Abstract
Circulating endothelial progenitor cells (EPCs) have been the focus of many clinical trials due to their roles in revascularisation following ischemic events such as acute myocardial infarction as well as their contribution to vascular repair during organ transplantation. Research on EPCs has been controversial due to the lack of distinct markers expressed at the cell surface and varying methods for isolation and culture have resulted in the identification of a multitude of cell types, with differing phenotype and function, all falling under the label of “EPCs”. The most widely documented EPCs isolated for cell therapy are adherent in nature and lacking the progenitor markers such as CD133 and therefore unlikely to represent a true circulating EPC, the cells mobilised in response to a vascular injury. We recently published the isolation and extensive characterisation of a population of non-adherent endothelial forming cells (naEFCs) (Appleby et al., 2012) (Figure 1). These cells expressed the progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ EPCs, the ‘late outgrowth EPC’ [more recently known as endothelial colony forming cells (ECFCs)] as well as mature endothelial cells (ECs). Figure 2A exemplifies the surface expression profile of the naEFCs. Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin (Figure 2A), (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature ECs increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human ECs and have contributed to various advances in scientific knowledge (Appleby et al., 2012; Barrett et al., 2011; Moldenhauer et al., 2015; Parham et al., 2015). Here, we describe the isolation and enrichment of a non-adherent CD133+ endothelial forming population of cells from human cord blood.
Keywords: Endothelial progenitor cells, Expansion, Human, CD133, Non-adherent
Figure 1. Enrichment of human naEFCs. A. Umbilical cord blood derived CD133+ enriched cells (naEFCs) at 4 days of culture and human umbilical vein endothelial cells (ECs) were compared for cell size by light microscopy. Scale bar=200 µm B. The cells were assessed for heterogeneity of enrichment process (0-10 days) via forward scatter and side scatter profiling using flow cytometric analysis and compared to mature ECs. Figure 2. Surface expression phenotype of human naEFCs. A. CD133+ enriched cells at 4 days of culture were assessed for progenitor and endothelial markers by flow cytometry. Histograms show a representative experiment from ≥3 biological replicates where grey dashed lines represent isotype controls and solid black lines represent cells stained with the indicated marker. B. The function of the naEFCs was assessed by flow cytometry and compared to mature ECs, detecting the ability of cells to uptake DiI labelled acetylated low density lipoprotein (Ac-LDL) and bind FITC labelled Ulex europaeus agglutinin I (UEA-1) lectin. Density plots represent stained cells of one representative experiment from ≥3 biological replicates.
Materials and Reagents
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Acknowledgments
We thank Dr. Sarah Appleby, Dr. Katie Tooley, Dr. Jeffrey Barrett and Samantha Escarbe for their assistance in developing this protocol; Dr. Rosalie Grivell, the staff and consenting donors at Women’s and Children’s Hospital and Burnside Memorial Hospital for collection of the umbilical cord blood. This project was funded by a Heart Foundation Fellowship to CSB (CR10A4983) as well as a project grant from the Co-operative Research Centre for Biomarker Translation (Trans Bio Ltd).
References
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