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