Background: The zebrafish embryo (Danio rerio) provides a number of advantages for in vivo study of vertebrate vasculogenesis and angiogenesis, including the possibility to observe in real-time the development of the embryonic vasculature as, for example, by confocal in vivo imaging of endothelial cells sprouting (Lawson et al., 2002). Furthermore, cell transplantation into the zebrafish embryo is a novel and interesting model system for the study of stem cells differentiation and plasticity. (Traver et al., 2003; Traver et al., 2004). Objective: The objective of the present study was 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 blastula and embryo. Methods and Results: hCD34+ cells were isolated from cord blood and subsequently labelled with Orange Cell Tracker™ dye (Invitrogen). hCD34+ cells (400-500) were injected into the zebrafish blastula and hCD14- cells were used as controls. Flow cytometry (FACS) analysis of injected embryos dissociated 24- and 44-hours after transplantation (28hpf and 48hpf stage) showed endothelial differentiation of hCD34+ cells, as demonstrated by increased expression levels of endothelial markers (KDR, CD105, CD146), whereas hematopoietic lineage markers (CD3, CD38, CD45 and CD48) were not affected. Further, immunofluorescence analysis showed KDR+ and VE-cadherin+ human cells localized in the dorsal artery and in the cardinal vein. The above changes were not observed in hCD14- cell – injected zebrafish. In other experiments 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 2 hours after transplantation showed circulating hCD34+ cells in developing vessels and no evidence of vascular occlusions. Further, at 12 hours 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 and ectopic 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. On the contrary, hCD14- cells injected into the embryo do not seem to affect vascular development. 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.
|Titolo:||Human CD34+ cell differentiation toward the endothelial lineage in the zebrafish embryo|
|Data di pubblicazione:||lug-2009|
|Settore Scientifico Disciplinare:||Settore BIO/06 - Anatomia Comparata e Citologia|
Settore BIO/05 - Zoologia
|Citazione:||Human CD34+ cell differentiation toward the endothelial lineage in the zebrafish embryo / O. Pozzoli, G. Iaffaldano, M. Lacovich, P. Devanna, C. Lora Lamia, F. Cotelli, U. Fascio, A. Biondi, M.C. Capogrossi, M. Pesce. ((Intervento presentato al 6. convegno European Zebrafish Genetics and Development Meeting tenutosi a Roma nel 2009.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|