Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of hPSC-derived definitive haemogenic endotheliuma, Experimental scheme. CD34+CD43− cells were isolated from embryoid bodies at day 8 of differentiation, reaggregated overnight in serum-free media supplemented with haematopoietic cytokines and then cultured for additional 6 days onto Matrigel-coated plates in the presence of haematopoietic cytokines to promote the endothelial-tohaematopoietic transition (EHT). This stage is referred to as the EHT culture. Following the EHT culture, the cells were assayed as indicated. b, Photomicrograph of day 8 CD34+ CD43− -derived cells following 1 (upper) and 4 days (lower) of EHT culture. Non-adherent (haematopoietic) cells are visible in the day 4 cultures. Scale bars: 100 μm. c, Representative flow cytometric analysis of the frequency of CD34+ and CD45+ cells in the day 8 CD34+-derived populations at the indicated days of EHT culture. d, Visualization of emerging round haematopoietic cells in EHT cultures by confocal imaging. Cells were stained for the endothelial marker CD144 (in green), the haematopoietic marker CD45 (in gray) and the EHT marker cKIT (in red). Scale bar: 5 μm. Dashed line demarcates a cell coexpressing CD144, CD45 and cKIT (white arrow). e, Gating strategy used to define the different CD34/CD45 fractions in the CD34+-derived population following 7 days of EHT culture. f, T cell potential of the different CD34/CD45 fractions indicated in e measured by the development of CD4+CD8+ cells within a CD45+CD56− gate following culture on OP9-DLL4 stromal cells for 24 days. g, Haematopoietic colony-forming potential of the different CD34/CD45 fractions indicated in e generated following 7 days of EHT culture of day 8 CD34+CD43− cells. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p = 0.002. BFU-E: progenitors that generate large segmented erythroid colonies, CFU-E: progenitors that give rise to small erythroid colonies, Myeloid: includes macrophage and mast cell progenitors. h, qRT-PCR analysis of MYB, RUNX1C, TAL1 and GATA2 expression in the different CD34/CD45 fractions isolated as in e. The day 8 CD34+CD43− population prior to culture is included as a control (ctrl). Cells were derived from H1 hESCs. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p < 0.0001. Images in b and plots in c are representative of 6 independent experiments, in f of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4551438&req=5

Figure 1: Characterization of hPSC-derived definitive haemogenic endotheliuma, Experimental scheme. CD34+CD43− cells were isolated from embryoid bodies at day 8 of differentiation, reaggregated overnight in serum-free media supplemented with haematopoietic cytokines and then cultured for additional 6 days onto Matrigel-coated plates in the presence of haematopoietic cytokines to promote the endothelial-tohaematopoietic transition (EHT). This stage is referred to as the EHT culture. Following the EHT culture, the cells were assayed as indicated. b, Photomicrograph of day 8 CD34+ CD43− -derived cells following 1 (upper) and 4 days (lower) of EHT culture. Non-adherent (haematopoietic) cells are visible in the day 4 cultures. Scale bars: 100 μm. c, Representative flow cytometric analysis of the frequency of CD34+ and CD45+ cells in the day 8 CD34+-derived populations at the indicated days of EHT culture. d, Visualization of emerging round haematopoietic cells in EHT cultures by confocal imaging. Cells were stained for the endothelial marker CD144 (in green), the haematopoietic marker CD45 (in gray) and the EHT marker cKIT (in red). Scale bar: 5 μm. Dashed line demarcates a cell coexpressing CD144, CD45 and cKIT (white arrow). e, Gating strategy used to define the different CD34/CD45 fractions in the CD34+-derived population following 7 days of EHT culture. f, T cell potential of the different CD34/CD45 fractions indicated in e measured by the development of CD4+CD8+ cells within a CD45+CD56− gate following culture on OP9-DLL4 stromal cells for 24 days. g, Haematopoietic colony-forming potential of the different CD34/CD45 fractions indicated in e generated following 7 days of EHT culture of day 8 CD34+CD43− cells. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p = 0.002. BFU-E: progenitors that generate large segmented erythroid colonies, CFU-E: progenitors that give rise to small erythroid colonies, Myeloid: includes macrophage and mast cell progenitors. h, qRT-PCR analysis of MYB, RUNX1C, TAL1 and GATA2 expression in the different CD34/CD45 fractions isolated as in e. The day 8 CD34+CD43− population prior to culture is included as a control (ctrl). Cells were derived from H1 hESCs. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p < 0.0001. Images in b and plots in c are representative of 6 independent experiments, in f of 3 independent experiments.

Mentions: We previously identified a definitive CD34+CD43− population that expresses HE markers (CD31+CD144+KDR+cKITlo) and displayed the capacity to generate T lymphoid, erythroid and myeloid cells following culture on stromal cells2, 10. To be able to monitor the EHT of this population, we isolated hESC-derived CD34+ cells and cultured them on Matrigel, in the presence of haematopoietic cytokines known to promote and sustain haematopoietic differentiation15-17 (EHT culture, Fig. 1a). Under these conditions, the cells rapidly formed an adhesive monolayer that underwent the EHT as demonstrated by the emergence of round cells within 3 to 4 days of culture and of a population of CD45+ cells by day 7 (Fig. 1b-c). Examination of the EHT cultures with time-lapse imaging revealed that the adherent cells gradually acquire CD45 expression and then give rise to non-adherent CD45+ haematopoietic cells (Supplementary Movie 1). Immunostaining analyses showed that the emerging round cells co-express endothelial (CD144) and haematopoietic (CD45) surface markers as well as cKIT, a marker indicative of EHT7, 18 (Fig. 1d, Supplementary Movie 2).


