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.

  • Article
  • Published:

Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages

Nature Cell Biology volume 17, pages 580–591 (2015)Cite this article

Subjects

Abstract

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34+CD73−CD184− fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C+ cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34+CD73medCD184+ and CD34+CD73hiCD184− fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Characterization of hPSC-derived definitive HE.
Figure 2: RUNX1C–EGFP is expressed during the EHT of the definitive HE.
Figure 3: Haematopoietic specification of definitive HE is NOTCH-dependent.
Figure 4: Expression of CD73 and CD184 distinguishes HE and VE in the day 8 CD34+CD43− population.
Figure 5: Engrafted CD73medCD184+ and CD73hiCD184− cells maintain their vascular identity.
Figure 6: The CD34+CD43−CD73−CD184− fraction contains both HE and VE progenitor cells.
Figure 7: The CD34+CD43−CD73−CD184−DLL4− HE fraction contains cells with multilineage potential.

Similar content being viewed by others

References

  1. Kaufman, D. S. Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells. Blood 114, 3513–3523 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Kennedy, M. et al. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep. 2, 1722–1735 (2012).

    CAS  PubMed  Google Scholar 

  3. Klimchenko, O. et al. Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions. Blood 117, 3065–3075 (2011).

    CAS  PubMed  Google Scholar 

  4. Takayama, N. et al. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. Blood 111, 5298–5306 (2008).

    CAS  PubMed  Google Scholar 

  5. Clements, W. K. & Traver, D. Signalling pathways that control vertebrate haematopoietic stem cell specification. Nat. Rev. Immunol. 13, 336–348 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Kissa, K. & Herbomel, P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464, 112–115 (2010).

    CAS  PubMed  Google Scholar 

  7. Bertrand, J. Y. et al. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464, 108–111 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Boisset, J. C. et al. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120 (2010).

    CAS  PubMed  Google Scholar 

  9. Rybtsov, S. et al. Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43− embryonic precursor. Stem Cell Rep. 3, 489–501 (2014).

    CAS  Google Scholar 

  10. Sturgeon, C. M., Ditadi, A., Awong, G., Kennedy, M. & Keller, G. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat. Biotechnol. 32, 554–561 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bertrand, J. Y., Cisson, J. L., Stachura, D. L. & Traver, D. Notch signaling distinguishes 2 waves of definitive hematopoiesis in the zebrafish embryo. Blood 115, 2777–2783 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Hadland, B. K. et al. A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development. Blood 104, 3097–3105 (2004).

    CAS  PubMed  Google Scholar 

  13. Kumano, K. et al. Notch1 but not Notch2 is essential for generating hematopoietic stem cells from endothelial cells. Immunity 18, 699–711 (2003).

    CAS  PubMed  Google Scholar 

  14. Robert-Moreno, A., Espinosa, L., de la Pompa, J. L. & Bigas, A. RBPjκ-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells. Development 132, 1117–1126 (2005).

    CAS  PubMed  Google Scholar 

  15. Peeters, M. et al. Ventral embryonic tissues and Hedgehog proteins induce early AGM hematopoietic stem cell development. Development 136, 2613–2621 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Robin, C. & Durand, C. The roles of BMP and IL-3 signaling pathways in the control of hematopoietic stem cells in the mouse embryo. Int. J. Dev. Biol. 54, 1189–1200 (2010).

    CAS  PubMed  Google Scholar 

  17. Zambidis, E. T. et al. Expression of angiotensin-converting enzyme (CD143) identifies and regulates primitive hemangioblasts derived from human pluripotent stem cells. Blood 112, 3601–3614 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Yokomizo, T. & Dzierzak, E. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos. Development 137, 3651–3661 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Uenishi, G. et al. Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions. Stem Cell Rep. 3, 1073–1084 (2014).

    CAS  Google Scholar 

  20. Bee, T. et al. Alternative Runx1 promoter usage in mouse developmental hematopoiesis. Blood Cells Mol. Dis. 43, 35–42 (2009).

    CAS  PubMed  Google Scholar 

  21. Choi, K. D. et al. Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures. Cell Rep. 2, 553–567 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Rafii, S. et al. Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition. Blood 121, 770–780 (2013).

