Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages (original) (raw)

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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.

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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
  2. Christopher M. Sturgeon
  3. Joanna Tober
  4. Geneve Awong
  5. Marion Kennedy
  6. Amanda D. Yzaguirre
  7. Lisa Azzola
  8. Elizabeth S. Ng
  9. Edouard G. Stanley
  10. Deborah L. French
  11. Xin Cheng
  12. Paul Gadue
  13. Nancy A. Speck
  14. Andrew G. Elefanty
  15. Gordon Keller

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 toGordon Keller.

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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-CFC_n_ = 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 _RUNX1C_GFP/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 _RUNX1C_GFP/w cells showing expression of the following surface markers associated with pluripotent cells, ECAD (CDH1), CD9, TRA 1 81 and GCTM2. c, _RUNX1C_GFP/w cells demonstrate a normal female karyotype. d, Pluritest (www.pluritest.org) analysis of transcriptional profiles of undifferentiated _RUNX1C_GFP/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 _RUNX1C_GFP/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-EGFP_expression (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.

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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

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