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)

Characterization of hPSC-derived definitive haemogenic endotheliuma, Experimental scheme. CD34+CD43− cells were isolated from embryoid bodies at day 8 of differentiation, reaggregated overnight in serum-free media supplemented with haematopoietic cytokines and then cultured for additional 6 days onto Matrigel-coated plates in the presence of haematopoietic cytokines to promote the endothelial-tohaematopoietic transition (EHT). This stage is referred to as the EHT culture. Following the EHT culture, the cells were assayed as indicated. b, Photomicrograph of day 8 CD34+ CD43− -derived cells following 1 (upper) and 4 days (lower) of EHT culture. Non-adherent (haematopoietic) cells are visible in the day 4 cultures. Scale bars: 100 μm. c, Representative flow cytometric analysis of the frequency of CD34+ and CD45+ cells in the day 8 CD34+-derived populations at the indicated days of EHT culture. d, Visualization of emerging round haematopoietic cells in EHT cultures by confocal imaging. Cells were stained for the endothelial marker CD144 (in green), the haematopoietic marker CD45 (in gray) and the EHT marker cKIT (in red). Scale bar: 5 μm. Dashed line demarcates a cell coexpressing CD144, CD45 and cKIT (white arrow). e, Gating strategy used to define the different CD34/CD45 fractions in the CD34+-derived population following 7 days of EHT culture. f, T cell potential of the different CD34/CD45 fractions indicated in e measured by the development of CD4+CD8+ cells within a CD45+CD56− gate following culture on OP9-DLL4 stromal cells for 24 days. g, Haematopoietic colony-forming potential of the different CD34/CD45 fractions indicated in e generated following 7 days of EHT culture of day 8 CD34+CD43− cells. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p = 0.002. BFU-E: progenitors that generate large segmented erythroid colonies, CFU-E: progenitors that give rise to small erythroid colonies, Myeloid: includes macrophage and mast cell progenitors. h, qRT-PCR analysis of MYB, RUNX1C, TAL1 and GATA2 expression in the different CD34/CD45 fractions isolated as in e. The day 8 CD34+CD43− population prior to culture is included as a control (ctrl). Cells were derived from H1 hESCs. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p < 0.0001. Images in b and plots in c are representative of 6 independent experiments, in f of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4551438&req=5

Figure 1: Characterization of hPSC-derived definitive haemogenic endotheliuma, Experimental scheme. CD34+CD43− cells were isolated from embryoid bodies at day 8 of differentiation, reaggregated overnight in serum-free media supplemented with haematopoietic cytokines and then cultured for additional 6 days onto Matrigel-coated plates in the presence of haematopoietic cytokines to promote the endothelial-tohaematopoietic transition (EHT). This stage is referred to as the EHT culture. Following the EHT culture, the cells were assayed as indicated. b, Photomicrograph of day 8 CD34+ CD43− -derived cells following 1 (upper) and 4 days (lower) of EHT culture. Non-adherent (haematopoietic) cells are visible in the day 4 cultures. Scale bars: 100 μm. c, Representative flow cytometric analysis of the frequency of CD34+ and CD45+ cells in the day 8 CD34+-derived populations at the indicated days of EHT culture. d, Visualization of emerging round haematopoietic cells in EHT cultures by confocal imaging. Cells were stained for the endothelial marker CD144 (in green), the haematopoietic marker CD45 (in gray) and the EHT marker cKIT (in red). Scale bar: 5 μm. Dashed line demarcates a cell coexpressing CD144, CD45 and cKIT (white arrow). e, Gating strategy used to define the different CD34/CD45 fractions in the CD34+-derived population following 7 days of EHT culture. f, T cell potential of the different CD34/CD45 fractions indicated in e measured by the development of CD4+CD8+ cells within a CD45+CD56− gate following culture on OP9-DLL4 stromal cells for 24 days. g, Haematopoietic colony-forming potential of the different CD34/CD45 fractions indicated in e generated following 7 days of EHT culture of day 8 CD34+CD43− cells. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p = 0.002. BFU-E: progenitors that generate large segmented erythroid colonies, CFU-E: progenitors that give rise to small erythroid colonies, Myeloid: includes macrophage and mast cell progenitors. h, qRT-PCR analysis of MYB, RUNX1C, TAL1 and GATA2 expression in the different CD34/CD45 fractions isolated as in e. The day 8 CD34+CD43− population prior to culture is included as a control (ctrl). Cells were derived from H1 hESCs. n = 4, independent experiments. (Mean ± SEM). ** ANOVA p < 0.0001. Images in b and plots in c are representative of 6 independent experiments, in f of 3 independent experiments.
Mentions: We previously identified a definitive CD34+CD43− population that expresses HE markers (CD31+CD144+KDR+cKITlo) and displayed the capacity to generate T lymphoid, erythroid and myeloid cells following culture on stromal cells2, 10. To be able to monitor the EHT of this population, we isolated hESC-derived CD34+ cells and cultured them on Matrigel, in the presence of haematopoietic cytokines known to promote and sustain haematopoietic differentiation15-17 (EHT culture, Fig. 1a). Under these conditions, the cells rapidly formed an adhesive monolayer that underwent the EHT as demonstrated by the emergence of round cells within 3 to 4 days of culture and of a population of CD45+ cells by day 7 (Fig. 1b-c). Examination of the EHT cultures with time-lapse imaging revealed that the adherent cells gradually acquire CD45 expression and then give rise to non-adherent CD45+ haematopoietic cells (Supplementary Movie 1). Immunostaining analyses showed that the emerging round cells co-express endothelial (CD144) and haematopoietic (CD45) surface markers as well as cKIT, a marker indicative of EHT7, 18 (Fig. 1d, Supplementary Movie 2).

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