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Three specific antigens to isolate endothelial progenitor cells from human liposuction material

  • Gudrun Hager
    Affiliations
    Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria

    Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria
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  • Wolfgang Holnthoner
    Correspondence
    Correspondence: Dr Wolfgang Holnthoner, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Donaueschingenstrasse 13, 1200 Vienna, Austria.
    Affiliations
    Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria

    Department of Biochemical Engineering, University of Applied Sciences Technikum Wien, Vienna, Austria
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  • Susanne Wolbank
    Affiliations
    Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria
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  • Anna-Maria Husa
    Affiliations
    Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria

    Department of Biochemical Engineering, University of Applied Sciences Technikum Wien, Vienna, Austria
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  • Kathrin Godthardt
    Affiliations
    Miltenyi Biotech, Bergisch Gladbach, Germany
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  • Heinz Redl
    Affiliations
    Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria
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  • Christian Gabriel
    Affiliations
    Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria
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      Abstract

      Background aims

      Human endothelial progenitor cells (EPC) play an important role in regenerative medicine and contribute to neovascularization on vessel injury. They are usually enriched from peripheral blood, cord blood and bone marrow. In human fat tissue, EPC are rare and their isolation remains a challenge.

      Methods

      Fat tissue was prepared by collagenase digestion, and the expression of specific marker proteins was evaluated by flow cytometry in the stromal vascular fraction (SVF). For enrichment, magnetic cell sorting was performed with the use of CD133 microbeads and EPC were cultured until colonies appeared. A second purification was performed with CD34; additional isolation steps were performed with the use of a combination of CD34 and CD31 microbeads. Enriched cells were investigated by flow cytometry for the expression of endothelial specific markers, by Matrigel assay and by the uptake of acetylated low-density lipoprotein.

      Results

      The expression pattern confirmed the heterogeneous nature of the SVF, with rare numbers of CD133+ detectable. EPC gained from the SVF by magnetic enrichment showed cobblestone morphology of outgrowth endothelial cells and expressed the specific markers CD31, CD144, vascular endothelial growth factor (VEGF)R2, CD146, CD73 and CD105. Functional integrity was confirmed by uptake of acetylated low-density lipoprotein and the formation of tube-like structures on Matrigel.

      Conclusions

      Rare EPC can be enriched from human fat tissue by magnetic cell sorting with the use of a combination of microbeads directed against CD133, an early EPC marker, CD34, a stem cell marker, and CD31, a typical marker for endothelial cells. In culture, they differentiate into EC and hence could have the potential to contribute to neovascularization in regenerative medicine.

      Key Words

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