Methodological Development of a Clonogenic Assay to Determine Endothelial Progenitor Cell Potential

Abstract

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133؉ cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133؉ cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology. (Circ Res. 2011;109:20-37.) Key Words: clonogenic assay Ⅲ differentiation Ⅲ endothelial progenitor cell Ⅲ vasculogenesis D espite significant efforts in research and development with respect to endothelial progenitor cell (EPC) biology during the past 10 years after their initial isolation, 1 EPC remain a controversial topic among researchers because there is no definitive delineation of EPC, no clear differentiation hierarchy, or any unambiguously defined isolation protocol.EPC have been quantified and qualified either as cell populations identified by cell surface markers such as CD34, CD133, vascular endothelial growth factor receptor-2 (VEGFR-2), [1][2][3][4][5][6][7][8] or as adhesive cells 6,9,10 and colonies 11 using conventional EPC culture methods to produce spindle-shape adherent cells from peripheral blood (PB), bone marrow (BM), or umbilical cord blood (UCB) mononuclear cells (MNC) with endothelial growth factors and cytokines. These assays using conventional EPC culture protocols were simple and satisfactory to speculate on the vasculogenic properties of EPC-enriched fractions but have recently been criticized. These assays further group heterogeneous EPC into one qualitative category: "adhesive cultured EPC" without any hierarchical discrimination ...

Development of Serum-Free Quality and Quantity Control Culture of Colony-Forming Endothelial Progenitor Cell for Vasculogenesis

“…In EPC-CFA of cultured cells in all Combs, as previously indicated in mouse or human EPC-CFA [32][33][34][35][36][37], two types of EPC colonies were morphologically detected: PEPC-CFU, formed of small, round cells, and DEPC-CFU, formed of large, spindle-like cells (supplemental online Fig. 1A).…”

“…Considering the necessity of creating a defined assay, a novel adhesive clonogenic assay for the quantitative and qualitative analysis of EPCs based on differentiation hierarchy has been developed recently [32][33][34][35][36][37]. The new EPC colony-forming assay (EPC-CFA) enabled the distinction and the definition of two different types of EPC-colony-forming units (EPC-CFUs), that is, primitive and definitive EPC-CFUs, composed of small and large cells, respectively.…”

“…1B), and immunohistochemical positivities of endothelial antigens, such as endothelial nitric oxide synthase, VEGFR-2, and vascular endothelial (VE) cadherin (supplemental online Fig. 1C) [37].…”

Loss of the Liver X Receptors Disrupts the Balance of Hematopoietic Populations, With Detrimental Effects on Endothelial Progenitor Cells

“…For the EPC colony formation assay, Lin − cells were resuspended in Iscove's modified Dulbecco's medium (Life Technologies) supplemented with 10% FBS. Then, they were plated in mouse serum/factor free methylcellulose‐based medium (StemCell Technologies) supplemented with 50 ng/mL vascular endothelial growth factor, 50 ng/mL basic fibroblast growth factor, 50 ng/mL endothelial growth factor, 50 ng/mL insulin‐like growth factor‐1, 100 ng/mL stem cell factor, 20 ng/mL interleukin‐3 (R&D Systems), and 2 U/mL heparin (Sigma‐Aldrich) 32. For the GM colony formation assay, Lin − cells were resuspended in Iscove's modified Dulbecco's medium supplemented with 2% FBS and plated in mouse methylcellulose‐based medium containing FBS, stem cell factor, interleukin‐3, interleukin‐6, and erythropoietin (StemCell Technologies), supplemented with 10% interleukin‐3 culture supplement (VWR) and 2 ng/mL GM colony‐stimulating factor (R&D Systems) 33.…”

A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization

“…27,28 The number of EPC colonies was assessed 16 days after seeding of Lnk siRNA and control siRNA-transfected KSL cells (1 Â 10 3 cells/well) in a six-well plate with methyl cellulose-containing medium M3236 (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 20 ng/ml SCF (Kirin, Tokyo, Japan), 50 ng/ml VEGF (R&D Systems, Minneapolis, MN), 20 ng/ml interleukin-3 (Kirin) and 50 ng/ml basic fibroblast growth factor (Wako, Osaka, Japan). Two different types of attaching cell colonies made of small EPCs and large EPCs were counted separately.…”