Submitted September 29, 2008
Accepted March 10, 2009
Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo
Andreas Reinisch, Nicole A. Hofmann, Anna C. Obenauf, Karl Kashofer, Eva Rohde, Katharina Schallmoser, Karin Flicker, Gerhard Lanzer, Werner Linkesch, Michael R. Speicher, and Dirk Strunk*
Stem Cell Research Unit, Medical University of Graz, Graz, Austria
Institute of Human Genetics, Medical University of Graz, Graz, Austria
Institute of Pathology, Medical University of Graz, Graz, Austria
University Clinic of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria
Department of Hematology and Stem Cell Transplantation, University Clinic of Internal Medicine, Medical University of Graz, Graz, Austria
* Corresponding author; email: dirk.strunk{at}klinikum-graz.at.
Endothelial progenitor cells (EPCs) are critically involved in essential biological processes such as vascular homeostasis, regeneration and tumor angiogenesis. Endothelial colony-forming cells (ECFCs) are EPCs with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum-free large-scale clinical-grade propagation of mesenchymal stromal cells we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of >30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation and genomic stability were maintained. For the first time, proliferative, functional and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.