    CAS  PubMed  Google Scholar 

  23. Yamamizu, K. et al. Convergence of Notch and β-catenin signaling induces arterial fate in vascular progenitors. J. Cell Biol. 189, 325–338 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Yurugi-Kobayashi, T. et al. Adrenomedullin/cyclic AMP pathway induces Notch activation and differentiation of arterial endothelial cells from vascular progenitors. Arterioscler. Thromb. Vasc. Biol. 26, 1977–1984 (2006).

    CAS  PubMed  Google Scholar 

  25. Tober, J., Yzaguirre, A. D., Piwarzyk, E. & Speck, N. A. Distinct temporal requirements for Runx1 in hematopoietic progenitors and stem cells. Development 140, 3765–3776 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Marcelo, K. L., Goldie, L. C. & Hirschi, K. K. Regulation of endothelial cell differentiation and specification. Circ. Res. 112, 1272–1287 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. You, L. R. et al. Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature 435, 98–104 (2005).

    CAS  PubMed  Google Scholar 

  28. Hong, C. C., Peterson, Q. P., Hong, J. Y. & Peterson, R. T. Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling. Curr. Biol. 16, 1366–1372 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lindskog, H. et al. Molecular identification of venous progenitors in the dorsal aorta reveals an aortic origin for the cardinal vein in mammals. Development 141, 1120–1128 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Benedito, R. & Duarte, A. Expression of Dll4 during mouse embryogenesis suggests multiple developmental roles. Gene Expr. Patterns 5, 750–755 (2005).

    CAS  PubMed  Google Scholar 

  31. Richard, C. et al. Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis. Dev. Cell 24, 600–611 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Ciau-Uitz, A., Wang, L., Patient, R. & Liu, F. ETS transcription factors in hematopoietic stem cell development. Blood Cells Mol. Dis. 51, 248–255 (2013).

    CAS  PubMed  Google Scholar 

  33. Swiers, G. et al. Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level. Nat. Commun. 4, 2924 (2013).

    PubMed  Google Scholar 

  34. Bigas, A., Robert-Moreno, A. & Espinosa, L. The Notch pathway in the developing hematopoietic system. Int. J. Dev. Biol. 54, 1175–1188 (2010).

    CAS  PubMed  Google Scholar 

  35. Kim, A. D. et al. Discrete Notch signaling requirements in the specification of hematopoietic stem cells. EMBO J. 33, 2363–2373 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Godin, I., Garcia-Porrero, J. A., Dieterlen-Lievre, F. & Cumano, A. Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites. J. Exp. Med. 190, 43–52 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Boiers, C. et al. Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells. Cell Stem Cell 13, 535–548 (2013).

    PubMed  Google Scholar 

  38. Luc, S. et al. The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential. Nat. Immunol. 13, 412–419 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Irion, S. et al. Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat. Biotechnol. 25, 1477–1482 (2007).

    CAS  PubMed  Google Scholar 

  40. Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C. & Bongso, A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20, 933–936 (2002).

    CAS  PubMed  Google Scholar 

  41. Davis, R. P. et al. A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis. Nat. Protoc. 3, 1550–1558 (2008).

    CAS  PubMed  Google Scholar 

  42. Livak, K. J. et al. Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells. Methods 59, 71–79 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Lorsbach, R. B. et al. Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression. Blood 103, 2522–2529 (2004).

    CAS  PubMed  Google Scholar 

  44. North, T. E. et al. Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity 16, 661–672 (2002).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank the SickKids–UHN Flow Cytometry Facility for their expert assistance with cell sorting, in particular A. Khandani, F. Xu at the Advanced Optical Microscopy Facility for the great help with the time-lapse and confocal imaging, and S. Zandi for assistance with single-cell qRT-PCR. This work was supported by the National Institutes of Health grant U01 HL100395 to G.K., SR00002303 to N.A.S. and by the Canadian Institutes of Health Research grants MOP93569 and EPS 127882 to G.K. Additional support to A.D. and C.M.S. was provided by the Magna-Golftown Post-Doctoral Fellowship and the McMurrich Post-Doctoral Fellowship, respectively. A.G.E. and E.G.S. are Senior Research Fellows of the National Health and Medical Research Council (NHMRC) of Australia. Their work was supported by Stem Cells Australia, the NHMRC and the Victorian Government’s Operational Infrastructure Support Program.

