induce hPSC-ECs differentiation. Mean values +/− s.d. of 3 independent experiments are shown. (E) Differentiation efficiency of hPSC-ECs on day 6 after sorting for CP21 and CHIR when used at their optimal concentration (defined in D). 3 wells per conditions of a single experiment. (F) Representative FACS plots showing the improvement of differentiation efficiency when BMP4 and forskolin are added to the media. This experiment was done once and 3 wells per conditions were analysed. (G) FACS analysis of CD144+ hPSC-ECs on day 10 showing the expression of ECs-specific markers (KDR, CD31, CD34, CD105) and the absence of hematopoietic markers (CD43, CD45). Representative results of 5 independent experiments. (H) The endothelial cell differentiation protocol produces a small amount of alpha smooth actin positive cells as shown by immunostaining on day 6 before MACS sorting. Representative image of 5 independent experiments. (I) Role of ActivinA and PDGF-BB in the differentiation efficiency of hPSC-VSMCs. 3 wells per conditions of a single experiment. (J) Differentiation efficiency of hPSC-VSMCs for the two GSK3β inhibitors CP21 and CHIR when used at their optimal concentration (1 μM and 6 μM, respectively). 3 wells per conditions of a single experiment. (K) Effect of BMP4 on hPSC-VSMCs differentiation, n = 3 wells of a single experiment. (K) Example of the efficiency of MACS sorting and the purity of the resulting hPSC-ECs population, representative result from 2 independent experiments.

Supplementary Figure 3 Global Transcriptome analysis during vascular wall cell differentiation.

(A) Principal component projections of transcriptomes coloured by sample type. The variability of the data set along Principal component 1 is 28.8% and along Principal component 2 is 24.1%. Note the clustering of precursor cells during the early time points and the clustering of differentiated vascular wall cells with their respective primary cells. (B) Dynamic gene expression of representative spatio-temporal regulated genes during the course of endothelial cell differentiation. The detection limit of the microarray platform is indicated by a dotted line; this signal is derived from 5000 random probes (60-mers of random nucleotides), which serve as a metric of non-specific annealing and background fluorescence. (C) Heat map of genes sets of biological processes (GO terms) significantly over- or under-represented in stem cell derived ECs, VSMCs, and primary cells in comparison to ESCs. Rows represent genes, and columns are samples. Row Z-score transformation was performed on log2 expression values for each gene with blue denoting a lower and red a higher expression level according to the average expression level. Hierarchical clustering of genes and samples is based on average linkage and correlation distance.

Supplementary Figure 4 In vitro characterization of hPSC-ECs and hPSC-VSMCs.

(A) Calcium imaging of SC-VSMCs at day 13 of differentiation. Stimulation with vasoconstrictive reagents resulted in increase in intracellular calcium. Time course of calcium flux after treatment. RFU was measured every second and average values of three independent experiments are shown. (B) Example of collagen gel contraction assay after 48 h with or without U46619. Gel surface areas were measured and further analysed using ImageJ. Scale bars: 1 cm. (C) Fibronectin production of hPSC-VSMCs on TGF-β treatment. Immunofluorescence staining of extracellular fibronectin depositions after 24 h of TGF-β treatment in the presence of absence of TFG-β inhibitors. Scale bars: 50 μM. Representative images of 3 independent experiments are shown.

Supplementary Figure 5 Whole mount view of fibrinogen implants.

Representative pictures of whole mount implants. This experiment was conducted once with 5 mice per conditions and 2 implants per mice (=10 implants per conditions) (A) hPSC-ECs only (B) HUVECs + MSCs (C) hPSC-ECs + MSCs and (D) hPSC-ECs + hPSC-VSMCs. Scale bars = 500 μM except A) scale bar = 5 mm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1228 kb)

Supplementary Table 1

Supplementary Information (XLSX 35 kb)

Supplementary Table 2

Supplementary Information (XLS 34 kb)

Supplementary Table 3

Supplementary Information (XLSX 128 kb)

Supplementary Table 4

Supplementary Information (XLSX 38 kb)

Supplementary Table 5

Supplementary Information (XLSX 38 kb)

Supplementary Table 6

Supplementary Information (XLSX 45 kb)

Supplementary Table 7

Supplementary Information (XLSX 40 kb)

Supplementary Table 8

Supplementary Information (XLSX 45 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Patsch, C., Challet-Meylan, L., Thoma, E. et al. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat Cell Biol 17, 994–1003 (2015). https://doi.org/10.1038/ncb3205

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3205

This article is cited by

Search

Quick links

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