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Blood-forming potential of vascular endothelium in the human embryo

INSERM Unité 506, Villejuif, France

View larger version (114K): [in a new window]   Fig. 1. Immunolocalization of endothelial and hematopoietic cells in the AGM region. (A) Transverse section through a 31-day human embryo immunostained for CD34. The boxed area is the AGM region, including the aorta, gonads and mesonephros. (B-D) Higher magnifications of the same AGM region stained for CD34, CD45 and CD31, respectively. (B) Anti-CD34 stains the endothelium lining the dorsal aorta (da), cardinal veins (cv) and capillaries around the mesonephros (mn). The arrowhead indicates the hematopoietic CD34+ cell progenitors associated with the ventral endothelium of the aorta. (C) CD45 is expressed on all hematopoietic cells present in the embryo, including the aortic stem cells (arrowhead), but is absent from endothelial cells. Rare CD45+ hematopoietic cells scattered in the mesoderm underlying the dorsal aorta are CD34-negative. (D) CD31 stains both endothelial cells and adherent intra-aortic hematopoietic cells. Scale bars: 100 µm; nt, neural tube; g, gonad.

View larger version (31K): [in a new window]   Fig. 2. Differential expression of CD34 and CD45 in human embryonic and fetal tissues. Dissociated cells were double-stained with FITC-anti-CD34 and PE-anti-CD45 MoAbs and analyzed by flow cytometry. Distinct CD34+ CD45+ and CD34+ CD45– cell subsets were observed in the 26-day yolk sac (B), 32-day AGM region (C), 32-day embryonic liver (D) and 24-week fetal bone marrow (E). In 52-day lungs, only CD34+ CD45– cells were present (A).

View larger version (24K): [in a new window]   Fig. 3. CD34+ CD45+ and CD34+CD45– cell sorting from the 40-day embryonic liver and re-analysis by flow cytometry. Dissociated cells were double-stained with FITC-anti-CD34 and PE-anti-CD45 MoAbs. CD34+ CD45+ and CD34+ CD45– cell subsets were sorted inside the gates (A) and immediately re-analyzed (B,C). A fraction of each sorted cell subset was then incubated, first, with biotinylated UEA-1 and then with APC-conjugated streptavidin. All sorted CD34+ CD45– cells were stained by UEA-1 (E), whereas none of the CD34+ CD45+ cells was (D).

View larger version (11K): [in a new window]   Fig. 4. RT-PCR analysis of CD45 expression within CD34+ CD45+ (+/+), CD34+ CD45– (+/–) and CD34– CD45– (–/–) cell subsets sorted from the human embryo. mRNA was extracted from CD34+ CD45– cells sorted from the 32-day yolk sac and 32-day AGM and from CD34+ CD45+, CD34+ CD45– and CD34– CD45– cells sorted from the 40-day embryonic liver. mRNA was also extracted from the whole (W) yolk sac, AGM and embryonic liver. mRNAs were then retro-transcribed into cDNAs and CD45 cDNAs were amplified by PCR. Amplified products were separated on a 1.5% agarose gel and visualized after BET staining. Product size was checked by running molecular weight markers. Signals obtained for ß-actin cDNA amplification were used as references to normalize differences between cDNA samples.

View larger version (97K): [in a new window]   Fig. 5. CD34+ CD45– cells sorted from embryonic tissues develop into endothelial cells in culture. Endothelial cells isolated from umbilical cord veins and CD34+ CD45– cells sorted from the 32-day yolk sac, 32-day AGM and 44-day embryonic liver were grown in EGM2 medium. Cells were observed directly by phase-contrast microscopy (A-D) or, after immunostaining, by fluorescence microscopy (E-T). Fixed cells were stained indirectly with biotinylated UEA-1 (I-L), with an antibody to CD31 (E-H) or with an antibody to vWF, after permeabilization (M- P). After 7 days of culture, confluent layers of cells with typical endothelial shape were obtained from CD34+ CD45– cells sorted from the yolk sac (A), AGM (B) and embryonic liver (C), when compared with HUVEC (D). Endothelial cells developed from CD34+ CD45– cells sorted from the yolk sac (E,I,M), AGM (F,J,N) or embryonic liver (G,K,O), when compared with HUVEC (H,L,P), all express CD31, UEA-1 ligand and vWF. vWF isotype controls on endothelial cells developed in culture from CD34+ CD45- cells sorted from the yolk sac (Q), AGM (R), embryonic liver (S) and from the umbilical vein (T). Scale bars: 50 µm in A-D; 5 µm in E-T.

View larger version (114K): [in a new window]   Fig. 6. Colony formation on MS-5 stroma by 24-week fetal bone marrow hematopoietic and endothelial cells. CD34+ CD45+ (A-C) and CD34+ CD45– (D-F) cells sorted from the fetal bone marrow gave rise to colonies of packed rounded hematopoietic cells on MS-5 cells from day 3 up to day 35 of co-culture. Phase-dark cobblestone-shaped colonies also developed underneath the stroma (arrows) from day 7 to day 35 of co-culture. Scale bars: 20 µm.

View larger version (33K): [in a new window]   Fig. 7. Endothelial and hematopoietic cells sorted from embryonic and fetal hematopoietic tissues yield identical blood cell progenies on MS-5 stroma. 26-day-old yolk sac CD34+ CD45+ and CD34+ CD45– cells and 28-day AGM or 16-week fetal bone marrow CD34+ CD45– cells were grown on MS-5 stromal cells. After 21 days, the whole content of co-cultures was harvested and double-stained with FITC-anti-CD34 and PE-anti-CD45 antibodies or simple-stained with PE-anti-CD33, FITC-anti-CD15, PE-anti-CD19 and PE-anti-CD56 antibodies. Immunostaining was analysed by flow cytometry. Hematopoietic cells were gated on forward and side scatter. YS, yolk sac; BM, bone marrow.

View larger version (31K): [in a new window]   Fig. 8. Frequency of blood-forming CD34+ CD45– endothelial cells in embryonic and fetal hematopoietic tissues. CD34+ CD45– cells sorted from the yolk sac, AGM, embryonic liver and fetal bone marrow were grown on MS-5 stromal cells in limiting dilution conditions. Hematopoietic cell colonies developing on or underneath MS-5 stromal cells were scored at week 5 under a phase-contrast inverted microscope. The number of cells seeded per well was plotted against the logarithm of the percentage of negative wells. As calculated from the line of best fit, the cell number at log 37% negative wells is the frequency of hemogenic cells in the starting population. D, days; W, weeks.

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