Generation of pure endothelial cells from transgenic embryonic stem cells exhibiting an endothelial cell-specific expression of green fluorescent protein upon differentiation
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Embryonic stem (ES) cell-derived endothelial cells (EC)s may be used as a therapeutic option in experimental models of diseases originating from vascular lesions. Moreover, studies on these cells may provide insight into EC development and differentiation in the human body. In this regard, it is fundamentally required to label, track, and finally isolate a pure population of ES-cell-derived ECs. In this study, a murine ES cell line was established, which expressed green fluorescent protein (GFP) as well as a zeocin resistance gene under the control of the murine Vascular Endothelial (VE)-cadherin promoter after lentiviral transduction of single ES cells. 192 ES colonies derived from single transduced ES cells were picked randomly and directed to differentiation. From day 6 of differentiation, 12.5% of the clones showed GFP-positive vessel-like structures. Immunofluorescence microscopy demonstrated the co-expression of various EC markers (VE-cadherin, CD31) on ES cell-derived vascular structures. Based on flow cytometry, the highest GFP expression level could be observed in embryoid bodies at differentiation day 8. Flow-cytometric cell sorting at this time point revealed a significantly higher level of expression of the majority of investigated EC markers in the GFP-positive compared to the GFP-negative population. In addition, magnetic beads were used for the isolation of ECs based on CD31 expression. The sorted cells were subsequently subjected to gene profiling, in order to determine the optimal time point for the isolation and subsequent culture of ECs. In the sorted cells on days 6 and 8 of differentiation, all investigated markers of EC differentiation and transcription factors of vasculogenesis demonstrated a markedly higher expression in the CD31-positive versus CD31-negative population. Cultured CD31-positive cells at differentiation day 6 developed a characteristic EC cobblestone morphology, co-expressed GFP and different endothelial markers, and eventually formed tube-like structures. In conclusion, generation of ES cell clones expressing GFP upon differentiation to ECs, and their sorting based on CD31, provides a feasible method for the production of pure labeled ECs. This system may serve as a powerful tool for studies on the differentiation of ECs from ES cells and induced pluripotent stem cells, as well as prospective cellular therapeutic approaches in various diseases associated with vascular damage.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : Laufersweiler