Author information

Author notes

  1. Christopher M. Sturgeon & Xin Cheng

    Present address: Present addresses: Department of Internal Medicine, Hematology Division, Washington University School of Medicine, St Louis, Missouri 63110, USA (C.M.S.); Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China (X.C.).,

Authors and Affiliations

  1. McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario M5G 1L7, Canada

    Andrea Ditadi, Christopher M. Sturgeon, Geneve Awong, Marion Kennedy & Gordon Keller

  2. Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    Joanna Tober, Amanda D. Yzaguirre & Nancy A. Speck

  3. Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Victoria 3052, Australia

    Lisa Azzola, Elizabeth S. Ng, Edouard G. Stanley & Andrew G. Elefanty

  4. Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University Clayton, Victoria 3052, Australia

    Elizabeth S. Ng, Edouard G. Stanley & Andrew G. Elefanty

  5. Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia

    Edouard G. Stanley & Andrew G. Elefanty

  6. Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA

    Deborah L. French, Xin Cheng & Paul Gadue

Authors
  1. Andrea Ditadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher M. Sturgeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joanna Tober
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Geneve Awong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marion Kennedy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Amanda D. Yzaguirre
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lisa Azzola
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Elizabeth S. Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Edouard G. Stanley
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Deborah L. French
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xin Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Paul Gadue
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Nancy A. Speck
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Andrew G. Elefanty
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Gordon Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.D., C.M.S., M.K. and G.K. all participated in the design of the experiments. C.M.S., A.D., G.A. and M.K. performed the experiments. J.T., A.D.Y. and N.A.S. generated the Runx1–GFP mouse data. L.A., E.S.N., E.G.S. and A.G.E. generated and provided the R1C–GFP cell line. D.L.F., X.C. and P.G. generated and provided the HES2-ICN1-ERtm cell line. A.D. and G.K. wrote the manuscript.

Corresponding author

Correspondence to Gordon Keller.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Haemoglobin genes expression analysis of the BFU-derived colonies generated from CD34+CD43− cells after EHT culture.

qRT-PCR analysis of globin gene expression in the BFU-derived erythroid colonies generated from the CD34med CD45+ cells and in EryP-CFC-derived colonies generated from day 8 CD43+ cells isolated from Activin A-induced EBs. FL: RNA from total fetal liver mononuclear cells, BM: adult bone marrow mononuclear cells. EryP-CFCn = 13 individual colonies from 4 independent experiments, BFU-E n = 7 individual colonies from 3 independent experiments, (Mean ± s.e.m.). Student’s t-test, ∗∗P = 0.0086.

Supplementary Figure 2 Generation and characterisation of RUNX1CGFP/w targeted hESCs.

a, Schematic depicting the organization of the RUNX1 genomic locus with exons shown as boxes. Non-coding exons are shown in white and coding exons in black. The distal (D) and proximal promoters (P) and the transcripts emanating from each are indicated. The targeting vector is shown with black triangles marking loxP sites. The targeted allele is shown before and after CRE recombinase mediated excision of the antibiotic resistance cassette (NEO). b, Flow cytometric analysis of undifferentiated RUNX1CGFP/w cells showing expression of the following surface markers associated with pluripotent cells, ECAD (CDH1), CD9, TRA 1 81 and GCTM2. c, RUNX1CGFP/w cells demonstrate a normal female karyotype. d, Pluritest (www.pluritest.org) analysis of transcriptional profiles of undifferentiated RUNX1CGFP/w cells, parental HES3 cells, H9 and MEL1 hESCs, scores all lines as pluripotent, with high pluripotency and low novelty scores. e, Stained haematoxylin and eosin stained paraffin sections of a teratoma derived from undifferentiated RUNX1CGFP/w cells injected under the kidney capsule of an immunocompromised mouse demonstrate diverse cell types derived from three germ layers within the same field, including pigmented epithelium (f), primitive muscle and mesenchyme (g), glandular epithelium (h) and neural rosettes (i). Scale bar, (e) 200 μM, (f)–(i) 50 μM. All images are representatives of three independent experiments.

Supplementary Figure 3 Kinetics of RUNX1C-EGFP expression.

a, Representative flow cytometric analysis of CD34 and RUNX1C-EGFP expression in day 4, 6 and 8 EBs. b, Representative flow cytometric analysis of CD34, CD43 or CD45 (upper panels) and RUNX1C-EGFPexpression (lower panels) in day 4, day 6 and day 8 EBs and after 7 days of EHT culture of day 8 CD34+CD43− cells in IWP2-induced cultures. All images are representatives of three independent experiments.

