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First published online 17 August 2005
doi: 10.1242/dev.01974

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Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver

Samir Taoudi^*, Aline M. Morrison^*, Hirofumi Inoue, Ruby Gribi, Janice Ure and Alexander Medvinsky

MRC Centre Development in Stem Cell Biology, Institute for Stem Cell Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

View larger version (20K): [in a new window]   Fig. 1. Candidate cell fractions from E11.5-E13.5 embryonic organs were purified by flow cytometry and transplanted into lethally irradiated adult recipients. High-level (>5%) long-term multi-lineage contribution to haematopoiesis was determined after 24 weeks. Path `a' represents the transplantation of freshly dissected organs, whereas path `b' indicates the additional 3-day explant culture step prior to transplantation.

View larger version (32K): [in a new window]   Fig. 2. (A) Flow cytometric analysis of E11.5 AGM region demonstrating endothelial (VE-cadherin+CD45-; R1), haematopoietic (VE-cadherin-CD45+; R3) and double-positive (DP) (VE-cadherin+CD45+; R2) cell populations. The contour plot shown is a representative example compiled from approximately 10e.e. of E11.5 AGM (1.5x106 cells). (B) The endothelial affiliation of the double-positive population extends beyond the expression of VE-cadherin, demonstrated by endothelial-like levels of TIE2, FLK1, PECAM1, Ac-LDL, CD34 and SCA1 expression. The DP and the endothelial populations largely share endothelial and stem cell markers. Isotype control (white) and specific antibody staining (grey) are presented. The analysis of DP, endothelial and haematopoietic populations was made using data from 200-700, 15,000-20,000 and 9000-30,000 live cells, respectively. All data are representative of two to four experiments for each marker analysed.

View larger version (76K): [in a new window]   Fig. 3. Morphology of E11.5 AGM endothelial, double-positive and haematopoietic populations. Cells with blast morphology are highly enriched within the VE-cadherin+CD45+ population (May-Grunwald-Giemsa staining of cytospin preparations).

View larger version (78K): [in a new window]   Fig. 4. In vitro endothelial and haematopoietic differentiation potential of E11.5 AGM fractions sorted according to VE-cadherin (VEcad) and CD45 expression. (A) The frequency of clonogenic myeloid progenitors is 14-fold higher within the DP fraction compared with the haematopoietic (VE-cadherin-CD45+) population. Tight (B) and diffuse colonies (C) are predominantly derived from VE-cadherin+CD45+ cells. (D) Clonal analysis of sorted VE-cadherin+CD45+ cells using OP9 assay reveals that haematopoietic progenitors are present at a frequency of 7.6%. (E) Cobble-stone areas (arrow) were frequently observed beneath the stoma and floating haematopoietic cells (asterisk), shown at higher magnification in F. (G) In vitro endothelial network formation was largely restricted to the VE-cadherin+CD45- population. After 4 days of co-culture with OP9 stromal cells, 60 PECAM1+ tubules (blue staining) were produced from 5000 endothelial cells (H) and extensive network formation was seen from 20,000 cells (I). All data were collected from a minimum of three replicate experiments.

View larger version (29K): [in a new window]   Fig. 5. (A) High-level chimerism could be detected in all major adult haematopoietic organs following the transplantation of VE-cadherin-expressing HSC candidates from the E11.5 AGM region, E12.5 peripheral blood and E13.5 foetal liver. There is significant contribution to short-lived granulocytes/monocytes (MAC1+GR1+) and pro/pre-B-cells (B220+CD43+) within the bone marrow, and immature CD4+CD8+ thymic T-cells 12 weeks after transplantation. (B) Successful haematopoietic reconstitution of secondary recipients demonstrates the long-term self-renewal potential of VE-cadherin+ stem cells. The percentage of cells in each gate/quadrant is indicated.

View larger version (56K): [in a new window]   Fig. 6. (A) VE-cadherin+CD45+ cells can be detected in all organs involved in the emergence (E12.5 yolk sac), migration (E12.5 blood) and expansion (E13.5 liver) of HSCs. The percentage of cells in each quadrant is indicated. Data are representative examples from six experiments. Each contour plot is composed from 1x106 YS, 1x105 (Ter119-) PB and 3x106 FL. Quadrants are based on appropriate isotype control staining (see Fig. S1 in the supplementary material). (B) Circulating double-positive cells of E12.5 peripheral blood show attenuated expression of endothelial markers. Each analysis was made using data from 80-110 DP, 1000-4000 endothelial or 8000-13,000 haematopoietic cells. (C) Within the E13.5 liver, some HSCs remain associated with the DP fraction. However, the majority of HSCs reside within the haematopoietic (VE-cadherin-CD45+) fraction. This is reflected in the upregulation of essential stem cells markers (TIE2, KIT, SCA1 and MAC1) in the haematopoietic population. Each analysis was made using data from 1000-4000 DP, 8000-20,000 endothelial or 30,000-70,000 haematopoietic cells. All data are representative of 2-4 experiments for each marker analysed. Isotype control (white) and specific antibody staining (grey) are presented.

View larger version (31K): [in a new window]   Fig. 7. VE-cadherin and CD45 expression within the E11.5 and E12.5 placental CD34+KIT+ population. (A) CD34 and KIT expression profile of the E11.5 placenta. CD34+KIT+ were gated (black box) and analysed for VE-cadherin and CD45 expression. Contour plot is composed from 1x105 cells. (B) All E11.5 CD34+KIT+ cells co-express VE-cadherin and CD45. (C) At E12.5, the placenta CD34+KIT+ population has markedly lost VE-cadherin expression. Analyses in B and C were made using data from from 2000-4000 gated cells. All data are representative of three experiments. Isotype control (white) and specific antibody staining (grey) are presented.