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. Author manuscript; available in PMC: 2015 Mar 17.
Published in final edited form as: Nat Commun. 2013;4:2924. doi: 10.1038/ncomms3924

Figure 2. Haemogenic endothelium is restricted to the 23GFP+ EC population.

Figure 2

(a) Experimental approach for data shown in Figs 2–5. (b) Generation of haematopoietic progeny by 23GFP+ or 23GFP– VE-Cadh+Ter119– CD45– CD41– ECs from E8.5 (5–12 sp) concepti or E10.5 (29–35 sp) AGM VU on OP9 stroma. Black arrowhead: round semi-adherent haematopoietic cells; white arrowhead: adherent cobblestone-like cells (scale bar, 50 μm). Flow cytometric analysis confirmed the presence of CD45+ haematopoietic cells. Data are representative of at least three independent cocultures. (c) Representative flow cytometric analysis of haemogenic cultures demonstrates both B-lymphoid (CD19) and myeloid (Mac1 or Gr1) cell generation. (d) May-Grünwald Giemsa stained cytospins of haemogenic cultures reveal myeloid (arrowhead, and ii–iv) and blast-like (arrow and v) haematopoietic cells. Asterisks point out OP9 stromal cells. Scale bar, 10 μm. (e) 23GFP+ or 23GFP– VE-Cadh+ Ter119– CD45– CD41– cells were isolated from E8.5 (5–12 sp) conceptus or E10.5 (29–35 sp) AGM + VU, and cultured on OP9 stroma to support endothelial tubule formation. Tubules were visualized by CD31 staining after 4 days of culture. Representative images and quantification (mean±s.d.) of endothelial tubules generated in OP9 cocultures by 23GFP+ or 23GFP– VE-Cadh+ Ter119– CD45– CD41– cells isolated from E8.5 (5–12 sp) conceptus or E10.5 (29–35 sp) AGM VU (n = 3). Only very limited endothelial tubule formation was observed in the 23GFP+ EC population at E10.5, shown is one of 11 tubules. Scale bar, 200 μm.