Evolutionary Conservation of Human CD34+ Cell Endothelial Differentiation in the Zebrafish Embryo

Background. We tested the zebrafish embryo as a novel system to study human CD34+ progenitor cells (hCD34+) differentiation toward the endothelial and hematopoietic lineages. Methods and Results. hCD34+ were isolated from cord blood and subsequently labelled either with orange cell trackerTM dye or infected with a lentivirus expressing GFP. Labelled hCD34+ (500–1000 cells) were injected into the sinus venosus of developing wt and Tg(fli1:EGFP) transgenic embryos prior to immune system development. Embryos transplanted with labelled hCD14– cells were used as controls. Time-lapse confocal microscopy at early time points (2 hours) showed hCD34+ circulation in developing vessels of the transgenic embryos and no vascular occlusions. Some hCD34+ integrated into the vessel wall, after their early adhesion to vascular structures. One day after transplantation, hCD34+ injection resulted in severe vascular defects, as increased diameter of dorsal artery (DA) and cardinal vein (CV) (DA=3.8±1.4um vs. 8.2±2.9um, p<0.05 n=11; CV=6.9±0.6um vs. 13±2.3um, p<0.01, in control and hCD34+ injected embryos, respectively), altered branching with ectopic sprouts in the growing vasculature (61/72 embryos), and abnormal angiogenesis in the yolk region (14/17 embryos). Further, it was found evidence of hCD34+ differentiation into mature endothelial hVWf - and hCD31 - positive cells. To investigate hCD34+ plasticity in the early events of vascular development, we performed hCD34+ transplantation into the zebrafish blastula. hCD34+ behaved as hemangioblast, homing to both the vascular and the hemato-poietic compartments. Finally, embryos injected with the specific Vegfc morpholino RNA, that display a vascular phenotype, were rescued by hCD34+ cells transplanted 2 hours later at blastula stage (29/32 embryos), suggesting that transplanted hCD34+ show a potent angiogenic activity that affects the developing vasculature of the Vegfc depleted embryos. Conclusions. Our in vivo study demonstrates the evolutionary conservation of hCD34+ differentiation mechanisms in the zebrafish embryo, supported by hCD34+ participation to the zebrafish blood vessels formation, and by hCD34+ production of angiogenic factors acting on resident embryonic vascular cells.