Supplementary Figure 4 Inhibition of NOTCH signalling by GSI during EHT inhibits T cell potential.

a, qRT-PCR analyses of expression of the Notch target genes HES1, HEY1 and HES5 in day 8 CD34+CD43− populations isolated from EBs treated with DMSO or GSI between days 3 and 8 of differentiation. Cells were derived from H1 hESCs. n = 3, independent experiments. (Mean ± s.e.m.). Student’s t-test, ∗∗P < 0.01. b, Quantification of the effect of GSI treatment during the indicated times on the generation of CD45+ cells at day 7 of EHT culture. n = 4, independent experiements. (Mean ± s.e.m.). ANOVA, ∗∗P < 0.01,∗P < 0.05.

Supplementary Figure 5 Expression of CD73 and CD184 distinguishes HE and VE CD34+CD43− cells derived from different hPSC lines.

a, Kinetic analysis of the expression of CD184 and CD73 in day 4 and day 6 H1-derived CD34+CD43− cells and gating strategy used for the isolation of the CD184+ and CD73+ fractions from the day 6 CD34+ CD43− population. b, Flow cytometric analyses of the frequency of CD34+ and CD45+ cells in populations generated after 7 days of EHT culture from the 3 H1-derived CD184/CD73 fractions isolated at day 6. c, T cell potential of the different H1-derived CD184/CD73 fractions measured by the development of CD4+ and CD8+ cells following culture on OP9-DLL4 stromal cells for 24 days. d, Haematopoietic colony-forming potential of CD184/CD73-derived populations following 7 days of EHT culture. The CD73− CD184− -derived population was treated with GSI during the EHT culture to evaluate NOTCH-dependency. n = 3, independent. (Mean ± s.e.m.).∗∗ ANOVA P < 0.01. e, Flow cytometric analyses of the frequency of CD34+ and CD45+ cells in populations generated from the 3 day 8 R1C-GFP-derived CD184/CD73 fractions following 7 days of EHT culture. f, T cell potential of the different R1C-GFP-derived CD184/CD73 fractions measured by the development of CD4+ and CD8+ cells following culture on OP9-DLL4 stromal cells for 24 days. g, Haematopoietic colony-forming potential of CD184/CD73-derived populations following 7 days of EHT culture. The CD73− CD184− -derived population was treated with GSI during the EHT culture to evaluate NOTCH-dependency. n = 3, independent. (Mean ± s.e.m.).∗∗ ANOVA P < 0.01. All images are representatives of three independent experiments.

Supplementary Figure 6 CD184 and CD73 expression on HE cells in vivo.

a–d, Representative flow cytometric analysis of the frequency of CD184+ and CD73+ cells in the E10.5 aorta-gonad-mesonephros (AGM) (a), E10.5 yolk sac (YS) (b), E8.5 (c) and E9.5 (d) para-aortic splanchnopleura (p-Sp) populations isolated from Runx1-GFP mouse embryos. The proportion of CD184+ and CD73+ cells was measured in the indicated CD31/Runx1-GFP fractions gated to exclude Ter119+ CD41+CD45+ cells (central panels). E10.5 embryos, n = 2; E8.5 and E9.5 embryos, n = 1.

Supplementary information

Supplementary Information

Supplementary Information (PDF 858 kb)

Time-lapse movie showing an adherent cell rounding up and gradually acquiring CD45 expression (in red) during EHT culture.

This cell undergoes EHT and cell division giving rise to a round and an adherent cell, both positive for CD45. Scale bars, 50 μm (MOV 7931 kb)

This movie shows the 3D reconstruction of confocal images of a cluster of emerging round haematopoietic cells in the EHT cultures.

Cells were stained for the endothelial marker CD144 (in green), the haematopoietic marker CD45 (in grey) and for the EHT marker cKIT (in red). Scale bar, 5 μm. (AVI 3961 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ditadi, A., Sturgeon, C., Tober, J. et al. Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages. Nat Cell Biol 17, 580–591 (2015). https://doi.org/10.1038/ncb3161

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3161

This article is cited by

Search

Advanced 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