title: Functional endothelial cells derived from embryonic stem cells labeled with HIV transactivator peptide-conjugated superparamagnetic nanoparticles.
abstract: BACKGROUND
  The development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo, for example, magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Although ES cells have been labeled with SPIO particles, the potential adverse effects of the label have not been fully examined. The objective of this study was to determine whether SPIO labeling affects murine ES cell viability, proliferation, or ability to differentiate into functional endothelial cells (ECs).
  
  
  METHODS
  Cross-linked iron oxide (CLIO, an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides, and murine ES cells were labeled with either CLIO-Tat, CLIO, or HIV-Tat. After labeling, ES cells were cultured for 4 days and Flk-1(+) ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS). Flk-1(+) cells were replated on fibronectin-coated dishes, and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF). ES cell viability was determined using trypan blue exclusion, and the proportion of SPIO(+) cells was evaluated using Prussian blue staining and transmission electron microscopy. After differentiation, the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction, flow cytometry, immunocytochemistry, DiI-labeled acetylated low-density lipoprotein (AcLDL) uptake, and Matrigel tube formation assay.
  
  
  RESULTS
  CLIO-Tat was a highly effective label for ES cells, with > 96% of cells incorporating the particles, and it did not alter the viability of the labeled cells. ECs derived from CLIO-Tat(+) ES cells were very similar to murine aortic ECs in their morphology, expression of endothelial cell markers, ability to form vascular-like channels, and scavenging of AcLDL from the culture medium.
  
  
  CONCLUSIONS
  CLIO-Tat is a highly effective label for ES cells and does not adversely affect cell viability, differentiation, or behavior. CLIO-Tat could be a useful marker for the non-invasive monitoring of transplanted stem cells.
authors.affiliation: Department of Vascular Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Gao
authors.forename: Wei-Guo
authors.initials: WG
authors.lastname: Fu
authors.forename: Zhi-Hui
authors.initials: ZH
authors.lastname: Dong
authors.forename: Zheng-Dong
authors.initials: ZD
authors.lastname: Fang
authors.forename: Zhen-Jie
authors.initials: ZJ
authors.lastname: Liu
authors.forename: Yi
authors.initials: Y
authors.lastname: Si
authors.forename: Xiang-Man
authors.initials: XM
authors.lastname: Zhang
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Wang
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects*; D005290:Ferric Compounds / Q000737:chemistry*; D005434:Flow Cytometry; D007150:Immunohistochemistry; D051379:Mice; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000009:adverse effects* / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D005290:Ferric Compounds