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Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages.

Ditadi A, Sturgeon CM, Tober J, Awong G, Kennedy M, Yzaguirre AD, Azzola L, Ng ES, Stanley EG, French DL, Cheng X, Gadue P, Speck NA, Elefanty AG, Keller G - Nat. Cell Biol. (2015)

Bottom Line: As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE).Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) 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.

View Article: PubMed Central - PubMed

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

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(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) 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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Expression of CD73 and CD184 distinguishes HE and VE in the day 8 CD34+ CD43− populationa, Gating strategy used for the isolation of the CD184+ and CD73+ fractions from the day 8 CD34+ CD43− population. b, Flow cytometric analyses of the frequency of CD34+ and CD45+ cells in populations generated from the 3 H1 hESC-derived CD184/CD73 fractions following 7 days of EHT culture. c, Representative flow cytometric analysis of the frequency of CD34+ and RUNX1C-EGFP+ cells generated from the 3 R1C-GFP hESC-derived CD184/CD73 fractions following 7 days of EHT culture. d, Haematopoietic colony-forming potential of the CD184/CD73-derived populations following 7 days of EHT culture. The CD73−CD184− -derived population was also treated with GSI during the EHT culture to evaluate NOTCH-dependency. n = 3, independent experiements. (Mean ± SEM). ** ANOVA p < 0.0001. e, T cell potential of the different H1-derived CD184/CD73 fractions measured by the development of CD4+ CD8+ cells following culture on OP9-DLL4 stromal cells for 24 days. f, qRT-PCR analysis of haematopoietic (MYB, TAL1 and RUNX1C), arterial (EFNB2, DLL4) and venous gene (NR2F2) expression in the different CD184/CD73 fractions isolated from a day 8 CD34+ EB population generated from H1 hESCs. n = 5, independent experiments. (Mean ± SEM). ** ANOVA MYB, TAL1, EFNB2 and NR2F2 p < 0.0001, RUNX1C p = 0.0003, DLL4 p = 0.0002. Plots in b are representative of 6 independent experiments, in c and e of 3 independent experiments.
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Figure 4: Expression of CD73 and CD184 distinguishes HE and VE in the day 8 CD34+ CD43− populationa, Gating strategy used for the isolation of the CD184+ and CD73+ fractions from the day 8 CD34+ CD43− population. b, Flow cytometric analyses of the frequency of CD34+ and CD45+ cells in populations generated from the 3 H1 hESC-derived CD184/CD73 fractions following 7 days of EHT culture. c, Representative flow cytometric analysis of the frequency of CD34+ and RUNX1C-EGFP+ cells generated from the 3 R1C-GFP hESC-derived CD184/CD73 fractions following 7 days of EHT culture. d, Haematopoietic colony-forming potential of the CD184/CD73-derived populations following 7 days of EHT culture. The CD73−CD184− -derived population was also treated with GSI during the EHT culture to evaluate NOTCH-dependency. n = 3, independent experiements. (Mean ± SEM). ** ANOVA p < 0.0001. e, T cell potential of the different H1-derived CD184/CD73 fractions measured by the development of CD4+ CD8+ cells following culture on OP9-DLL4 stromal cells for 24 days. f, qRT-PCR analysis of haematopoietic (MYB, TAL1 and RUNX1C), arterial (EFNB2, DLL4) and venous gene (NR2F2) expression in the different CD184/CD73 fractions isolated from a day 8 CD34+ EB population generated from H1 hESCs. n = 5, independent experiments. (Mean ± SEM). ** ANOVA MYB, TAL1, EFNB2 and NR2F2 p < 0.0001, RUNX1C p = 0.0003, DLL4 p = 0.0002. Plots in b are representative of 6 independent experiments, in c and e of 3 independent experiments.

Mentions: To enrich for HE within the CD34+ population, we next analysed it for expression of CD73 as recent studies showed that this marker distinguishes haemogenic and non-haemogenic progenitors at early stages in hPSC differentiation cultures20-22. We also monitored CD184 expression, which demarcates arterial endothelium in the mouse embryo and in mESC-derived endothelial populations23, 24. The expression of these 2 markers resolved the following 3 fractions in the day 8 CD34+ population: CD73−CD184−, CD73medCD184+ and CD73hiCD184− (Fig. 4a). When cultured under EHT conditions, all 3 populations generated an adhesive monolayer consisting of cells with endothelial morphology. However, only the CD73−CD184−-derived population underwent EHT and gave rise to CD45+ cells (Fig. 4b), CD34+RUNX1C+cells (Fig. 4c) and myeloid/erythroid (Fig. 4d) and T cell progenitors (Fig. 4e). Limiting-dilution analyses revealed a frequency of 1/35 T cell progenitors in the CD73−CD184− population compared to 1/700 in the nonfractionated CD34+ population, indicating a significant enrichment in HE. As observed with the unfractionated CD34+ population, the transition of the CD73−CD184− cells to a haematopoietic fate appears to be a NOTCH dependent event, as addition of GSI during the EHT culture strongly reduced the development of the myeloid and erythroid progenitors (Fig. 4d).


Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages.

