Abstract 5425: Human CD34+ Cell Differentiation toward the Endothelial Lineage in the Zebrafish Embryo
Background. The zebrafish embryo provides a number of advantages for in vivo studies of vertebrate angiogenesis, including the possibility to observe in real-time the development of the embryonic vasculature. Furthermore, cell transplantation into the zebrafish embryo is an interesting model system for the study of stem cells differentiation.
Objective. The objective of the present work is to determine whether human CD34+ progenitor (hCD34+) cells differentiate toward the endothelial lineage and contribute to new blood vessels development after transplantation into the zebrafish embryo.
Methods and Results. hCD34+ cells were isolated from cord blood, labelled and injected into the zebrafish blastula (400 cells). Human CD14− cells were used as controls. Flow cytometry analysis of injected embryos dissociated 24- and 44-hours after transplantation showed endothelial differentiation of hCD34+ cells, as demonstrated by increased expression levels of endothelial markers (KDR, CD105, CD146), whereas hematopoietic lineage markers were not affected. Further, we showed KDR+ and Endothelin+ human cells localized in the dorsal artery and in the cardinal vein. hCD34+ and hCD14− cells (500–1000) were injected into the sinus venosus of developing Tg(fli1:EGFP) transgenic embryos (48hpf), prior to immune system development. Time-lapse confocal analysis performed 2h after transplantation showed circulating hCD34+ cells in developing vessels. At 12h after injection, some hCD34+ cells had incorporated into the vessel wall. One day after transplantation, hCD34+- injected embryos exhibited altered blood vessels sprouting in the growing tail vasculature as well as enhanced angiogenesis at the level of the subintestinal vein; these results raised the possibility that hCD34+ cells may synthesize angiogenic factors. In agreement with this hypothesis it was found that hCD34+ cell injection into the zebrafish blastula rescued the vascular phenotype caused by Vegfc knock-down. The above results were not observed in hCD14− cell - injected zebrafish.
Conclusions. The present study demonstrates the evolutionary conservation of hCD34+ cells differentiation mechanisms toward the endothelial lineage and their angiogenic properties in the zebrafish embryo.