Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial p
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ORIGINAL PAPER
Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial progenitor cells from peripheral blood and in vitro formation of primitive endothelial networks Wei-Li Fu . Zhou Xiang . Fu-Guo Huang . Shi-Qiang Cen . Gang Zhong . Xin Duan . Ming Liu . Frankie Leung
Received: 25 March 2015 / Accepted: 28 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Endothelial progenitor cells (EPC) derived from the circulation may be used to enhance neovascularization. Since the combination of granulocyte colonystimulating factor (GCSF) and CXCR4 antagonist AMD3100 efficiently mobilizes hematopoietic stem cells into peripheral circulation, it may increase the pool of endogenously circulating EPC. We tested this hypothesis by administering GCSF and AMD3100 to adult rabbits and rats, isolating mononuclear cells from peripheral blood by Ficoll density gradient centrifugation, and characterizing the blood-derived EPC based on morphology, immunophenotyping, gene expression and other functional analyses. These EPC showed clonal growth similar to that of human umbilical vein endothelial cells when cultured in complete EGM-2 medium on collagen I-precoated culture plates. The EPC exhibited a typical cobblestone-like morphology and were relatively homogeneous by the third passage. The cells expressed the typical endothelial marker CD31 based on flow cytometry and fluorescence microscopy, formed capillary-like structures when
W.-L. Fu Z. Xiang (&) F.-G. Huang S.-Q. Cen G. Zhong X. Duan M. Liu Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu 610041, China e-mail: [email protected] F. Leung Department of Orthopaedics and Traumatology, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong
cultured in Matrigel, internalized DiI-acetylated lowdensity lipoprotein, bound Ulex europaeus agglutinin-1, and expressed CD31 and several other endothelial markers (VEGFR2, VE-cadherin, Tie-2, eNOS, vWF) at significantly higher levels than bone marrow-derived mesenchymal stem cells. These results suggest that the combination of GCSF and AMD3100 can efficiently release stem cells into peripheral circulation and generate EPC that show the desired morphological, immunophenotypic and functional characteristics. This minimally invasive approach may be useful for autologous cell transplantation for postnatal neovasculogenesis and tissue repair. Keywords Endothelial progenitor cells Circulating endothelial progenitor cells Stem cell mobilization peripheral blood stem cell transplantation
Introduction Regenerative medicine relies on cell-based therapies to repair and restore damaged tissues or organs, but current techniques are severely limited by low survival of implanted cells in vivo. For example, up to 99 % of mesenchymal stem cells (MSC) die within 3–4 days after transplantation into the ischemic heart (Das et al. 2010). Survival in vivo depends on angiogenesis, leading many investigators to explore the
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