figure legend: 3D model of the Trans Golgi Network (TGN) with a mature and an immature Weibel-Palade body (WPB), connected by a small bridge-like structure with the TGN (arrow).
scientific background:
A main topic in the field of applied vascular biology concerns differentiation of stem cells into mature endothelial cells in respect to their role in repairing damaged blood vessels. Differentiating stem cells undergo several maturation stages characterized by a reorganization of the Golgi complex and the Trans Golgi Network (TGN). During maturation of stem cells into endothelial progenitor cells and early matured endothelial cells, the formation of Weibel-Palade bodies (WPBs) starts. These endothelium-specific exocytotic organelles serve apart from other cell functions as storage compartment for the von Willebrand factor (vWF) which plays an important role in hemostasis. WPBs are formed as dilatations of the TGN which pinch off after incorporation of vWF in a crystalline and microtubular structure
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method: CD133 positive stem cells were isolated from human cord blood using density gradient centrifugation and separation with antibody-coated magnetic particles. Cells were seeded onto fibronectin-coated flasks in cytokine-supplemented culture medium in order to initiate differentiation. Clusters of adherent endothelial progenitor cells differentiated into early mature endothelial cells form a significant amount of WPBs. Cells were chemically fixed and conventionally prepared for electron microscopy. Semithin sections (200 nm) were prepared and electron tomography was performed using a Tecnai-20 transmission electron microscope (FEI, Eindhoven, The Netherlands) equipped with a tilting stage. Series of tilted images (range: -65° to + 65°) were acquired with a tilt increment of 1° by the help of the Explore 3D software (FEI). Using an alignment and reconstruction software (Inspect 3D, FEI) the 200 nm volume of the section could be reconstructed into serial slices from which a 3D model could be performed using the Amira 3.0 software (Mercury Computer Systems, Merignac Cedex, France).
method: CD133 positive stem cells were isolated from human cord blood using density gradient centrifugation and separation with antibody-coated magnetic particles. Cells were seeded onto fibronectin-coated flasks in cytokine-supplemented culture medium in order to initiate differentiation. Clusters of adherent endothelial progenitor cells differentiated into early mature endothelial cells form a significant amount of WPBs. Cells were chemically fixed and conventionally prepared for electron microscopy. Semithin sections (200 nm) were prepared and electron tomography was performed using a Tecnai-20 transmission electron microscope (FEI, Eindhoven, The Netherlands) equipped with a tilting stage. Series of tilted images (range: -65° to + 65°) were acquired with a tilt increment of 1° by the help of the Explore 3D software (FEI). Using an alignment and reconstruction software (Inspect 3D, FEI) the 200 nm volume of the section could be reconstructed into serial slices from which a 3D model could be performed using the Amira 3.0 software (Mercury Computer Systems, Merignac Cedex, France).
contact: josef.neumueller@meduniwien.ac.at