Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using human umbilical cord blood

Abstract

Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells (EPCs), for a variety of cell therapies. Recently, EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition, we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde- High IntroductionEndothelial progenitor cells (EPCs) were originally identified as a population of stem cells in human peripheral blood (PB) and characterized by the expression of CD34, KDR (VEGFR-2), and CD133 markers. 1-3 Subsequently, EPCs have been isolated from other sources, such as bone marrow (BM), fetal liver, and umbilical cord blood (UCB). [4][5][6] Recent studies have shown that EPCs are a potential tool for therapeutic angiogenesis in the treatment of patients suffering from severe limb ischemia or myocardial infarction. 7 EPCs have been identified as contributors to vessel development in both normal physiological processes such as wound healing and pathological processes such as cancer. 8 The definition of an EPC has been controversial and hence the method to isolate EPCs has been variable among investigators. 9 Several studies have demonstrated that there are 2 distinct types of EPCs, the so-called early and late EPCs, which appear sequentially. 10 Early EPCs, likely originating from monocytic/dendritic cells, are characterized by the expression of CD45 and CD14, together with some endothelial cell (EC) markers, and have a short lifespan of 3 to 4 weeks. On the other hand, late EPCs rapidly grow out from mononuclear cells with a cobblestone-like morphology and are characterized by EC markers such as CD31, CD34, VEGFR2, and VE-cadherin, but are negative for myeloid markers. 10 However, Yoder et al have recently demonstrated that progeny of the CD45 ϩ CD14 ϩ cells that coexpress EC markers are not endothelial progenitor cells but hematopoietic-derived myeloid progenitor cells. 11 Alternatively, Ingram et al divided ECs into several subpopulations according to their clonogenic and proliferative potential. 12 They identified a population of highly proliferative endothelial potential-colony-forming cells (HPP-ECFCs), which form secondary and ternary colonies in human UCB. Given the therapeutic usefulness of EPCs, the effective isolation of highly proliferative EPCs is centrally important for the generation of reliable and safe cell-based therapy.Aldehyde dehydrogenase (ALDH) is an enzyme responsible for oxidizing intercellular aldehydes. 13 This ...

Human Endothelial Progenitor Cells

“…The cord blood ECFC display high telomerase activity and vigorous in vivo human vessel formation when suspended in a matrix and implanted into immunodeficient mice (Ingram et al 2004). The ability of these ECFCs to display spontaneous vasculogenic properties, to integrate long-term into the systemic vasculature of the host animal, and to remodel into arteries and veins in vivo distinguishes this EPC from all other types of cells that have been given this term (Bompais et al 2004;Gulati et al 2004;Hur et al 2004;Ingram et al 2004Ingram et al , 2005Yoon et al 2005;Guven et al 2006;Shepherd et al 2006;Melero-Martin et al 2007;Nagano et al 2007;Timmermans et al 2007;Au et al 2008).…”

“…Subsequent transcriptome, proteomic, and functional analyses have determined that CFU-Hill are more closely related to human hematopoietic cells than to primary endothelial cells. Another assay system identifies outgrowth endothelial cells (OECs) possessing clonal endothelial colony-forming cell (ECFC) ability within 1-3 wk of culture, when blood cells are plated on matrix coated dishes with added growth factors (Gulati et al 2003(Gulati et al , 2004Bompais et al 2004;Hur et al 2004;Ingram et al 2004Ingram et al , 2005Yoon et al 2005;Guven et al 2006;Shepherd et al 2006;Melero-Martin et al 2007;Nagano et al 2007;Timmermans et al 2007;Au et al 2008). A hierarchy of clonal proliferative potential is displayed by the ECFC with some colonies growing to more than 10,000 progeny from a single cell plated 14 þ cells (dark blue gate in B).…”

Rapid Anastomosis of Endothelial Progenitor Cell–Derived Vessels with Host Vasculature Is Promoted by a High Density of Cotransplanted Fibroblasts

“…For example, transplanted EPCs have been shown to induce angiogenesis and restore the function of ischemic tissue. 21,22 Importantly, like other ECs, EPCs can form robust vessel networks in engineered tissue constructs in the presence of stromal cells, and can be functional in vivo for up to 28 days. [17][18][19] However, a study addressing the critical issue of the rate of anastomosis using engineered vessels formed by EPCs has yet to be reported.…”

The Role of CCL5 in the Ability of Adipose Tissue-Derived Mesenchymal Stem Cells to Support Repair of Ischemic Regions

“…A higher number of lectin-binding EC was observed at the femoral region in the AT-MSC and endothelial progenitor cell (EPC) [31] (positive control) groups on day 14 (Fig. 4B).…”

“…The capillary density at the thigh muscle was assessed by immunofluorescence using Banderiraea simplicifolia lectin I-TRITC (0.1 mg/mL; Sigma-Aldrich) as reported previously [31]. The frozen sections were mounted and observed under a microscope equipped with the appropriate filters (Olympus, Tokyo, Japan).…”