Augmented Effects on Neovascularization by Mixing the Conditioned Medium from Early EPCs into Late EPCs Derived from Human Cord Blood.

Recently, we have reported on two different types of EPCs from human umbilical cord blood(ASH 2005, Abstract #1706). These EPCs had different biologic properties in angiogenic capabilities during the HCB ex vivo expansion. In this present study, the aim is to examine the synergism by mixing the conditioned medium from the early EPCs into the late EPCs on neovascularization. The mononuclear cells from the HCB were cultures using an EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. We found early spindle-shaped cells(early EPCs), which were grown in the first week of the culture and late cobblestone shaped cells(late EPCs) which peaked in their growth in the third week of the culture. First, we compared the two types of cells in terms of phenotypic expressions and migration ability. Next, we examined their proliferation capacity and tube formations in the Matrigel plate under the conditions that the early outgrowing cells-contained medium was added to the cobblestone shaped cells. The late-appearing cobblestone shaped cells were positive for VEGFR2, VE-cadherin, CD31, CD34 and CXCR-4 but not for CD14 and CD54. These late outgrowing cells expressed high levels of mRNA on the endothelial marker genes and effectively formed capillary tubes in the Matrigel plates. The early spindle cells excreted more angiogenic cytokines and had more migratory ability. When the early spindle-shaped cell-conditioned medium was added to the late cobblestone shaped outgrowing cells, significantly higher proliferation and tube formation measured by the area and length of tubes were found. These results suggested that the two types of cells raised with different biologic properties during the ex vivo HCB expansion and the angiogenic capacity of late EPCs were augmented by a mutual interaction via the excreted cytokines from early EPCs. These finding may have potential applications for a “cell therapy” in situations such as vascular injuries (ie, hindlimb ischemia/myocardial infarction). Murine models are being tested to see whether the injections of two different EPCs will result in synergic noevascularization in our Lab.