Article | Published:

Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages

Nature Cell Biology volume 17, pages 580591 (2015) | Download Citation

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+CD73CD184 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.

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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

    • Christopher M. Sturgeon
    •  & Xin Cheng

    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.).

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

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Gordon Keller.

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Videos

  1. 1.

    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

  2. 2.

    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.

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DOI

https://doi.org/10.1038/ncb3161

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