Human Bone Marrow Mononuclear Cells, CD34+ Cells and CD133+ Cells Do Not Form Vascular Structures in Vivo

The clinical results of using bone marrow-derived progenitor cells to repair ischemic tissue in coronary disease are controversial since some studies demonstrate improvement of cardiac function, whereas others do not. In these studies both unmanipulated mononuclear bone marrow cells (MNC) and highly enriched stem cells such as CD34+, CD133+ or mesenchymal cells (MSC) were used. In this study we have examined the in vitro and in vivo endothelial progenitor properties of these populations. MNC were obtained from whole BM. CD34+ cells and CD133+ cells were isolated using magnetic bead technology (MACS, Miltenyi). MSC was established from BM, and only second passages from MSC were used. Cytokine profiles of MSC were measured on a Bio-Plex system (BIO-RAD Laboratories). In vitro studies were performed by culturing cells in either complete EGM-2 medium to identify EPC, or in M199 with endothelial growth factors to investigate endothelial differentiation. We observed little or no endothelial differentiation when culturing CD133+ or CD34+ cells in M199 media. Furthermore, we did not observe EPC colony formation of MNC, CD133+ cells, or CD34+ cells cultured in EGM-2 medium. To study the in vivo effect of MNC, CD34+, CD133+ as well as MSC, we adoptively transferred human cells into immunodeficient mice and examined their endothelial potential assessing the formation of blood vessels as readout (Skovseth et al Am J of Pathol 2002, Blood 2005). Human umbilical cord-derived endothelial cells (HUVEC) served as positive controls. MNC only grew as round shaped clustered cells with no signs of vessel formation. Neither CD34+ cells nor CD133+ cells demonstrated signs of endothelial development in vivo. MSCs were found to ex vivo secrete endothelial growth factors, including VEGF, bFGF and EGF. When Matrigel plugs with MSCs were examined, an accumulation of smooth muscle actin-positive cells could be detected, but no vessel like endothelial structures derived from human cells could be found. If these putative smooth muscle cells are differentiated from the MSCs, or are murine host cells attracted to the human MSCs due to the cytokine secretion effects of the MSCs is not clear. Currently, we are exploring if co-cultivation of MSC in combination with MNC, CD133+ cells or CD34+ cells will facilitate endothelial development. In conclusion, our experiments did not show endothelial cell differentiation or vessel formation from human BM-MNC, CD34+ cells or CD133+ cells suggesting that such cells have a limited vasculogenic effect. Our findings with the MSCs are interesting and the mechanism of actions is under further investigation.