Ditadi A, Sturgeon CM, Tober J, Awong G, Kennedy M, Yzaguirre AD, Azzola L, Ng ES, Stanley EG, French DL, Cheng X, Gadue P, Speck NA, Elefanty AG, Keller G - Nat. Cell Biol. (2015)

Expression of CD73 and CD184 distinguishes HE and VE in the day 8 CD34+ CD43− populationa, Gating strategy used for the isolation of the CD184+ and CD73+ fractions from the day 8 CD34+ CD43− population. b, Flow cytometric analyses of the frequency of CD34+ and CD45+ cells in populations generated from the 3 H1 hESC-derived CD184/CD73 fractions following 7 days of EHT culture. c, Representative flow cytometric analysis of the frequency of CD34+ and RUNX1C-EGFP+ cells generated from the 3 R1C-GFP hESC-derived CD184/CD73 fractions following 7 days of EHT culture. d, Haematopoietic colony-forming potential of the CD184/CD73-derived populations following 7 days of EHT culture. The CD73−CD184− -derived population was also treated with GSI during the EHT culture to evaluate NOTCH-dependency. n = 3, independent experiements. (Mean ± SEM). ** ANOVA p < 0.0001. e, T cell potential of the different H1-derived CD184/CD73 fractions measured by the development of CD4+ CD8+ cells following culture on OP9-DLL4 stromal cells for 24 days. f, qRT-PCR analysis of haematopoietic (MYB, TAL1 and RUNX1C), arterial (EFNB2, DLL4) and venous gene (NR2F2) expression in the different CD184/CD73 fractions isolated from a day 8 CD34+ EB population generated from H1 hESCs. n = 5, independent experiments. (Mean ± SEM). ** ANOVA MYB, TAL1, EFNB2 and NR2F2 p < 0.0001, RUNX1C p = 0.0003, DLL4 p = 0.0002. Plots in b are representative of 6 independent experiments, in c and e of 3 independent experiments.
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Figure 4: Expression of CD73 and CD184 distinguishes HE and VE in the day 8 CD34+ CD43− populationa, Gating strategy used for the isolation of the CD184+ and CD73+ fractions from the day 8 CD34+ CD43− population. b, Flow cytometric analyses of the frequency of CD34+ and CD45+ cells in populations generated from the 3 H1 hESC-derived CD184/CD73 fractions following 7 days of EHT culture. c, Representative flow cytometric analysis of the frequency of CD34+ and RUNX1C-EGFP+ cells generated from the 3 R1C-GFP hESC-derived CD184/CD73 fractions following 7 days of EHT culture. d, Haematopoietic colony-forming potential of the CD184/CD73-derived populations following 7 days of EHT culture. The CD73−CD184− -derived population was also treated with GSI during the EHT culture to evaluate NOTCH-dependency. n = 3, independent experiements. (Mean ± SEM). ** ANOVA p < 0.0001. e, T cell potential of the different H1-derived CD184/CD73 fractions measured by the development of CD4+ CD8+ cells following culture on OP9-DLL4 stromal cells for 24 days. f, qRT-PCR analysis of haematopoietic (MYB, TAL1 and RUNX1C), arterial (EFNB2, DLL4) and venous gene (NR2F2) expression in the different CD184/CD73 fractions isolated from a day 8 CD34+ EB population generated from H1 hESCs. n = 5, independent experiments. (Mean ± SEM). ** ANOVA MYB, TAL1, EFNB2 and NR2F2 p < 0.0001, RUNX1C p = 0.0003, DLL4 p = 0.0002. Plots in b are representative of 6 independent experiments, in c and e of 3 independent experiments.
Mentions: To enrich for HE within the CD34+ population, we next analysed it for expression of CD73 as recent studies showed that this marker distinguishes haemogenic and non-haemogenic progenitors at early stages in hPSC differentiation cultures20-22. We also monitored CD184 expression, which demarcates arterial endothelium in the mouse embryo and in mESC-derived endothelial populations23, 24. The expression of these 2 markers resolved the following 3 fractions in the day 8 CD34+ population: CD73−CD184−, CD73medCD184+ and CD73hiCD184− (Fig. 4a). When cultured under EHT conditions, all 3 populations generated an adhesive monolayer consisting of cells with endothelial morphology. However, only the CD73−CD184−-derived population underwent EHT and gave rise to CD45+ cells (Fig. 4b), CD34+RUNX1C+cells (Fig. 4c) and myeloid/erythroid (Fig. 4d) and T cell progenitors (Fig. 4e). Limiting-dilution analyses revealed a frequency of 1/35 T cell progenitors in the CD73−CD184− population compared to 1/700 in the nonfractionated CD34+ population, indicating a significant enrichment in HE. As observed with the unfractionated CD34+ population, the transition of the CD73−CD184− cells to a haematopoietic fate appears to be a NOTCH dependent event, as addition of GSI during the EHT culture strongly reduced the development of the myeloid and erythroid progenitors (Fig. 4d).

Bottom Line: As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE).Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) 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.

View Article: PubMed Central - PubMed

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

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(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) 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.

Show MeSH
Related in: MedlinePlus